U.S. patent application number 10/994726 was filed with the patent office on 2005-07-07 for lyme disease vaccines.
Invention is credited to Choi, Gil H., Erwin, Alice L., Hanson, Mark S., Lathigra, Raju.
Application Number | 20050147999 10/994726 |
Document ID | / |
Family ID | 34624035 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050147999 |
Kind Code |
A1 |
Choi, Gil H. ; et
al. |
July 7, 2005 |
Lyme disease vaccines
Abstract
The present invention relates to novel vaccines for the
prevention or attenuation of Lyme disease. The invention further
relates to isolated nucleic acid molecules encoding antigenic
polypeptides of Borrelia burgdorferi. Antigenic polypeptides are
also provided, as are vectors, host cells and recombinant methods
for producing the same. The invention additionally relates to
diagnostic methods for detecting Borrelia gene expression.
Inventors: |
Choi, Gil H.; (Rockville,
MD) ; Erwin, Alice L.; (Seattle, WA) ; Hanson,
Mark S.; (Clarksville, MD) ; Lathigra, Raju;
(Germantown, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Family ID: |
34624035 |
Appl. No.: |
10/994726 |
Filed: |
November 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10994726 |
Nov 23, 2004 |
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09830230 |
Sep 27, 2001 |
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09830230 |
Sep 27, 2001 |
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PCT/US98/12718 |
Jun 18, 1998 |
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60053377 |
Jul 22, 1997 |
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60050359 |
Jun 20, 1997 |
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60053344 |
Jul 22, 1997 |
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60057483 |
Sep 3, 1997 |
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Current U.S.
Class: |
435/6.16 ;
435/252.3; 435/320.1; 435/69.3; 435/7.32; 530/350; 530/388.4;
536/23.7 |
Current CPC
Class: |
Y02A 50/401 20180101;
Y02A 50/30 20180101; C07K 2319/01 20130101; C07K 2319/30 20130101;
A61K 38/00 20130101; A61K 39/00 20130101; C07K 14/20 20130101 |
Class at
Publication: |
435/006 ;
435/069.3; 435/252.3; 435/320.1; 530/350; 530/388.4; 536/023.7;
435/007.32 |
International
Class: |
C12Q 001/68; G01N
033/554; G01N 033/569; C07H 021/04; C07K 014/195; C07K 016/12; C12N
015/74 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding any one of the amino acid
sequences of the polypeptides shown in Table 1; (b) a nucleotide
sequence complementary to any one of the nucleotide sequences in
(a); (c) a nucleotide sequence at least 95% identical to any one of
the nucleotide sequences shown in Table 1; and (d) a nucleotide
sequence at least 95% identical to a nucleotide sequence
complementary to any one of the nucleotide sequences shown in Table
1.
2. An isolated nucleic acid molecule of claim 1 comprising a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
identical to a nucleotide sequence in (a) or (b) of claim 1.
3. An isolated nucleic acid molecule of claim 1 comprising a
polynucleotide which encodes an epitope-bearing portion of a
polypeptide in (a) of claim 1.
4. The isolated nucleic acid molecule of claim 3, wherein said
epitope-bearing portion of a polypeptide comprises an amino acid
sequence listed in Table 4.
5. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
6. A recombinant vector produced by the method of claim 5.
7. A host cell comprising the vector of claim 6.
8. A method of producing a polypeptide comprising: (a) growing the
host cell of claim 7 such that the protein is expressed by the
cell; and (b) recovering the expressed polypeptide.
9. An isolated polypeptide comprising a polypeptide selected from
the group consisting of: (a) a polypeptide consisting of one of the
complete amino acid sequences of Table 1; (b) a polypeptide
consisting of one the complete amino acid sequences of Table 1
except the N-terminal residue; (c) a fragment of the polypeptide of
(a) having biological activity; and (d) a fragment of the
polypeptide of (a) which binds to an antibody specific for the
polypeptide of (a).
10. An isolated antibody specific for the polypeptide of claim
9.
11. A polypeptide produced according to the method of claim 8.
12. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to the polypeptide of claim 9.
13. An isolated polypeptide antigen comprising an amino acid
sequence of an B. burgdorferi epitope shown in Table 4.
14. An isolated nucleic acid molecule comprising a polynucleotide
with a nucleotide sequence encoding a polypeptide of claim 9.
15. A hybridoma which produces an antibody of claim 10.
16. A vaccine, comprising: (1) one or more B. burgdorferi
polypeptides selected from the group consisting of a polypeptide of
claim 9; and (2) a pharmaceutically acceptable diluent, carrier, or
excipient; wherein said polypeptide is present, in an amount
effective to elicit protective antibodies in an animal to a member
of the Borrelia genus.
17. A method of preventing or attenuating an infection caused by a
member of the Borrelia genus in an animal, comprising administering
to said animal a polypeptide of claim 9, wherein said polypeptide
is administered in an amount effective to prevent or attenuate said
infection.
18. A method of detecting Borrelia nucleic acids in a biological
sample obtained from an animal, comprising a process selected from
the group consisting of: (a) contacting the sample with one or more
nucleic acids of claim 1, under conditions such that hybridization
occurs, and detecting hybridization of said nucleic acids to the
one or more Borrelia nucleic acid sequences present in the
biological sample; and (b) amplifying one or more Borrelia nucleic
acid sequences in said sample using polymerase chain reaction, and
detecting said amplified Borrelia nucleic acid.
19. A kit for detecting Borrelia antibodies in a biological sample
obtained from an animal, comprising (a) a polypeptide of claim 9
attached to a solid support; and (b) detecting means.
20. A method of detecting Borrelia antibodies in a biological
sample obtained from an animal, comprising (a) contacting the
sample with a polypeptide of claim 9; and (b) detecting
antibody-antigen complexes.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/830,230, filed Sep. 27, 2001, which is the national stage of
International Application No. PCT/US98/12718, filed Jun. 18, 1998,
which claims benefit of U.S. Provisional Application No.
60/057,483, filed Sep. 3, 1997, 60/053,344, filed Jul. 22, 1997,
60/053,377, filed Jul. 22, 1997, and 60/050,359, filed Jun. 20,
1997. U.S. Provisional Application No. 60/057,483 is incorporated
by reference herein in its entirety.
REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as an electronic document on two identical
compact discs (CD-R), labeled "Copy 1" and "Copy 2." These compact
discs each contain the file "PB481D1.ST25.txt" (1,364,231 bytes,
created on Nov. 23, 2004), which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to novel vaccines for the
prevention or attenuation of Lyme disease. The invention further
relates to isolated nucleic acid molecules encoding antigenic
polypeptides of Borrelia burgdorferi. Antigenic polypeptides are
also provided, as are vectors, host cells and recombinant methods
for producing the same. The invention additionally relates to
diagnostic methods for detecting Borrelia gene expression.
BACKGROUND OF THE INVENTION
[0004] Lyme disease (Steere, A. C., Proc. Natl. Acad. Sci. USA 91:
2378-2383 (1991)), or Lyme borreliosis, is presently the most
common human disease in the United States transmitted by an
arthropod vector (Center for Disease Control, Morbid. Mortal.
Weekly Rep. 46(23): 531-535 (1997)). Further, infection of
house-hold pets, such as dogs, is a considerable problem.
[0005] While initial symptoms often include a rash at the infection
point, Lyme disease is a multisystemic disorder that may include
arthritic, carditic, and neurological manifestations. While
antibiotics are currently used to treat active cases of Lyme
disease, B. burgdorferi persists even after prolonged antibiotic
treatment. Further, B. burgdorferi can persist for years in a
mammalian host in the presence of an active immune response
(Straubinger, R. et al., J. Clin. Microbiol. 35: 111-116 (1997);
Steere, A., N. Engl. J. Med. 321: 586-596 (1989)).
[0006] Lyme disease is caused by the related tick-borne spirochetes
classified as Borrelia burgdorferi sensu lato (including B.
burgdorferi sensu stricto, B. afzelii, B. garinii). Although
substantial progress has been made in the biochemical,
ultrastructural, and genetic characterization of the organism, the
spirochetal factors responsible for infectivity, immune evasion and
disease pathogenesis remain largely obscure.
[0007] A number of antigenic B. burgdorferi cell surface proteins
have been identified. These include the outer membrane surface
proteins (Osp) OspA, OspB, OspC and OspD. OspA and OspB are encoded
by tightly linked tandem genes which are transcribed as a single
transcriptional unit (Brusca, J. et al., J. Bacteriol. 173:
8004-8008 (1991)). The most-studied B. burgdorferi membrane protein
is OspA, a lipoprotein antigen expressed by borreliae in resting
ticks and the most abundant protein expressed in vitro by most
borrelial isolates (Barbour, A. G., et al., Infection &
Immunity 41: 795-804 (1983); Howe, T. R., et al., Science 227: 645
(1985)).
[0008] A number of different types of Lyme disease vaccines have
been shown to induce immunological responses. Whole-cell B.
burgdorferi vaccines, for example, have been shown to induce both
immunological responses and protective immunity in several animal
models (Reviewed in Wormser, G., Clin. Infect. Dis. 21: 1267-1274
(1995)). Further, passive immunity has been demonstrated in both
humans and other animals using B. burgdorferi specific
antisera.
[0009] While whole-cell Lyme disease vaccines confer protective
immunity in animal models, use of such vaccines presents the risk
that responsive antibodies will produce an autoimmune response
(Reviewed in Wormser, G., supra). This problem is at least partly
the result of the production of B. burgdorferi specific antibodies
which cross-react with hepatocytes and both muscle and nerve cells.
B. burgdorferi heat shock proteins and the 41-kd flagellin subunit
are believed to contain antigens which elicit production of these
cross-reactive antibodies.
[0010] Single protein subunit vaccines for Lyme disease have also
been tested. The cell surface proteins of B. burgdorferi are
potential candidates for use in such vaccines and several have been
shown to elicit protective immune responses in mammals (Probert, W.
et al., Vaccine 15: 15-19 (1997); Fikrig, E. et al., Infect. Immun.
63: 1658-1662 (1995); Langerman S. et al., Nature 372: 552-556
(1994); Fikrig, E. et al., J. Immunol. 148: 2256-2260 (1992)).
Experimental OspA vaccines, for example, have demonstrated efficacy
in several animal models (Fikrig, E., et al., Proc. Natl. Acad.
Sci. USA 89: 5418-5421 (1992); Johnson, B. J., et al., Vaccine 13:
1086-1094 (1996); Fikrig, E., et al., Infect. Immun. 60: 657-661
(1992); Chang, Y. F., et al., Infection & Immunity 63:
3543-3549 (1995)), and OspA vaccines for human use are under
clinical evaluation (Keller, D., et al., J. Am. Med. Assoc. 271:
1764-1768 (1994); Van Hoecke, C., et al., Vaccine 14: 1620-1626
(1996)). Passive immunity is also conferred by antisera containing
antibodies specific for the full-length OspA protein. Further,
vaccination with plasmid DNA encoding OspA has been demonstrated to
elicit protective immune responses in mice (Luke, C. et al., J.
Infect. Dis. 175: 91-97 (1997); Zhong, W. et al., Eur. J. Immunol.
26: 2749-2757 (1996)).
[0011] Recent immunofluorescence assay observations indicate that
during tick engorgement the expression of OspA by borreliae
diminishes (deSilva, A. M., et al., J. Exp. Med. 183: 271-275
(1996)) while expression of other proteins, exemplified by OspC,
increases (Schwan, T. G., et al., Proc. Natl. Acad. Sci. USA 92:
2909-2913 (1985)). By the time of transmission to hosts,
spirochetes in the tick salivary glands express little or no OspA.
This down-modulation of OspA appears to explain the difficulties in
demonstrating immune responses to this antigen early in infection
following tick bites (Kalish, R. A., et al., Infect. Immun. 63:
2228-2235 (1995); Gern, L., et al., J. Infect. Dis. 167: 971-975
(1993); Schiable, U. E., et al., Immunol. Lett. 36: 219-226 (1993))
or following challenge with limiting doses of cultured borreliae
(Schiable, U. E., et al., Immunol. Lett. 36: 219-226 (1993);
Barthold, S. W. and Bockenstedt, L. K., Infect. Immun. 61:
4696-4702 (1993)).
[0012] Furthermore, OspA-specific antibodies are ineffective if
administered after a borrelial challenge delivered by syringe
(Schiable, U. E., et al., Proc. Natl. Acad. Sci. USA 87: 3768-3772
(1990)) or tick bite (deSilva, A. M., et al., J. Exp. Med. 183:
271-275 (1996)). To be efficacious, OspA vaccines must elicit
protective levels of antibody which are maintained throughout
periods of tick exposure in order to block borrelia transmission
from the arthropod vector.
[0013] Vaccines in current use against other pathogens include in
vivo-expressed antigens which could boost anamnestic responses upon
infection, potentiate the action of immune effector cells and
complement, and inhibit key virulence mechanisms. OspC is both
expressed during infection (Montgomery, R. R., et al., J. Exp. Med.
183: 261-269 (1996)) and a target for protective immunity (Gilmore,
R. D., et al., Infect. Immun. 64: 2234-2239 (1996); Probert, W. S.
and LeFebvre, R. B., Infect. Immun. 62: 1920-1926 (1994);
Preac-Mursic, V., et al., Infection 20: 342-349 (1992)), but mice
immunized with this protein were only protected against challenge
with the homologous borrelial isolate (Probert, W. S., et al., J.
Infect. Dis. 175: 400-405 (1997)). Identification of in
vivo-expressed, and broadly protective, antigens of B. burgdorferi
has remained elusive.
SUMMARY OF THE INVENTION
[0014] The present invention provides isolated nucleic acid
molecules comprising polynucleotides encoding the B. burgdorferi
peptides having the amino acid sequences shown in Table 1. Thus,
one aspect of the invention provides isolated nucleic acid
molecules comprising polynucleotides having a nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding any of the amino acid sequences of the full-length
polypeptides shown in Table 1; (b) a nucleotide sequence encoding
any of the amino acid sequences of the full-length polypeptides
shown in Table 1 but minus the N-terminal methionine residue, if
present; (c) a nucleotide sequence encoding any of the amino acid
sequences of the truncated polypeptides shown in Table 1; and (d) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), or (c) above.
[0015] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), or (d) above, or a polynucleotide which
hybridizes under stringent hybridization conditions to a
polynucleotide in (a), (b), (c), or (d) above. This polynucleotide
which hybridizes does not hybridize under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
consisting of only A residues or of only T residues. Additional
nucleic acid embodiments of the invention relate to isolated
nucleic acid molecules comprising polynucleotides which encode the
amino acid sequences of epitope-bearing portions of a B.
burgdorferi polypeptide having an amino acid sequence in (a), (b),
or (c) above.
[0016] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using these vectors for the production of B. burgdorferi
polypeptides or peptides by recombinant techniques.
[0017] The invention further provides isolated B. burgdorferi
polypeptides having an amino acid sequence selected from the group
consisting of: (a) an amino acid sequence of any of the full-length
polypeptides shown in Table 1; (b) an amino acid sequence of any of
the full-length polypeptides shown in Table 1 but minus the
N-terminal methionine residue, if present; (c) an amino acid
sequence of any of the truncated polypeptides shown in Table 1; and
(d) an amino acid sequence of an epitope-bearing portion of any one
of the polypeptides of (a), (b), or (c).
[0018] The polypeptides of the present invention also include
polypeptides having an amino acid sequence with at least 70%
similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% similarity to those described in (a), (b),
(c), or (d) above, as well as polypeptides having an amino acid
sequence at least 70% identical, more preferably at least 75%
identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to those above; as well as isolated nucleic
acid molecules encoding such polypeptides.
[0019] The present invention further provides a vaccine, preferably
a multi-component vaccine comprising one or more of the B.
burgdorferi polypeptides shown in Table 1, or fragments thereof,
together with a pharmaceutically acceptable diluent, carrier, or
excipient, wherein the B. burgdorferi polypeptide(s) are present in
an amount effective to elicit an immune response to members of the
Borrelia genus in an animal. The B. burgdorferi polypeptides of the
present invention may further be combined with one or more
immunogens of one or more other borrelial or non-borrelial
organisms to produce a multi-component vaccine intended to elicit
an immunological response against members of the Borrelia genus
and, optionally, one or more non-borrelial organisms.
[0020] The vaccines of the present invention can be administered in
a DNA form, e.g., "naked" DNA, wherein the DNA encodes one or more
borrelial polypeptides and, optionally, one or more polypeptides of
a non-borrelial organism. The DNA encoding one or more polypeptides
may be constructed such that these polypeptides are expressed
fusion proteins.
[0021] The vaccines of the present invention may also be
administered as a component of a genetically engineered organism.
Thus, a genetically engineered organism which expresses one or more
B. burgdorferi polypeptides may be administered to an animal. For
example, such a genetically engineered organism may contain one or
more B. burgdorferi polypeptides of the present invention
intracellularly, on its cell surface, or in its periplasmic space.
Further, such a genetically engineered organism may secrete one or
more B. burgdorferi polypeptides.
[0022] The vaccines of the present invention may be co-administered
to an animal with an immune system modulator (e.g., CD86 and
GM-CSF).
[0023] The invention also provides a method of inducing an
immunological response in an animal to one or more members of the
Borrelia genus, e.g., B. burgdorferi sensu stricto, B. afzelii, and
B. garinii, comprising administering to the animal a vaccine as
described above.
[0024] The invention further provides a method of inducing a
protective immune response in an animal, sufficient to prevent or
attenuate an infection by members of the Borrelia genus, comprising
administering to the animal a composition comprising one or more of
the polypeptides shown in Table 1, or fragments thereof. Further,
these polypeptides, or fragments thereof, may be conjugated to
another immunogen and/or administered in admixture with an
adjuvant.
[0025] The invention further relates to antibodies elicited in an
animal by the administration of one or more B. burgdorferi
polypeptides of the present invention.
[0026] The invention also provides diagnostic methods for detecting
the expression of genes of members of the Borrelia genus in an
animal. One such method involves assaying for the expression of a
gene encoding Borrelia peptides in a sample from an animal. This
expression may be assayed either directly (e.g., by assaying
polypeptide levels using antibodies elicited in response to amino
acid sequences shown in Table 1) or indirectly (e.g., by assaying
for antibodies having specificity for amino acid sequences shown in
Table 1). An example of such a method involves the use of the
polymerase chain reaction (PCR) to amplify and detect Borrelia
nucleic acid sequences.
[0027] The present invention also relates to nucleic acid probes
having all or part of a nucleotide sequence shown in Table 1 which
are capable of hybridizing under stringent conditions to Borrelia
nucleic acids. The invention further relates to a method of
detecting one or more Borrelia nucleic acids in a biological sample
obtained from an animal, said one or more nucleic acids encoding
Borrelia polypeptides, comprising:
[0028] a) contacting the sample with one or more of the
above-described nucleic acid probes, under conditions such that
hybridization occurs, and
[0029] b) detecting hybridization of said one or more probes to the
Borrelia nucleic acid present in the biological sample.
DETAILED DESCRIPTION
[0030] The present invention relates to recombinant antigenic B.
burgdorferi polypeptides and fragments thereof. The invention also
relates to methods for using these polypeptides to produce
immunological responses and to confer immunological protection to
disease caused by members of the genus Borrelia. The invention
further relates to nucleic acid sequences which encode antigenic B.
burgdorferi polypeptides and to methods for detecting Borrelia
nucleic acids and polypeptides in biological samples. The invention
also relates to Borrelia specific antibodies and methods for
detecting such antibodies produced in a host animal.
[0031] Definitions
[0032] The following definitions are provided to clarify the
subject matter which the inventors consider to be the present
invention.
[0033] As used herein, the phrase "pathogenic agent" means an agent
which causes a disease state or affliction in an animal. Included
within this definition, for examples, are bacteria, protozoans,
fungi, viruses and metazoan parasites which either produce a
disease state or render an animal infected with such an organism
susceptible to a disease state (e.g., a secondary infection).
Further included are species and strains of the genus Borrelia
which produce disease states in animals.
[0034] As used herein, the term "organism" means any living
biological system, including viruses, regardless of whether it is a
pathogenic agent.
[0035] As used herein, the term "Borrelia" means any species or
strain of bacteria which is members of the genus Borrelia. Included
within this definition are Borrelia burgdorferi sensu lato
(including B. burgdorferi sensu stricto, B. afzelii, B. garinii),
B. andersonii, B. anserina, B. japonica, B. coriaceae, and other
members of the genus Borrelia regardless of whether they are known
pathogenic agents.
[0036] As used herein, the phrase "one or more B. burgdorferi
polypeptides of the present invention" means the amino acid
sequence of one or more of the B. burgdorferi polypeptides
disclosed in Table 1. These polypeptides may be expressed as fusion
proteins wherein the B. burgdorferi polypeptides of the present
invention are linked to additional amino acid sequences which may
be of borrelial or non-borrelial origin. This phrase further
includes fragments of the B. burgdorferi polypeptides of the
present invention.
[0037] As used herein, the phrase "full-length amino acid sequence"
and "full-length polypeptide" refer to an amino acid sequence or
polypeptide encoded by a full-length open reading frame (ORF). An
ORF may be defined as a nucleotide sequence bounded by stop codons
which encodes a putative polypeptide. An ORF may also be defined as
a nucleotide sequence within a stop codon bounded sequence which
contains an initiation codon (e.g., a methionine or valine codon)
on the 5' end and a stop codon on the 3' end.
[0038] As used herein, the phrase "truncated amino acid sequence"
and "truncated polypeptide" refer to a sub-sequence of a
full-length amino acid sequence or polypeptide. Several criteria
may also be used to define the truncated amino acid sequence or
polypeptide. For example, a truncated polypeptide may be defined as
a mature polypeptide (e.g., a polypeptide which lacks a leader
sequence). A truncated polypeptide may also be defined as an amino
acid sequence which is a portion of a longer sequence that has been
selected for ease of expression in a heterologous system but
retains regions which render the polypeptide useful for use in
vaccines (e.g., antigenic regions which are expected to elicit a
protective immune response).
[0039] Additional definitions are provided throughout the
specification.
[0040] Explanation of Table 1
[0041] Table 1 lists B. burgdorferi nucleotide and amino acid
sequences of the present invention. The nomenclature used therein
is as follows:
[0042] "nt" refers to nucleotide sequences;
[0043] "aa" refers to amino acid sequences;
[0044] "f" refers to full-length nucleotide or amino acid
sequences; and
[0045] "t" refers to truncated nucleotide or amino acid
sequences.
[0046] Thus, for example, the designation "f101.aa" refers to the
full-length amino acid sequence of B. burgdorferi polypeptide
number 101. Further, "f101.nt" refers to the full-length nucleotide
sequence encoding the full-length amino acid sequence of B.
burgdorferi polypeptide number 101.
[0047] Explanation of Table 2
[0048] Table 2 lists accession numbers for the closest matching
sequences between the polypeptides of the present invention and
those available through GenBank and GeneSeq databases. These
reference numbers are the database entry numbers commonly used by
those of skill in the art, who will be familiar with their
denominations. The descriptions of the nomenclature for GenBank are
available from the National Center for Biotechnology Information.
Column 1 lists the gene or ORF of the present invention. Column 2
lists the accession number of a "match" gene sequence in GenBank or
GeneSeq databases. Column 3 lists the description of the "match"
gene sequence. Columns 4 and 5 are the high score and smallest sum
probability, respectively, calculated by BLAST. Polypeptides of the
present invention that do not share significant identity/similarity
with any polypeptide sequences of GenBank and GeneSeq are not
represented in Table 2. Polypeptides of the present invention that
share significant identity/similarity with more than one of the
polypeptides of GenBank and GeneSeq are represented more than
once.
[0049] Explanation of Table 3.
[0050] The B. burgdorferi polypeptides of the present invention may
include one or more conservative amino acid substitutions from
natural mutations or human manipulation as indicated in Table 3.
Changes are preferably of a minor nature, such as conservative
amino acid substitutions that do not significantly affect the
folding or activity of the protein. Residues from the following
groups, as indicated in Table 3, may be substituted for one
another: Aromatic, Hydrophobic, Polar, Basic, Acidic, and
Small,
[0051] Explanation of Table 4
[0052] Table 4 lists residues comprising antigenic epitopes of
antigenic epitope-bearing fragments present in each of the full
length B. burgdorferi polypeptides described in Table 1 as
predicted by the inventors using the algorithm of Jameson and Wolf,
(1988) Comp. Appl. Biosci. 4: 181-186. The Jameson-Wolf antigenic
analysis was performed using the computer program PROTEAN (Version
3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street
Madison, Wis.). B. burgdorferi polypeptide shown in Table 1 may one
or more antigenic epitopes comprising residues described in Table
4. It will be appreciated that depending on the analytical criteria
used to predict antigenic determinants, the exact address of the
determinant may vary slightly. The residues and locations shown
described in Table 4 correspond to the amino acid sequences for
each full length gene sequence shown in Table 1 and in the Sequence
Listing. Polypeptides of the present invention that do not have
antigenic epitopes recognized by the Jameson-Wolf algorithm are not
represented in Table 2.
[0053] Selection of Nucleic Acid Sequences Encoding Antigenic B.
burgdorferi Polypeptides
[0054] The present invention provides a select number of ORFs from
those presented in the fragments of the Borrelia burgdorferi genome
which may prove useful for the generation of a protective immune
response. The sequenced B. burgdorferi genomic DNA was obtained
from a sub-cultured isolate of ATCC Deposit No. 35210. The
sub-cultured isolate was deposited on Aug. 8, 1997 at the American
Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md.
20852, and given accession number 202012.
[0055] Some ORFs contained in the subset of fragments of the B.
burgdorferi genome disclosed herein were derived through the use of
a number of screening criteria detailed below. The ORFs are
generally bounded at the amino terminus by a methionine residue and
at the carboxy terminus by a stop codon.
[0056] Many of the selected sequences do not consist of complete
ORFs. Although a polypeptide representing a complete ORF may be the
closest approximation of a protein native to an organism, it is not
always preferred to express a complete ORF in a heterologous
system. It may be challenging to express and purify a highly
hydrophobic protein by common laboratory methods. Some of the
polypeptide vaccine candidates described herein have been modified
slightly to simplify the production of recombinant protein. For
example, nucleotide sequences which encode highly hydrophobic
domains, such as those found at the amino terminal signal sequence,
have been excluded from some constructs used for in vitro
expression of the polypeptides. Furthermore, any highly hydrophobic
amino acid sequences occurring at the carboxy terminus have also
been excluded from the recombinant expression constructs. Thus, in
one embodiment, a polypeptide which represents a truncated or
modified ORF may be used as an antigen.
[0057] While numerous methods are known in the art for selecting
potentially immunogenic polypeptides, many of the ORFs disclosed
herein were selected on the basis of screening all theoretical
Borrelia burgdorferi ORFs for several aspects of potential
immunogenicity. One set of selection criteria are as follows:
[0058] 1. Type I signal sequence: An amino terminal type I signal
sequence generally directs a nascent protein across the plasma and
outer membranes to the exterior of the bacterial cell. Experimental
evidence obtained from studies with Escherichia coli suggests that
the typical type I signal sequence consists of the following
biochemical and physical attributes (Izard, J. W. and Kendall, D.
A. Mol. Microbiol. 13: 765-773 (1994)). The length of the type I
signal sequence is approximately 15 to 25 primarily hydrophobic
amino acid residues with a net positive charge in the extreme amino
terminus. In addition, the central region of the signal sequence
adopts an alpha-helical conformation in a hydrophobic environment.
Finally, the region surrounding the actual site of cleavage is
ideally six residues long, with small side-chain amino acids in the
-1 and -3 positions.
[0059] 2. Type IV signal sequence: The type IV signal sequence is
an example of the several types of functional signal sequences
which exist in addition to the type I signal sequence detailed
above. Although functionally related, the type IV signal sequence
possesses a unique set of biochemical and physical attributes
(Strom, M. S. and Lory, S., J. Bacteriol. 174: 7345-7351 (1992)).
These are typically six to eight amino acids with a net basic
charge followed by an additional sixteen to thirty primarily
hydrophobic residues. The cleavage site of a type IV signal
sequence is typically after the initial six to eight amino acids at
the extreme amino terminus. In addition, type IV signal sequences
generally contain a phenylalanine residue at the +1 site relative
to the cleavage site.
[0060] 3. Lipoprotein: Studies of the cleavage sites of twenty-six
bacterial lipoprotein precursors has allowed the definition of a
consensus amino acid sequence for lipoprotein cleavage. Nearly
three-fourths of the bacterial lipoprotein precursors examined
contained the sequence L-(A,S)-(G,A)-C at positions -3 to +1,
relative to the point of cleavage (Hayashi, S. and Wu, H. C., J.
Bioenerg. Biomembr. 22: 451-471 (1990)).
[0061] 4. LPXTG motif: It has been experimentally determined that
most anchored proteins found on the surface of gram-positive
bacteria possess a highly conserved carboxy terminal sequence. More
than fifty such proteins from organisms such as S. pyogenes, S.
mutans, B. burgdorferi, S. pneumoniae, and others, have been
identified based on their extracellular location and carboxy
terminal amino acid sequence (Fischetti, V. A., ASM News 62:
405-410 (1996)). The conserved region consists of six charged amino
acids at the extreme carboxy terminus coupled to 15-20 hydrophobic
amino acids presumed to function as a transmembrane domain.
Immediately adjacent to the transmembrane domain is a six amino
acid sequence conserved in nearly all proteins examined. The amino
acid sequence of this region is L-P-X-T-G-X, where X is any amino
acid.
[0062] An algorithm for selecting antigenic and immunogenic
Borrelia burgdorferi polypeptides including the foregoing criteria
was developed. The algorithm is similar to that described in U.S.
patent application Ser. No. 08/781,986, filed Jan. 3, 1997, which
is fully incorporated by reference herein. Use of the algorithm by
the inventors to select immunologically useful Borrelia burgdorferi
polypeptides resulted in the selection of a number of the disclosed
ORFs. Polypeptides comprising the polypeptides identified in this
group may be produced by techniques standard in the art and as
further described herein.
[0063] Nucleic Acid Molecules
[0064] The present invention provides isolated nucleic acid
molecules comprising polynucleotides encoding the B. burgdorferi
polypeptides having the amino acid sequences shown in Table 1,
which were determined by sequencing the genome of B. burgdorferi
deposited as ATCC deposit no. 202012 and selected as putative
immunogens.
[0065] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of DNA sequences determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0066] Unless otherwise indicated, each "nucleotide sequence" set
forth herein is presented as a sequence of deoxyribonucleotides
(abbreviated A, G, C and T). However, by "nucleotide sequence" of a
nucleic acid molecule or polynucleotide is intended, for a DNA
molecule or polynucleotide, a sequence of deoxyribonucleotides, and
for an RNA molecule or polynucleotide, the corresponding sequence
of ribonucleotides (A, G, C and U), where each thymidine
deoxyribonucleotide (T) in the specified deoxyribonucleotide
sequence is replaced by the ribonucleotide uridine (U). For
instance, reference to an RNA molecule having a sequence of Table 1
set forth using deoxyribonucleotide abbreviations is intended to
indicate an RNA molecule having a sequence in which each
deoxyribonucleotide A, G or C of Table 1 has been replaced by the
corresponding ribonucleotide A, G or C, and each
deoxyribonucleotide T has been replaced by a ribonucleotide U.
[0067] Nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including,
for instance, cDNA and genomic DNA obtained by cloning or produced
synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known as
the sense strand, or it may be the non-coding strand, also referred
to as the anti-sense strand.
[0068] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0069] In addition, isolated nucleic acid molecules of the
invention include DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode a B.
burgdorferi polypeptides and peptides of the present invention
(e.g. polypeptides of Table 1). That is, all possible DNA sequences
that encode the B. burgdorferi polypeptides of the present
invention. This includes the genetic code and species-specific
codon preferences known in the art. Thus, it would be routine for
one skilled in the art to generate the degenerate variants
described above, for instance, to optimize codon expression for a
particular host (e.g., change codons in the bacteria mRNA to those
preferred by a mammalian or other bacterial host such as E.
coli).
[0070] The invention further provides isolated nucleic acid
molecules having the nucleotide sequence shown in Table 1 or a
nucleic acid molecule having a sequence complementary to one of the
above sequences. Such isolated molecules, particularly DNA
molecules, are useful as probes for gene mapping and for
identifying B. burgdorferi in a biological sample, for instance, by
PCR, Southern blot, Northern blot, or other form of hybridization
analysis.
[0071] The present invention is further directed to nucleic acid
molecules encoding portions or fragments of the nucleotide
sequences described herein. Fragments include portions of the
nucleotide sequences of Table 1 at least 10 contiguous nucleotides
in length selected from any two integers, one of which representing
a 5' nucleotide position and a second of which representing a 3'
nucleotide position, where the first nucleotide for each nucleotide
sequence in Table 1 is position 1. That is, every combination of a
5' and 3' nucleotide position that a fragment at least 10
contiguous nucleotides in length could occupy is included in the
invention. "At least" means a fragment may be 10 contiguous
nucleotide bases in length or any integer between 10 and the length
of an entire nucleotide sequence of Table 1 minus 1. Therefore,
included in the invention are contiguous fragments specified by any
5' and 3' nucleotide base positions of a nucleotide sequences of
Table 1 wherein the contiguous fragment is any integer between 10
and the length of an entire nucleotide sequence minus 1.
[0072] Further, the invention includes polynucleotides comprising
fragments specified by size, in nucleotides, rather than by
nucleotide positions. The invention includes any fragment size, in
contiguous nucleotides, selected from integers between 10 and the
length of an entire nucleotide sequence minus 1. Preferred sizes of
contiguous nucleotide fragments include 20 nucleotides, 30
nucleotides, 40 nucleotides, 50 nucleotides. Other preferred sizes
of contiguous nucleotide fragments, which may be useful as
diagnostic probes and primers, include fragments 50-300 nucleotides
in length which include, as discussed above, fragment sizes
representing each integer between 50-300. Larger fragments are also
useful according to the present invention corresponding to most, if
not all, of the nucleotide sequences shown in Table 1 or of the B.
burgdorferi nucleotide sequences of the plasmid clones listed in
Table 1. The preferred sizes are, of course, meant to exemplify not
limit the present invention as all size fragments, representing any
integer between 10 and the length of an entire nucleotide sequence
minus 1, are included in the invention. Additional preferred
nucleic acid fragments of the present invention include nucleic
acid molecules encoding epitope-bearing portions of B. burgdorferi
polypeptides identified in Table 4.
[0073] The present invention also provides for the exclusion of any
fragment, specified by 5' and 3' base positions or by size in
nucleotide bases as described above for any nucleotide sequence of
Table 1 or the plasmid clones listed in Table 1. Any number of
fragments of nucleotide sequences in Table 1 or the plasmid clones
listed in Table 1, specified by 5' and 3' base positions or by size
in nucleotides, as described above, may be excluded from the
present invention.
[0074] Preferred nucleic acid fragments of the present invention
also include nucleic acid molecules encoding epitope-bearing
portions of the B. burgdorferi polypeptides shown in Table 1. Such
nucleic acid fragments of the present invention include, for
example, nucleic acid molecules encoding polypeptide fragments
comprising from about the amino terminal residue to about the
carboxy terminal residue of each fragment shown in Table 4. The
above referred to polypeptide fragments are antigenic regions of
particular B. burgdorferi polypeptides shown in Table 1. Methods
for determining other such epitope-bearing portions for the
remaining polypeptides described in Table 1 are well known in the
art and are described in detail below.
[0075] In another aspect, the invention provides isolated nucleic
acid molecules comprising polynucleotides which hybridize under
stringent hybridization conditions to a portion of a polynucleotide
in a nucleic acid molecule of the invention described above, for
instance, a nucleic acid sequence shown in Table 1. By "stringent
hybridization conditions" is intended overnight incubation at 42 C
in a solution comprising: 50% formamide, 5.times.SSC (150 mM NaCl,
15 mM trisodium citrate), 0.50 mM sodium phosphate (pH 7.6),
5.times. Denhardt's solution, 10% dextran sulfate, and 20 g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65 C.
[0076] By polynucleotides which hybridize to a "portion" of a
polynucleotide is intended polynucleotides (either DNA or RNA)
which hybridize to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0077] Of course, polynucleotides hybridizing to a larger portion
of the reference polynucleotide, for instance, a portion 50-100 nt
in length, or even to the entire length of the reference
polynucleotide, are also useful as probes according to the present
invention, as are polynucleotides corresponding to most, if not
all, of a nucleotide sequence as shown in Table 1. By a portion of
a polynucleotide of "at least 20 nt in length," for example, is
intended 20 or more contiguous nucleotides from the nucleotide
sequence of the reference polynucleotide (e.g., a nucleotide
sequences as shown in Table 1). As noted above, such portions are
useful diagnostically either as probes according to conventional
DNA hybridization techniques or as primers for amplification of a
target sequence by PCR, as described, for instance, in Molecular
Cloning, A Laboratory Manual, 2nd. edition, Sambrook, J., Fritsch,
E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989), the entire disclosure of which is
hereby incorporated herein by reference.
[0078] Since nucleic acid sequences encoding the B. burgdorferi
polypeptides of the present invention are provided in Table 1,
generating polynucleotides which hybridize to portions of these
sequences would be routine to the skilled artisan. For example, the
hybridizing polynucleotides of the present invention could be
generated synthetically according to known techniques.
[0079] As indicated, nucleic acid molecules of the present
invention which encode B. burgdorferi polypeptides of the present
invention may include, but are not limited to those encoding the
amino acid sequences of the polypeptides by themselves; and
additional coding sequences which code for additional amino acids,
such as those which provide additional functionalities. Thus, the
sequences encoding these polypeptides may be fused to a marker
sequence, such as a sequence encoding a peptide which facilitates
purification of the fused polypeptide. In certain preferred
embodiments of this aspect of the invention, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (Qiagen, Inc.), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86: 821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the resulting fusion
protein.
[0080] Thus, the present invention also includes genetic fusions
wherein the B. burgdorferi nucleic acid sequences coding sequences
provided in Table 1 are linked to additional nucleic acid sequences
to produce fusion proteins. These fusion proteins may include
epitopes of borrelial or non-borrelial origin designed to produce
proteins having enhanced immunogenicity. Further, the fusion
proteins of the present invention may contain antigenic
determinants known to provide helper T-cell stimulation, peptides
encoding sites for post-translational modifications which enhance
immunogenicity (e.g., acylation), peptides which facilitate
purification (e.g., histidine "tag"), or amino acid sequences which
target the fusion protein to a desired location (e.g., a
heterologous leader sequence). For instance, hexa-histidine
provides for convenient purification of the fusion protein. See
Gentz et al. (1989) Proc. Natl. Acad. Sci. 86: 821-24. The "HA" tag
is another peptide useful for purification which corresponds to an
epitope derived from the influenza hemagglutinin protein. See
Wilson et al. (1984) Cell 37: 767. As discussed below, other such
fusion proteins include the B. burgdorferi polypeptides of the
present invention fused to Fc at the N- or C-terminus.
[0081] Post-translational modification of the full-length B.
burgdorferi OspA protein expressed in E. coli is believed to
increase the immunogenicity of this protein. Erdile, L. et al.,
Infect. Immun. 61: 81-90 (1993). B. burgdorferi OspA when expressed
in E. coli, for example, is post-translationally modified in at
least two ways. First, a signal peptide is cleaved; second, lipid
moieties are attached. The presence of these lipid moieties is
believed to confer enhanced immunogenicity and results in the
elicitation of a strong protective immunological response.
[0082] Variant and Mutant Polynucleotides
[0083] The present invention thus includes nucleic acid molecules
and sequences which encode fusion proteins comprising one or more
B. burgdorferi polypeptides of the present invention fused to an
amino acid sequence which allows for post-translational
modification to enhance immunogenicity. This post-translational
modification may occur either in vitro or when the fusion protein
is expressed in vivo in a host cell. An example of such a
modification is the introduction of an amino acid sequence which
results in the attachment of a lipid moiety. Such a lipid moiety
attachment site of OspA, which is lipidated upon expression in E.
coli, has been identified. Bouchon, B. et al., Anal. Biochem. 246:
52-61 (1997).
[0084] Thus, as indicated above, the present invention includes
genetic fusions wherein a B. burgdorferi nucleic acid sequence
provided in Table 1 is linked to a nucleotide sequence encoding
another amino acid sequence. These other amino acid sequences may
be of borrelial origin (e.g., another sequence selected from Table
1) or non-borrelial origin. An example of such a fusion protein is
reported in Fikrig, E. et al., Science 250: 553-556 (1990) where an
OspA-glutathione-5-transferase fusion protein was produced and
shown to elicit protective immunity against Lyme disease in immune
competent mice.
[0085] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the B. burgdorferi polypeptides
shown in Table 1. Variants may occur naturally, such as a natural
allelic variant. By an "allelic variant" is intended one of several
alternate forms of a gene occupying a given locus on a chromosome
of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,
New York (1985). Non-naturally occurring variants may be produced
using art-known mutagenesis techniques.
[0086] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. These variants
may be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the B. burgdorferi polypeptides disclosed herein or
portions thereof. Also especially preferred in this regard are
conservative substitutions.
[0087] The present application is further directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleic acid sequence shown in Table 1. The above nucleic acid
sequences are included irrespective of whether they encode a
polypeptide having B. burgdorferi activity. This is because even
where a particular nucleic acid molecule does not encode a
polypeptide having B. burgdorferi activity, one of skill in the art
would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe. Uses of the nucleic acid
molecules of the present invention that do not encode a polypeptide
having B. burgdorferi activity include, inter alia, isolating an B.
burgdorferi gene or allelic variants thereof from a DNA library,
and detecting B. burgdorferi mRNA expression samples, environmental
samples, suspected of containing B. burgdorferi by Northern Blot
analysis.
[0088] Embodiments of the invention include isolated nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence
at least 90% identical, and more preferably at least 95%, 96%, 97%,
98% or 99% identical to (a) a nucleotide sequence encoding any of
the amino acid sequences of the full-length polypeptides shown in
Table 1; (b) a nucleotide sequence encoding any of the amino acid
sequences of the full-length polypeptides shown in Table 1 but
minus the N-terminal methionine residue, if present; (c) a
nucleotide sequence encoding any of the amino acid sequences of the
truncated polypeptides shown in Table 1; and (d) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), or (c) above.
[0089] Preferred, are nucleic acid molecules having sequences at
least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in Table 1, which do, in fact, encode a polypeptide
having B. burgdorferi protein activity By "a polypeptide having B.
burgdorferi activity" is intended polypeptides exhibiting activity
similar, but not necessarily identical, to an activity of the B.
burgdorferi protein of the invention, as measured in a particular
biological assay suitable for measuring activity of the specified
protein.
[0090] Due to the degeneracy of the genetic code, one of ordinary
skill in the art will immediately recognize that a large number of
the nucleic acid molecules having a sequence at least 90%, 95%,
96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown
in Table 1 will encode a polypeptide having B. burgdorferi protein
activity. In fact, since degenerate variants of these nucleotide
sequences all encode the same polypeptide, this will be clear to
the skilled artisan even without performing the above described
comparison assay. It will be further recognized in the art that,
for such nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode a polypeptide having B.
burgdorferi protein activity. This is because the skilled artisan
is fully aware of amino acid substitutions that are either less
likely or not likely to significantly effect protein function
(e.g., replacing one aliphatic amino acid with a second aliphatic
amino acid), as further described below.
[0091] The biological activity or function of the polypeptides of
the present invention are expected to be similar or identical to
polypeptides from other bacteria that share a high degree of
structural identity/similarity. Tables 2 lists accession numbers
and descriptions for the closest matching sequences of polypeptides
available through GenBank and Derwent databases. It is therefore
expected that the biological activity or function of the
polypeptides of the present invention will be similar or identical
to those polypeptides from other bacterial genuses, species, or
strains listed in Table 2.
[0092] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence of
the present invention, it is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the B. burgdorferi polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% (5 of
100) of the nucleotides in the reference sequence may be deleted,
inserted, or substituted with another nucleotide. The query
sequence may be an entire sequence shown in Table 1, the ORF (open
reading frame), or any fragment specified as described herein.
[0093] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99%
identical to a nucleotide sequence of the presence invention can be
determined conventionally using known computer programs. A
preferred method for determining the best overall match between a
query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be
determined using the FASTDB computer program based on the algorithm
of Brutlag et al. See Brutlag et al. (1990) Comp. App. Biosci. 6:
237-245. In a sequence alignment the query and subject sequences
are both DNA sequences. An RNA sequence can be compared by first
converting U's to T's. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB
alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject
nucleotide sequence, whichever is shorter.
[0094] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only nucleotides outside the 5' and 3'
nucleotides of the subject sequence, as displayed by the FASTDB
alignment, which are not matched/aligned with the query sequence,
are calculated for the purposes of manually adjusting the percent
identity score.
[0095] For example, a 90 nucleotide subject sequence is aligned to
a 100 nucleotide query sequence to determine percent identity. The
deletions occur at the 5' end of the subject sequence and
therefore, the FASTDB alignment does not show a matched/alignment
of the first 10 nucleotides at 5' end. The 10 unpaired nucleotides
represent 10% of the sequence (number of nucleotides at the 5' and
3' ends not matched/total number of nucleotides in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 nucleotides
were perfectly matched the final percent identity would be 90%. In
another example, a 90 nucleotide subject sequence is compared with
a 100 nucleotide query sequence. This time the deletions are
internal deletions so that there are no nucleotides on the 5' or 3'
of the subject sequence which are not matched/aligned with the
query. In this case the percent identity calculated by FASTDB is
not manually corrected. Once again, only nucleotides 5' and 3' of
the subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0096] Vectors and Host Cells
[0097] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of B. burgdorferi polypeptides or fragments thereof
by recombinant techniques.
[0098] Recombinant constructs may be introduced into host cells
using well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0099] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0100] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0101] In certain preferred embodiments in this regard, the vectors
provide for specific expression, which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0102] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0103] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating site at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0104] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0105] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A
available from Stratagene; pET series of vectors available from
Novagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available
from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. Other suitable
vectors will be readily apparent to the skilled artisan.
[0106] Among known bacterial promoters suitable for use in the
present invention include the E. coli lacI and lacZ promoters, the
T3 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0107] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0108] Transcription of DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act to increase transcriptional activity of a promoter in a given
host cell-type. Examples of enhancers include the SV40 enhancer,
which is located on the late side of the replication origin at bp
100 to 270, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
[0109] For secretion of the translated polypeptide into the lumen
of the endoplasmic reticulum, into the periplasmic space or into
the extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0110] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, hIL5-receptor has been fused with Fc portions for the purpose
of high-throughput screening assays to identify antagonists of
hIL-5. See Bennett, D. et al., J. Molec. Recogn. 8: 52-58 (1995)
and Johanson, K. et al., J. Biol. Chem. 270 (16): 9459-9471
(1995).
[0111] The B. burgdorferi polypeptides can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, lectin chromatography and high performance liquid
chromatography ("HPLC") is employed for purification. Polypeptides
of the present invention include naturally purified products,
products of chemical synthetic procedures, and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells.
[0112] Polypeptides and Fragments
[0113] The invention further provides isolated polypeptides having
the amino acid sequences in Table 1, and peptides or polypeptides
comprising portions of the above polypeptides. The terms "peptide"
and "oligopeptide" are considered synonymous (as is commonly
recognized) and each term can be used interchangeably as the
context requires to indicate a chain of at least to amino acids
coupled by peptidyl linkages. The word "polypeptide" is used herein
for chains containing more than ten amino acid residues. All
oligopeptide and polypeptide formulas or sequences herein are
written from left to right and in the direction from amino terminus
to carboxy terminus.
[0114] As discussed in detail below, immunization using B.
burgdorferi sensu stricto isolate B31 decorin-binding protein
elicits the production of antiserum which confers passive immunity
against Borrelia species and strains which express divergent forms
of this protein. Cassatt, D. et al., Protection of Borrelia
burgdorferi Infection by Antibodies to Decorin-binding Protein, in
VACCINES97, Cold Spring Harbor Press (1997), pages 191-195. Thus,
some amino acid sequences of the B. burgdorferi polypeptides shown
in Table 1 can be varied without significantly effecting the
antigenicity of the polypeptides. If such differences in sequence
are contemplated, it should be remembered that there will be
critical areas on the polypeptide which determine antigenicity. In
general, it is possible to replace residues which do not form part
of an antigenic epitope without significantly effecting the
antigenicity of a polypeptide.
[0115] Variant and Mutant Polypeptides
[0116] To improve or alter the characteristics of B. burgdorferi
polypeptides of the present invention, protein engineering may be
employed. Recombinant DNA technology known to those skilled in the
art can be used to create novel mutant proteins or muteins
including single or multiple amino acid substitutions, deletions,
additions, or fusion proteins. Such modified polypeptides can show,
e.g., enhanced activity or increased stability. In addition, they
may be purified in higher yields and show better solubility than
the corresponding natural polypeptide, at least under certain
purification and storage conditions.
[0117] N-Terminal and C-Terminal Deletion Mutants
[0118] It is known in the art that one or more amino acids may be
deleted from the N-terminus or C-terminus without substantial loss
of biological function. For instance, Ron et al. J. Biol. Chem.,
268: 2984-2988 (1993), reported modified KGF proteins that had
heparin binding activity even if 3, 8, or 27 N-terminal amino acid
residues were missing. Accordingly, the present invention provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the B. burgdorferi
polypeptides shown in Table 1, and polynucleotides encoding such
polypeptides.
[0119] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance, Interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein See,
e.g., Dobeli, et al. (1988) J. Biotechnology 7: 199-216.
Accordingly, the present invention provides polypeptides having one
or more residues from the carboxy terminus of the amino acid
sequence of the B. burgdorferi polypeptides shown in Table 1. The
invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini as described
below.
[0120] The present invention is further directed to polynucleotide
encoding portions or fragments of the amino acid sequences
described herein as well as to portions or fragments of the
isolated amino acid sequences described herein. Fragments include
portions of the amino acid sequences of Table 1, are at least 5
contiguous amino acid in length, are selected from any two
integers, one of which representing a N-terminal position. The
initiation codon of the polypeptides of the present inventions
position 1. Every combination of a N-terminal and C-terminal
position that a fragment at least 5 contiguous amino acid residues
in length could occupy, on any given amino acid sequence of Table 1
is included in the invention. At least means a fragment may be 5
contiguous amino acid residues in length or any integer between 5
and the number of residues in a full length amino acid sequence
minus 1. Therefore, included in the invention are contiguous
fragments specified by any N-terminal and C-terminal positions of
amino acid sequence set forth in Table 1 wherein the contiguous
fragment is any integer between 5 and the number of residues in a
full length sequence minus 1.
[0121] Further, the invention includes polypeptides comprising
fragments specified by size, in amino acid residues, rather than by
N-terminal and C-terminal positions. The invention includes any
fragment size, in contiguous amino acid residues, selected from
integers between 5 and the number of residues in a full length
sequence minus 1. Preferred sizes of contiguous polypeptide
fragments include about 5 amino acid residues, about 10 amino acid
residues, about 20 amino acid residues, about 30 amino acid
residues, about 40 amino acid residues, about 50 amino acid
residues, about 100 amino acid residues, about 200 amino acid
residues, about 300 amino acid residues, and about 400 amino acid
residues. The preferred sizes are, of course, meant to exemplify,
not limit, the present invention as all size fragments representing
any integer between 5 and the number of residues in a full length
sequence minus 1 are included in the invention. The present
invention also provides for the exclusion of any fragments
specified by N-terminal and C-terminal positions or by size in
amino acid residues as described above. Any number of fragments
specified by N-terminal and C-terminal positions or by size in
amino acid residues as described above may be excluded.
[0122] The above fragments need not be active since they would be
useful, for example, in immunoassays, in epitope mapping, epitope
tagging, to generate antibodies to a particular portion of the
protein, as vaccines, and as molecular weight markers.
[0123] Other Mutants
[0124] In addition to N- and C-terminal deletion forms of the
protein discussed above, it also will be recognized by one of
ordinary skill in the art that some amino acid sequences of the B.
burgdorferi polypeptide can be varied without significant effect of
the structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity.
[0125] Thus, the invention further includes variations of the B.
burgdorferi polypeptides which show substantial B. burgdorferi
polypeptide activity or which include regions of B. burgdorferi
protein such as the protein portions discussed below. Such mutants
include deletions, insertions, inversions, repeats, and type
substitutions selected according to general rules known in the art
so as to have little effect on activity. For example, guidance
concerning how to make phenotypically silent amino acid
substitutions is provided. There are two main approaches for
studying the tolerance of an amino acid sequence to change. See,
Bowie, J. U. et al. (1990), Science 247: 1306-1310. The first
method relies on the process of evolution, in which mutations are
either accepted or rejected by natural selection. The second
approach uses genetic engineering to introduce amino acid changes
at specific positions of a cloned gene and selections or screens to
identify sequences that maintain functionality.
[0126] These studies have revealed that proteins are surprisingly
tolerant of amino acid substitutions. The studies indicate which
amino acid changes are likely to be permissive at a certain
position of the protein. For example, most buried amino acid
residues require nonpolar side chains, whereas few features of
surface side chains are generally conserved. Other such
phenotypically silent substitutions are described by Bowie et al.
(supra) and the references cited therein. Typically seen as
conservative substitutions are the replacements, one for another,
among the aliphatic amino acids Ala, Val, Leu and Ile; interchange
of the hydroxyl residues Ser and Thr, exchange of the acidic
residues Asp and Glu, substitution between the amide residues Asn
and Gln, exchange of the basic residues Lys and Arg and
replacements among the aromatic residues Phe, Tyr.
[0127] Thus, the fragment, derivative, analog, or homolog of the
polypeptide of Table 1, or that encoded by the plasmids listed in
Table 1, may be: (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code: or (ii) one in which one or more of the amino acid
residues includes a substituent group: or (iii) one in which the B.
burgdorferi polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol): or (iv) one in which the additional amino
acids are fused to the above form of the polypeptide, such as an
IgG Fc fusion region peptide or leader or secretory sequence or a
sequence which is employed for purification of the above form of
the polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0128] Thus, the B. burgdorferi polypeptides of the present
invention may include one or more amino acid substitutions,
deletions, or additions, either from natural mutations or human
manipulation. As indicated, changes are preferably of a minor
nature, such as conservative amino acid substitutions that do not
significantly affect the folding or activity of the protein (see
Table 3).
[0129] Amino acids in the B. burgdorferi proteins of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis. See, e.g., Cunningham et al. (1989)
Science 244: 1081-1085. The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity using
assays appropriate for measuring the function of the particular
protein.
[0130] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic. See, e.g., Pinckard et al., (1967)
Clin. Exp. Immunol. 2: 331-340; Robbins, et al., (1987) Diabetes
36: 838-845; Cleland, et al., (1993) Crit. Rev. Therapeutic Drug
Carrier Systems 10: 307-377.
[0131] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the B. burgdorferi
polypeptide can be substantially purified by the one-step method
described by Smith et al. (1988) Gene 67: 31-40. Polypeptides of
the invention also can be purified from natural or recombinant
sources using antibodies directed against the polypeptides of the
invention in methods which are well known in the art of protein
purification.
[0132] The invention further provides for isolated B. burgdorferi
polypeptides comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of a full-length
B. burgdorferi polypeptide having the complete amino acid sequence
shown in Table 1; (b) the amino acid sequence of a full-length B.
burgdorferi polypeptide having the complete amino acid sequence
shown in Table 1 excepting the N-terminal methionine; (c) the
complete amino acid sequence encoded by the plasmids listed in
Table 1; and (d) the complete amino acid sequence excepting the
N-terminal methionine encoded by the plasmids listed in Table 1.
The polypeptides of the present invention also include polypeptides
having an amino acid sequence at least 80% identical, more
preferably at least 90% identical, and still more preferably 95%,
96%, 97%, 98% or 99% identical to those described in (a), (b), (c),
and (d) above.
[0133] Further polypeptides of the present invention include
polypeptides which have at least 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above.
[0134] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of a B.
burgdorferi polypeptide having an amino acid sequence which
contains at least one conservative amino acid substitution, but not
more than 50 conservative amino acid substitutions, not more than
40 conservative amino acid substitutions, not more than 30
conservative amino acid substitutions, and not more than 20
conservative amino acid substitutions. Also provided are
polypeptides which comprise the amino acid sequence of a B.
burgdorferi polypeptide, having at least one, but not more than 10,
9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid
substitutions.
[0135] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, (indels) or substituted with
another amino acid. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0136] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequences shown in Table 1 or to the amino acid
sequence encoded by the plasmids listed in Table 1 can be
determined conventionally using known computer programs. A
preferred method for determining the best overall match between a
query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be
determined using the FASTDB computer program based on the algorithm
of Brutlag et al., (1990) Comp. App. Biosci. 6: 237-245. In a
sequence alignment the query and subject sequences are both amino
acid sequences. The result of said global sequence alignment is in
percent identity. Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window
Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[0137] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, the results, in percent identity, must be manually
corrected. This is because the FASTDB program does not account for
N- and C-terminal truncations of the subject sequence when
calculating global percent identity. For subject sequences
truncated at the N- and C-termini, relative to the query sequence,
the percent identity is corrected by calculating the number of
residues of the query sequence that are N- and C-terminal of the
subject sequence, which are not matched/aligned with a
corresponding subject residue, as a percent of the total bases of
the query sequence. Whether a residue is matched/aligned is
determined by results of the FASTDB sequence alignment. This
percentage is then subtracted from the percent identity, calculated
by the above FASTDB program using the specified parameters, to
arrive at a final percent identity score. This final percent
identity score is what is used for the purposes of the present
invention. Only residues to the N- and C-termini of the subject
sequence, which are not matched/aligned with the query sequence,
are considered for the purposes of manually adjusting the percent
identity score. That is, only query amino acid residues outside the
farthest N- and C-terminal residues of the subject sequence.
[0138] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not match/align
with the first 10 residues at the N-terminus. The 10 unpaired
residues represent 10% of the sequence (number of residues at the
N- and C-termini not matched/total number of residues in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 residues were
perfectly matched the final percent identity would be 90%. In
another example, a 90 residue subject sequence is compared with a
100 residue query sequence. This time the deletions are internal so
there are no residues at the N- or C-termini of the subject
sequence which are not matched/aligned with the query. In this case
the percent identity calculated by FASTDB is not manually
corrected. Once again, only residue positions outside the N- and
C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequence
are manually corrected. No other manual corrections are to made for
the purposes of the present invention.
[0139] The above polypeptide sequences are included irrespective of
whether they have their normal biological activity. This is because
even where a particular polypeptide molecule does not have
biological activity, one of skill in the art would still know how
to use the polypeptide, for instance, as a vaccine or to generate
antibodies. Other uses of the polypeptides of the present invention
that do not have B. burgdorferi activity include, inter alia, as
epitope tags, in epitope mapping, and as molecular weight markers
on SDS-PAGE gels or on molecular sieve gel filtration columns using
methods known to those of skill in the art.
[0140] As described below, the polypeptides of the present
invention can also be used to raise polyclonal and monoclonal
antibodies, which are useful in assays for detecting B. burgdorferi
protein expression or as agonists and antagonists capable of
enhancing or inhibiting B. burgdorferi protein function. Further,
such polypeptides can be used in the yeast two-hybrid system to
"capture" B. burgdorferi protein binding proteins which are also
candidate agonists and antagonists according to the present
invention. See, e.g., Fields et al. (1989) Nature 340: 245-246.
[0141] Epitope-Bearing Portions
[0142] In another aspect, the invention provides peptides and
polypeptides comprising epitope-bearing portions of the B.
burgdorferi polypeptides of the present invention. These epitopes
are immunogenic or antigenic epitopes of the polypeptides of the
present invention. An "immunogenic epitope" is defined as a part of
a protein that elicits an antibody response when the whole protein
or polypeptide is the immunogen. These immunogenic epitopes are
believed to be confined to a few loci on the molecule. On the other
hand, a region of a protein molecule to which an antibody can bind
is defined as an "antigenic determinant" or "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, e.g., Geysen, et al.
(1983) Proc. Natl. Acad. Sci. USA 81: 3998-4002. Predicted
antigenic epitopes are shown in Table 4, below. It is pointed out
that Table 4 only lists amino acid residues comprising epitopes
predicted to have the highest degree of antigenicity. The
polypeptides not listed in Table 4 and portions of polypeptides not
listed in Table 4 are not considered non-antigenic. This is because
they may still be antigenic in vivo but merely not recognized as
such by the particular algorithm used. Thus, Table 4 lists the
amino acid residues comprising preferred antigenic epitopes but not
a complete list. Amino acid residues comprising other antigenic
epitopes may be determined by algorithms similar to the
Jameson-Wolf analysis or by in vivo testing for an antigenic
response using the methods described herein or those known in the
art.
[0143] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, e.g.,
Sutcliffe, et al., (1983) Science 219: 660-666. Peptides capable of
eliciting protein-reactive sera are frequently represented in the
primary sequence of a protein, can be characterized by a set of
simple chemical rules, and are confined neither to immunodominant
regions of intact proteins (i.e., immunogenic epitopes) nor to the
amino or carboxyl terminals. Peptides that are extremely
hydrophobic and those of six or fewer residues generally are
ineffective at inducing antibodies that bind to the mimicked
protein; longer, peptides, especially those containing proline
residues, usually are effective. See, Sutcliffe, et al., supra, p.
661. For instance, 18 of 20 peptides designed according to these
guidelines, containing 8-39 residues covering 75% of the sequence
of the influenza virus hemagglutinin HA1 polypeptide chain, induced
antibodies that reacted with the HA1 protein or intact virus; and
12/12 peptides from the MuLV polymerase and 18/18 from the rabies
glycoprotein induced antibodies that precipitated the respective
proteins.
[0144] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Thus, a high proportion of hybridomas obtained by
fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide generally secrete antibody reactive with
the native protein. See Sutcliffe, et al., supra, p. 663. The
antibodies raised by antigenic epitope-bearing peptides or
polypeptides are useful to detect the mimicked protein, and
antibodies to different peptides may be used for tracking the fate
of various regions of a protein precursor which undergoes
post-translational processing. The peptides and anti-peptide
antibodies may be used in a variety of qualitative or quantitative
assays for the mimicked protein, for instance in competition assays
since it has been shown that even short peptides (e.g., about 9
amino acids) can bind and displace the larger peptides in
immunoprecipitation assays. See, e.g., Wilson, et al., (1984) Cell
37: 767-778. The anti-peptide antibodies of the invention also are
useful for purification of the mimicked protein, for instance, by
adsorption chromatography using methods known in the art.
[0145] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines preferably
contain a sequence of at least seven, more preferably at least nine
and most preferably between about 10 to about 50 amino acids (i.e.
any integer between 7 and 50) contained within the amino acid
sequence of a polypeptide of the invention. However, peptides or
polypeptides comprising a larger portion of an amino acid sequence
of a polypeptide of the invention, containing about 50 to about 100
amino acids, or any length up to and including the entire amino
acid sequence of a polypeptide of the invention, also are
considered epitope-bearing peptides or polypeptides of the
invention and also are useful for inducing antibodies that react
with the mimicked protein. Preferably, the amino acid sequence of
the epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues and highly hydrophobic sequences
are preferably avoided); and sequences containing proline residues
are particularly preferred.
[0146] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate an Borrelia-specific immune response
or antibodies include portions of the amino acid sequences
identified in Table 1. More specifically, Table 4 discloses a list
of non-limiting residues that are involved in the antigenicity of
the epitope-bearing fragments of the present invention. Therefore,
the present inventions provides for isolated and purified antigenic
epitope-bearing fragments of the polypeptides of the present
invention comprising a peptide sequences of Table 4. The antigenic
epitope-bearing fragments comprising a peptide sequence of Table 4
preferably contain a sequence of at least seven, more preferably at
least nine and most preferably between about 10 to about 50 amino
acids (i.e. any integer between 7 and 50) of a polypeptide of the
present invention. That is, included in the present invention are
antigenic polypeptides between the integers of 7 and 50 amino acid
in length comprising one or more of the sequences of Table 4.
Therefore, in most cases, the polypeptides of Table 4 make up only
a portion of the antigenic polypeptide. All combinations of
sequences between the integers of 7 and 50 amino acid in length
comprising one or more of the sequences of Table 4 are included.
The antigenic epitope-bearing fragments may be specified by either
the number of contiguous amino acid residues or by specific
N-terminal and C-terminal positions as described above for the
polypeptide fragments of the present invention, wherein the
initiation codon is residue 1. Any number of the described
antigenic epitope-bearing fragments of the present invention may
also be excluded from the present invention in the same manner.
[0147] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, an epitope-bearing
amino acid sequence of the present invention may be fused to a
larger polypeptide which acts as a carrier during recombinant
production and purification, as well as during immunization to
produce anti-peptide antibodies. Epitope-bearing peptides also may
be synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by ELISA-type binding studies) in less than four
weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82: 5131-5135
(1985)). This "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Pat. No. 4,631,211 to Houghten
and coworkers (1986). In this procedure the individual resins for
the solid-phase synthesis of various peptides are contained in
separate solvent-permeable packets, enabling the optimal use of the
many identical repetitive steps involved in solid-phase methods. A
completely manual procedure allows 500-1000 or more syntheses to be
conducted simultaneously (Houghten et al. (1985) Proc. Natl. Acad.
Sci. 82: 5131-5135 at 5134.
[0148] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, e.g., Sutcliffe, et al., supra; Wilson, et al.,
supra; and Bittle, et al. (1985) J. Gen. Virol. 66: 2347-2354.
Generally, animals may be immunized with free peptide; however,
anti-peptide antibody titer may be boosted by coupling of the
peptide to a macromolecular carrier, such as keyhole limpet
hemacyanin (KLH) or tetanus toxoid. For instance, peptides
containing cysteine may be coupled to carrier using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g peptide or carrier protein and
Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0149] Immunogenic epitope-bearing peptides of the invention, i.e.,
those parts of a protein that elicit an antibody response when the
whole protein is the immunogen, are identified according to methods
known in the art. For instance, Geysen, et al., supra, discloses a
procedure for rapid concurrent synthesis on solid supports of
hundreds of peptides of sufficient purity to react in an ELISA.
Interaction of synthesized peptides with antibodies is then easily
detected without removing them from the support. In this manner a
peptide bearing an immunogenic epitope of a desired protein may be
identified routinely by one of ordinary skill in the art. For
instance, the immunologically important epitope in the coat protein
of foot-and-mouth disease virus was located by Geysen et al. supra
with a resolution of seven amino acids by synthesis of an
overlapping set of all 208 possible hexapeptides covering the
entire 213 amino acid sequence of the protein. Then, a complete
replacement set of peptides in which all 20 amino acids were
substituted in turn at every position within the epitope were
synthesized, and the particular amino acids conferring specificity
for the reaction with antibody were determined. Thus, peptide
analogs of the epitope-bearing peptides of the invention can be
made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen
(1987) further describes this method of identifying a peptide
bearing an immunogenic epitope of a desired protein.
[0150] Further still, U.S. Pat. No. 5,194,392, to Geysen (1990),
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope") which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092, also
to Geysen (1989), describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) discloses linear
C.sub.1-C.sub.7-alkyl peralkylated oligopeptides and sets and
libraries of such peptides, as well as methods for using such
oligopeptide sets and libraries for determining the sequence of a
peralkylated oligopeptide that preferentially binds to an acceptor
molecule of interest. Thus, non-peptide analogs of the
epitope-bearing peptides of the invention also can be made
routinely by these methods. The entire disclosure of each document
cited in this section on "Polypeptides and Fragments" is hereby
incorporated herein by reference.
[0151] As one of skill in the art will appreciate, the polypeptides
of the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased
half-life in vivo. This has been shown, e.g., for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EPA 0,394,827; Traunecker et
al. (1988) Nature 331: 84-86. Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG part can also be
more efficient in binding and neutralizing other molecules than a
monomeric B. burgdorferi polypeptide or fragment thereof alone. See
Fountoulakis et al. (1995) J. Biochem. 270: 3958-3964. Nucleic
acids encoding the above epitopes of B. burgdorferi polypeptides
can also be recombined with a gene of interest as an epitope tag to
aid in detection and purification of the expressed polypeptide.
[0152] Antibodies
[0153] B. burgdorferi protein-specific antibodies for use in the
present invention can be raised against the intact B. burgdorferi
protein or an antigenic polypeptide fragment thereof, which may be
presented together with a carrier protein, such as an albumin, to
an animal system (such as rabbit or mouse) or, if it is long enough
(at least about 25 amino acids), without a carrier.
[0154] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules, single chain
whole antibodies, and antibody fragments. Antibody fragments of the
present invention include Fab and F(ab')2 and other fragments
including single-chain Fvs (scFv) and disulfide-linked Fvs (sdFv).
Also included in the present invention are chimeric and humanized
monoclonal antibodies and polyclonal antibodies specific for the
polypeptides of the present invention. The antibodies of the
present invention may be prepared by any of a variety of methods.
For example, cells expressing a polypeptide of the present
invention or an antigenic fragment thereof can be administered to
an animal in order to induce the production of sera containing
polyclonal antibodies. For example, a preparation of B. burgdorferi
polypeptide or fragment thereof is prepared and purified to render
it substantially free of natural contaminants. Such a preparation
is then introduced into an animal in order to produce polyclonal
antisera of greater specific activity.
[0155] In a preferred method, the antibodies of the present
invention are monoclonal antibodies or binding fragments thereof.
Such monoclonal antibodies can be prepared using hybridoma
technology. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS
563-681 (Elsevier, N.Y., 1981). Fab and F(ab)2 fragments may be
produced by proteolytic cleavage, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab)2 fragments).
Alternatively, B. burgdorferi polypeptide-binding fragments,
chimeric, and humanized antibodies can be produced through the
application of recombinant DNA technology or through synthetic
chemistry using methods known in the art.
[0156] Alternatively, additional antibodies capable of binding to
the polypeptide antigen of the present invention may be produced in
a two-step procedure through the use of anti-idiotypic antibodies.
Such a method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
B. burgdorferi polypeptide-specific antibodies are used to immunize
an animal, preferably a mouse. The splenocytes of such an animal
are then used to produce hybridoma cells, and the hybridoma cells
are screened to identify clones which produce an antibody whose
ability to bind to the B. burgdorferi polypeptide-specific antibody
can be blocked by the B. burgdorferi polypeptide antigen. Such
antibodies comprise anti-idiotypic antibodies to the B. burgdorferi
polypeptide-specific antibody and can be used to immunize an animal
to induce formation of further B. burgdorferi polypeptide-specific
antibodies.
[0157] Antibodies and fragments thereof of the present invention
may be described by the portion of a polypeptide of the present
invention recognized or specifically bound by the antibody.
Antibody binding fragments of a polypeptide of the present
invention may be described or specified in the same manner as for
polypeptide fragments discussed above., i.e., by N-terminal and
C-terminal positions or by size in contiguous amino acid residues.
Any number of antibody binding fragments, of a polypeptide of the
present invention, specified by N-terminal and C-terminal positions
or by size in amino acid residues, as described above, may also be
excluded from the present invention. Therefore, the present
invention includes antibodies the specifically bind a particularly
described fragment of a polypeptide of the present invention and
allows for the exclusion of the same.
[0158] Antibodies and fragments thereof of the present invention
may also be described or specified in terms of their
cross-reactivity. Antibodies and fragments that do not bind
polypeptides of any other species of Borrelia other than B.
burgdorferi are included in the present invention. Likewise,
antibodies and fragments that bind only species of Borrelia, i.e.
antibodies and fragments that do not bind bacteria from any genus
other than Borrelia, are included in the present invention.
[0159] Diagnostic Assays
[0160] The present invention further relates to methods for
assaying staphylococcal infection in an animal by detecting the
expression of genes encoding staphylococcal polypeptides of the
present invention. The methods comprise analyzing tissue or body
fluid from the animal for Borrelia-specific antibodies, nucleic
acids, or proteins. Analysis of nucleic acid specific to Borrelia
is assayed by PCR or hybridization techniques using nucleic acid
sequences of the present invention as either hybridization probes
or primers. See, e.g., Sambrook et al. Molecular cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.,
1989, page 54 reference); Eremeeva et al. (1994) J. Clin.
Microbiol. 32: 803-810 (describing differentiation among spotted
fever group Rickettsiae species by analysis of restriction fragment
length polymorphism of PCR-amplified DNA) and Chen et al. 1994 J.
Clin. Microbiol. 32: 589-595 (detecting B. burgdorferi nucleic
acids via PCR).
[0161] Where diagnosis of a disease state related to infection with
Borrelia has already been lade, the present invention is useful for
monitoring progression or regression of the disease state whereby
patients exhibiting enhanced Borrelia gene expression will
experience a worse clinical outcome relative to patients expressing
these gene(s) at a lower level.
[0162] By "biological sample" is intended any biological sample
obtained from an animal, cell line, tissue culture, or other source
which contains Borrelia polypeptide, mRNA, or DNA. Biological
samples include body fluids (such as saliva, blood, plasma, urine,
mucus, synovial fluid, etc.) tissues (such as muscle, skin, and
cartilage) and any other biological source suspected of containing
Borrelia polypeptides or nucleic acids. Methods for obtaining
biological samples such as tissue are well known in the art.
[0163] The present invention is useful for detecting diseases
related to Borrelia infections in animals. Preferred animals
include monkeys, apes, cats, dogs, birds, cows, pigs, mice, horses,
rabbits and humans. Particularly preferred are humans.
[0164] Total RNA can be isolated from a biological sample using any
suitable technique such as the single-step
guanidinium-thiocyanate-phenol- -chloroform method described in
Chomczynski et al. (1987) Anal. Biochem. 162: 156-159. mRNA
encoding Borrelia polypeptides having sufficient homology to the
nucleic acid sequences identified in Table 1 to allow for
hybridization between complementary sequences are then assayed
using any appropriate method. These include Northern blot analysis,
S1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0165] Northern blot analysis can be performed as described in
Harada et al. (1990) Cell 63: 303-312. Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. A B. burgdorferi
polynucleotide sequence shown in Table 1 labeled according to any
appropriate method (such as the .sup.32P-multiprimed DNA labeling
system (Amersham)) is used as probe. After hybridization overnight,
the filter is washed and exposed to x-ray film. DNA for use as
probe according to the present invention is described in the
sections above and will preferably at least 15 nucleotides in
length.
[0166] S1 mapping can be performed as described in Fujita et al.
(1987) Cell 49: 357-367. To prepare probe DNA for use in S1
mapping, the sense strand of an above-described B. burgdorferi DNA
sequence of the present invention is used as a template to
synthesize labeled antisense DNA. The antisense DNA can then be
digested using an appropriate restriction endonuclease to generate
further DNA probes of a desired length. Such antisense probes are
useful for visualizing protected bands corresponding to the target
mRNA (i.e., mRNA encoding Borrelia polypeptides).
[0167] Levels of mRNA encoding Borrelia polypeptides are assayed,
for e.g., using the RT-PCR method described in Makino et al. (1990)
Technique 2: 295-301. By this method, the radioactivities of the
"amplicons" in the polyacrylamide gel bands are linearly related to
the initial concentration of the target mRNA. Briefly, this method
involves adding total RNA isolated from a biological sample in a
reaction mixture containing a RT primer and appropriate buffer.
After incubating for primer annealing, the mixture can be
supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and
reverse transcriptase. After incubation to achieve reverse
transcription of the RNA, the RT products are then subject to PCR
using labeled primers. Alternatively, rather than labeling the
primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR amplification can be performed in a DNA thermal cycler
according to conventional techniques. After a suitable number of
rounds to achieve amplification, the PCR reaction mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate bands (corresponding to the mRNA
encoding the Borrelia polypeptides of the present invention) are
quantified using an imaging analyzer. RT and PCR reaction
ingredients and conditions, reagent and gel concentrations, and
labeling methods are well known in the art. Variations on the
RT-PCR method will be apparent to the skilled artisan. Other PCR
methods that can detect the nucleic acid of the present invention
can be found in PCR PRIMER: A LABORATORY MANUAL (C. W. Dieffenbach
et al. eds., Cold Spring Harbor Lab Press, 1995).
[0168] The polynucleotides of the present invention, including both
DNA and RNA, may be used to detect polynucleotides of the present
invention or Borrelia species including B. burgdorferi using bio
chip technology. The present invention includes both high density
chip arrays (>1000 oligonucleotides per cm.sup.2) and low
density chip arrays (<1000 oligonucleotides per cm.sup.2). Bio
chips comprising arrays of polynucleotides of the present invention
may be used to detect Borrelia species, including B. burgdorferi,
in biological and environmental samples and to diagnose an animal,
including humans, with an B. burgdorferi or other Borrelia
infection. The bio chips of the present invention may comprise
polynucleotide sequences of other pathogens including bacteria,
viral, parasitic, and fungal polynucleotide sequences, in addition
to the polynucleotide sequences of the present invention, for use
in rapid differential pathogenic detection and diagnosis. The bio
chips can also be used to monitor an B. burgdorferi or other
Borrelia infections and to monitor the genetic changes (deletions,
insertions, mismatches, etc.) in response to drug therapy in the
clinic and drug development in the laboratory. The bio chip
technology comprising arrays of polynucleotides of the present
invention may also be used to simultaneously monitor the expression
of a multiplicity of genes, including those of the present
invention. The polynucleotides used to comprise a selected array
may be specified in the same manner as for the fragments, i.e., by
their 5' and 3' positions or length in contiguous base pairs and
include from. Methods and particular uses of the polynucleotides of
the present invention to detect Borrelia species, including B.
burgdorferi, using bio chip technology include those known in the
art and those of: U.S. Pat. Nos. 5,510,270, 5,545,531, 5,445,934,
5,677,195, 5,532,128, 5,556,752, 5,527,681, 5,451,683, 5,424,186,
5,60,7646, 5,658,732 and World Patent Nos. WO/9710365, WO/9511995,
WO/9743447, WO/9535505, each incorporated herein in their
entireties.
[0169] Biosensors using the polynucleotides of the present
invention may also be used to detect, diagnose, and monitor B.
burgdorferi or other Borrelia species and infections thereof.
Biosensors using the polynucleotides of the present invention may
also be used to detect particular polynucleotides of the present
invention. Biosensors using the polynucleotides of the present
invention may also be used to monitor the genetic changes
(deletions, insertions, mismatches, etc.) in response to drug
therapy in the clinic and drug development in the laboratory.
Methods and particular uses of the polynucleotides of the present
invention to detect Borrelia species, including B. burgdorferi,
using biosensors include those known in the art and those of: U.S.
Pat. Nos. 5,721,102, 5,658,732, 5631170, and World Patent Nos.
WO97/35011, WO/9720203, each incorporated herein in their
entireties.
[0170] Thus, the present invention includes both bio chips and
biosensors comprising polynucleotides of the present invention and
methods of their use.
[0171] Assaying Borrelia polypeptide levels in a biological sample
can occur using any art-known method, such as antibody-based
techniques. For example, Borrelia polypeptide expression in tissues
can be studied with classical immunohistological methods. In these,
the specific recognition is provided by the primary antibody
(polyclonal or monoclonal) but the secondary detection system can
utilize fluorescent, enzyme, or other conjugated secondary
antibodies. As a result, an immunohistological staining of tissue
section for pathological examination is obtained. Tissues can also
be extracted, e.g., with urea and neutral detergent, for the
liberation of Borrelia polypeptides for Western-blot or dot/slot
assay. See, e.g., Jalkanen, M. et al. (1985) J. Cell. Biol. 101:
976-985; Jalkanen, M. et al. (1987) J. Cell. Biol. 105: 3087-3096.
In this technique, which is based on the use of cationic solid
phases, quantitation of a Borrelia polypeptide can be accomplished
using an isolated Borrelia polypeptide as a standard. This
technique can also be applied to body fluids.
[0172] Other antibody-based methods useful for detecting Borrelia
polypeptide gene expression include immunoassays, such as the ELISA
and the radioimmunoassay (RIA). For example, a Borrelia
polypeptide-specific monoclonal antibodies can be used both as an
immunoabsorbent and as an enzyme-labeled probe to detect and
quantify a Borrelia polypeptide. The amount of a Borrelia
polypeptide present in the sample can be calculated by reference to
the amount present in a standard preparation using a linear
regression computer algorithm. Such an ELISA is described in
Iacobelli et al. (1988) Breast Cancer Research and Treatment 11:
19-30. In another ELISA assay, two distinct specific monoclonal
antibodies can be used to detect Borrelia polypeptides in a body
fluid. In this assay, one of the antibodies is used as the
immunoabsorbent and the other as the enzyme-labeled probe.
[0173] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting the Borrelia polypeptide with immobilized antibody and,
without washing, contacting the mixture with the labeled antibody.
The "two-step" assay involves washing before contacting the mixture
with the labeled antibody. Other conventional methods may also be
employed as suitable. It is usually desirable to immobilize one
component of the assay system on a support, thereby allowing other
components of the system to be brought into contact with the
component and readily removed from the sample. Variations of the
above and other immunological methods included in the present
invention can also be found in Harlow et al., ANTIBODIES: A
LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988).
[0174] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase is particularly
preferred as it has good stability and its substrate (glucose) is
readily available. Activity of an oxidase label may be assayed by
measuring the concentration of hydrogen peroxide formed by the
enzyme-labeled antibody/substrate reaction. Besides enzymes, other
suitable labels include radioisotopes, such as iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulphur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0175] Further suitable labels for the Borrelia
polypeptide-specific antibodies of the present invention are
provided below. Examples of suitable enzyme labels include malate
dehydrogenase, Borrelia nuclease, delta-5-steroid isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, and acetylcholine esterase.
[0176] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, etc. .sup.111In is a preferred isotope where in vivo
imaging is used since its avoids the problem of dehalogenation of
the .sup.125I or .sup.131I-labeled monoclonal antibody by the
liver. In addition, this radionucleotide has a more favorable gamma
emission energy for imaging. See, e.g., Perkins et al. (1985) Eur.
J. Nucl. Med. 10: 296-301; Carasquillo et al. (1987) J. Nucl. Med.
28: 281-287. For example, .sup.111In coupled to monoclonal
antibodies with 1-(P-isothiocyanatobenzy- l)-DPTA has shown little
uptake in non-tumors tissues, particularly the liver, and therefore
enhances specificity of tumor localization. See, Esteban et al.
(1987) J. Nucl. Med. 28: 861-870.
[0177] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
[0178] Examples of suitable fluorescent labels include an
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0179] Examples of suitable toxin labels include, Pseudomonas
toxin, diphtheria toxin, ricin, and cholera toxin.
[0180] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
[0181] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and iron.
[0182] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al. (1976) Clin. Chim. Acta
70: 1-31, and Schurs et al. (1977) Clin. Chim. Acta 81: 1-40.
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy- -succinimide ester method, all of
which methods are incorporated by reference herein.
[0183] In a related aspect, the invention includes a diagnostic kit
for use in screening serum containing antibodies specific against
B. burgdorferi infection. Such a kit may include an isolated B.
burgdorferi antigen comprising an epitope which is specifically
immunoreactive with at least one anti-B. burgdorferi antibody. Such
a kit also includes means for detecting the binding of said
antibody to the antigen. In specific embodiments, the kit may
include a recombinantly produced or chemically synthesized peptide
or polypeptide antigen. The peptide or polypeptide antigen may be
attached to a solid support.
[0184] In a more specific embodiment, the detecting means of the
above-described kit includes a solid support to which said peptide
or polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the B. burgdorferi antigen
can be detected by binding of the reporter labeled antibody to the
anti-B. burgdorferi polypeptide antibody.
[0185] In a related aspect, the invention includes a method of
detecting B. burgdorferi infection in a subject. This detection
method includes reacting a body fluid, preferably serum, from the
subject with an isolated B. burgdorferi antigen, and examining the
antigen for the presence of bound antibody. In a specific
embodiment, the method includes a polypeptide antigen attached to a
solid support, and serum is reacted with the support. Subsequently,
the support is reacted with a reporter-labeled anti-human antibody.
The support is then examined for the presence of reporter-labeled
antibody.
[0186] The solid surface reagent employed in the above assays and
kits is prepared by known techniques for attaching protein material
to solid support material, such as polymeric beads, dip sticks,
96-well plates or filter material. These attachment methods
generally include non-specific adsorption of the protein to the
support or covalent attachment of the protein, typically through a
free amine group, to a chemically reactive group on the solid
support, such as an activated carboxyl, hydroxyl, or aldehyde
group. Alternatively, streptavidin coated plates can be used in
conjunction with biotinylated antigen(s).
[0187] The polypeptides and antibodies of the present invention,
including fragments thereof, may be used to detect Borrelia species
including B. burgdorferi using bio chip and biosensor technology.
Bio chip and biosensors of the present invention may comprise the
polypeptides of the present invention to detect antibodies, which
specifically recognize Borrelia species, including B. burgdorferi.
Bio chip and biosensors of the present invention may also comprise
antibodies which specifically recognize the polypeptides of the
present invention to detect Borrelia species, including B.
burgdorferi or specific polypeptides of the present invention. Bio
chips or biosensors comprising polypeptides or antibodies of the
present invention may be used to detect Borrelia species, including
B. burgdorferi, in biological and environmental samples and to
diagnose an animal, including humans, with an B. burgdorferi or
other Borrelia infection. Thus, the present invention includes both
bio chips and biosensors comprising polypeptides or antibodies of
the present invention and methods of their use.
[0188] The bio chips of the present invention may further comprise
polypeptide sequences of other pathogens including bacteria, viral,
parasitic, and fungal polypeptide sequences, in addition to the
polypeptide sequences of the present invention, for use in rapid
differential pathogenic detection and diagnosis. The bio chips of
the present invention may further comprise antibodies or fragments
thereof specific for other pathogens including bacteria, viral,
parasitic, and fungal polypeptide sequences, in addition to the
antibodies or fragments thereof of the present invention, for use
in rapid differential pathogenic detection and diagnosis. The bio
chips and biosensors of the present invention may also be used to
monitor an B. burgdorferi or other Borrelia infection and to
monitor the genetic changes (amino acid deletions, insertions,
substitutions, etc.) in response to drug therapy in the clinic and
drug development in the laboratory. The bio chip and biosensors
comprising polypeptides or antibodies of the present invention may
also be used to simultaneously monitor the expression of a
multiplicity of polypeptides, including those of the present
invention. The polypeptides used to comprise a bio chip or
biosensor of the present invention may be specified in the same
manner as for the fragments, i.e., by their N-terminal and
C-terminal positions or length in contiguous amino acid residue.
Methods and particular uses of the polypeptides and antibodies of
the present invention to detect Borrelia species, including B.
burgdorferi, or specific polypeptides using bio chip and biosensor
technology include those known in the art, those of the U.S. Patent
Nos. and World Patent Nos. listed above for bio chips and
biosensors using polynucleotides of the present invention, and
those of: U.S. Pat. Nos. 5,658,732, 5,135,852, 5567301, 5,677,196,
5,690,894 and World Patent Nos. WO9729366, WO9612957, each
incorporated herein in their entireties.
[0189] Treatment:
[0190] Agonists and Antagonists--Assays and Molecules
[0191] The invention also provides a method of screening compounds
to identify those which enhance or block the biological activity of
the B. burgdorferi polypeptides of the present invention. The
present invention further provides where the compounds kill or slow
the growth of B. burgdorferi. The ability of B. burgdorferi
antagonists, including B. burgdorferi ligands, to prophylactically
or therapeutically block antibiotic resistance may be easily tested
by the skilled artisan. See, e.g., Straden et al. (1997) J.
Bacteriol. 179(1): 9-16.
[0192] An agonist is a compound which increases the natural
biological function or which functions in a manner similar to the
polypeptides of the present invention, while antagonists decrease
or eliminate such functions. Potential antagonists include small
organic molecules, peptides, polypeptides, and antibodies that bind
to a polypeptide of the invention and thereby inhibit or extinguish
its activity.
[0193] The antagonists may be employed for instance to inhibit
peptidoglycan cross bridge formation. Antibodies against B.
burgdorferi may be employed to bind to and inhibit B. burgdorferi
activity to treat antibiotic resistance. Any of the above
antagonists may be employed in a composition with a
pharmaceutically acceptable carrier.
[0194] Vaccines
[0195] The present invention also provides vaccines comprising one
or more polypeptides of the present invention. Heterogeneity in the
composition of a vaccine may be provided by combining B.
burgdorferi polypeptides of the present invention. Multi-component
vaccines of this type are desirable because they are likely to be
more effective in eliciting protective immune responses against
multiple species and strains of the Borrelia genus than single
polypeptide vaccines. Thus, as discussed in detail below, a
multi-component vaccine of the present invention may contain one or
more, preferably 2 to about 20, more preferably 2 to about 15, and
most preferably 3 to about 8, of the B. burgdorferi polypeptides
shown in Table 1, or fragments thereof.
[0196] Multi-component vaccines are known in the art to elicit
antibody production to numerous immunogenic components. Decker, M.
and Edwards, K., J. Infect. Dis. 174: S270-275 (1996). In addition,
a hepatitis B, diphtheria, tetanus, pertussis tetravalent vaccine
has recently been demonstrated to elicit protective levels of
antibodies in human infants against all four pathogenic agents.
Aristegui, J. et al., Vaccine 15: 7-9 (1997).
[0197] The present invention thus also includes multi-component
vaccines. These vaccines comprise more than one polypeptide,
immunogen or antigen. An example of such a multi-component vaccine
would be a vaccine comprising more than one of the B. burgdorferi
polypeptides shown in Table 1. A second example is a vaccine
comprising one or more, for example 2 to 10, of the B. burgdorferi
polypeptides shown in Table 1 and one or more, for example 2 to 10,
additional polypeptides of either borrelial or non-borrelial
origin. Thus, a multi-component vaccine which confers protective
immunity to both a borrelial infection and infection by another
pathogenic agent is also within the scope of the invention.
[0198] As indicated above, the vaccines of the present invention
are expected to elicit a protective immune response against
infections caused by species and strains of Borrelia other than B.
burgdorferi sensu stricto isolate B31 (ATCC Accession No. 35210).
Immunizations using decorin-binding protein and OspA derived from
one strain of B. burgdorferi has been shown to elicit the
production of antiserum which confers passive immunity against
other strains of B. burgdorferi. Cassatt, D. et al., Protection of
Borrelia burgdorferi Infection by Antibodies to Decorin-binding
Protein, in VACCINES97, Cold Spring Harbor Press (1997), pages
191-195. Further, the inventors have found using an in vitro assay
that antiserum produced in response to B. burgdorferi
decorin-binding protein will kill several species of Borrelia. The
amino acid sequences of decorin-binding protein expressed by
different strains of B. burgdorferi are believed to diverge by as
much as 25%. Thus, antisera elicited against decorin-binding
proteins confers passive immunity against Borrelia expressing
proteins having only 75% or less amino acid sequence
similarity.
[0199] Further within the scope of the invention are whole cell and
whole viral vaccines. Such vaccines may be produced recombinantly
and involve the expression of one or more of the B. burgdorferi
polypeptides shown in Table 1. For example, the B. burgdorferi
polypeptides of the present invention may be either secreted or
localized intracellular, on the cell surface, or in the periplasmic
space. Further, when a recombinant virus is used, the B.
burgdorferi polypeptides of the present invention may, for example,
be localized in the viral envelope, on the surface of the capsid,
or internally within the capsid. Whole cells vaccines which employ
cells expressing heterologous proteins are known in the art. See,
e.g., Robinson, K. et al., Nature Biotech. 15: 653-657 (1997);
Sirard, J. et al., Infect. Immun. 65: 2029-2033 (1997);
Chabalgoity, J. et al., Infect. Immun. 65: 2402-2412 (1997). These
cells may be administered live or may be killed prior to
administration. Chabalgoity, J. et al., supra, for example, report
the successful use in mice of a live attenuated Salmonella vaccine
strain which expresses a portion of a platyhelminth fatty
acid-binding protein as a fusion protein on its cells surface.
[0200] A multi-component vaccine can also be prepared using
techniques known in the art by combining one or more B. burgdorferi
polypeptides of the present invention, or fragments thereof, with
additional non-borrelial components (e.g., diphtheria toxin or
tetanus toxin, and/or other compounds known to elicit an immune
response). Such vaccines are useful for eliciting protective immune
responses to both members of the Borrelia genus and non-borrelial
pathogenic agents.
[0201] The vaccines of the present invention also include DNA
vaccines. DNA vaccines are currently being developed for a number
of infectious diseases. Boyer, J et al., Nat. Med. 3: 526-532
(1997); reviewed in Spier, R., Vaccine 14: 1285-1288 (1996). Such
DNA vaccines contain a nucleotide sequence encoding one or more B.
burgdorferi polypeptides of the present invention oriented in a
manner that allows for expression of the subject polypeptide. The
direct administration of plasmid DNA encoding OspA has been shown
to elicit protective immunity in mice against borrelial challenge.
Luke, C. et al., J. Infect. Dis. 175: 91-97 (1997).
[0202] The present invention also relates to the administration of
a vaccine which is co-administered with a molecule capable of
modulating immune responses. Kim, J. et al., Nature Biotech. 15:
641-646 (1997), for example, report the enhancement of immune
responses produced by DNA immunizations when DNA sequences encoding
molecules which stimulate the immune response are co-administered.
In a similar fashion, the vaccines of the present invention may be
co-administered with either nucleic acids encoding immune
modulators or the immune modulators themselves. These immune
modulators include granulocyte macrophage colony stimulating factor
(GM-CSF) and CD86.
[0203] The vaccines of the present invention may be used to confer
resistance to borrelial infection by either passive or active
immunization. When the vaccines of the present invention are used
to confer resistance to borrelial infection through active
immunization, a vaccine of the present invention is administered to
an animal to elicit a protective immune response which either
prevents or attenuates a borrelial infection. When the vaccines of
the present invention are used to confer resistance to borrelial
infection through passive immunization, the vaccine is provided to
a host animal (e.g., human, dog, or mouse), and the antisera
elicited by this antisera is recovered and directly provided to a
recipient suspected of having an infection caused by a member of
the Borrelia genus.
[0204] The ability to label antibodies, or fragments of antibodies,
with toxin molecules provides an additional method for treating
borrelial infections when passive immunization is conducted. In
this embodiment, antibodies, or fragments of antibodies, capable of
recognizing the B. burgdorferi polypeptides disclosed herein, or
fragments thereof, as well as other Borrelia proteins, are labeled
with toxin molecules prior to their administration to the patient.
When such toxin derivatized antibodies bind to Borrelia cells,
toxin moieties will be localized to these cells and will cause
their death.
[0205] The present invention thus concerns and provides a means for
preventing or attenuating a borrelial infection resulting from
organisms which have antigens that are recognized and bound by
antisera produced in response to the polypeptides of the present
invention. As used herein, a vaccine is said to prevent or
attenuate a disease if its administration to an animal results
either in the total or partial attenuation (i.e., suppression) of a
symptom or condition of the disease, or in the total or partial
immunity of the animal to the disease.
[0206] The administration of the vaccine (or the antisera which it
elicits) may be for either a "prophylactic" or "therapeutic"
purpose. When provided prophylactically, the compound(s) are
provided in advance of any symptoms of borrelial infection. The
prophylactic administration of the compound(s) serves to prevent or
attenuate any subsequent infection. When provided therapeutically,
the compound(s) is provided upon or after the detection of symptoms
which indicate that an animal may be infected with a member of the
Borrelia genus. The therapeutic administration of the compound(s)
serves to attenuate any actual infection. Thus, the B. burgdorferi
polypeptides, and fragments thereof, of the present invention may
be provided either prior to the onset of infection (so as to
prevent or attenuate an anticipated infection) or after the
initiation of an actual infection.
[0207] The polypeptides of the invention, whether encoding a
portion of a native protein or a functional derivative thereof, may
be administered in pure form or may be coupled to a macromolecular
carrier. Example of such carriers are proteins and carbohydrates.
Suitable proteins which may act as macromolecular carrier for
enhancing the immunogenicity of the polypeptides of the present
invention include keyhole limpet hemacyanin (KLH) tetanus toxoid,
pertussis toxin, bovine serum albumin, and ovalbumin. Methods for
coupling the polypeptides of the present invention to such
macromolecular carriers are disclosed in Harlow et al., Antibodies:
A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1988), the entire disclosure of which is
incorporated by reference herein.
[0208] A composition is said to be "pharmacologically acceptable"
if its administration can be tolerated by a recipient animal and is
otherwise suitable for administration to that animal. Such an agent
is said to be administered in a "therapeutically effective amount"
if the amount administered is physiologically significant. An agent
is physiologically significant if its presence results in a
detectable change in the physiology of a recipient patient.
[0209] While in all instances the vaccine of the present invention
is administered as a pharmacologically acceptable compound, one
skilled in the art would recognize that the composition of a
pharmacologically acceptable compound varies with the animal to
which it is administered. For example, a vaccine intended for human
use will generally not be co-administered with Freund's adjuvant.
Further, the level of purity of the B. burgdorferi polypeptides of
the present invention will normally be higher when administered to
a human than when administered to a non-human animal.
[0210] As would be understood by one of ordinary skill in the art,
when the vaccine of the present invention is provided to an animal,
it may be in a composition which may contain salts, buffers,
adjuvants, or other substances which are desirable for improving
the efficacy of the composition. Adjuvants are substances that can
be used to specifically augment a specific immune response. These
substances generally perform two functions: (1) they protect the
antigen(s) from being rapidly catabolized after administration and
(2) they nonspecifically stimulate immune responses.
[0211] Normally, the adjuvant and the composition are mixed prior
to presentation to the immune system, or presented separately, but
into the same site of the animal being immunized. Adjuvants can be
loosely divided into several groups based upon their composition.
These groups include oil adjuvants (for example, Freund's complete
and incomplete), mineral salts (for example, AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH.sub.4(SO.sub.4), silica, kaolin, and
carbon), polynucleotides (for example, poly IC and poly AU acids),
and certain natural substances (for example, wax D from
Mycobacterium tuberculosis, as well as substances found in
Corynebacterium parvum, or Bordetella pertussis, and members of the
genus Brucella. Other substances useful as adjuvants are the
saponins such as, for example, Quil A. (Superfos A/S, Denmark).
Preferred adjuvants for use in the present invention include
aluminum salts, such as AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2,
and AlNH.sub.4(SO.sub.4). Examples of materials suitable for use in
vaccine compositions are provided in Remington's Pharmaceutical
Sciences (Osol, A, Ed, Mack Publishing Co, Easton, Pa., pp.
1324-1341 (1980), which reference is incorporated herein by
reference).
[0212] The therapeutic compositions of the present invention can be
administered parenterally by injection, rapid infusion,
nasopharyngeal absorption (intranasopharangeally), dermoabsorption,
or orally. The compositions may alternatively be administered
intramuscularly, or intravenously. Compositions for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Carriers
or occlusive dressings can be used to increase skin permeability
and enhance antigen absorption. Liquid dosage forms for oral
administration may generally comprise a liposome solution
containing the liquid dosage form. Suitable forms for suspending
liposomes include emulsions, suspensions, solutions, syrups, and
elixirs containing inert diluents commonly used in the art, such as
purified water. Besides the inert diluents, such compositions can
also include adjuvants, wetting agents, emulsifying and suspending
agents, or sweetening, flavoring, or perfuming agents.
[0213] Therapeutic compositions of the present invention can also
be administered in encapsulated form. For example, intranasal
immunization of mice against Bordetella pertussis infection using
vaccines encapsulated in biodegradable microsphere composed of
poly(DL-lactide-co-glycolide) has been shown to stimulate
protective immune responses. Shahin, R. et al., Infect. Immun. 63:
1195-1200 (1995). Similarly, orally administered encapsulated
Salmonella typhimurium antigens have also been shown to elicit
protective immunity in mice. Allaoui-Attarki, K. et al., Infect.
Immun. 65: 853-857 (1997). Encapsulated vaccines of the present
invention can be administered by a variety of routes including
those involving contacting the vaccine with mucous membranes (e.g.,
intranasally, intracolonicly, intraduodenally).
[0214] Many different techniques exist for the timing of the
immunizations when a multiple administration regimen is utilized.
It is possible to use the compositions of the invention more than
once to increase the levels and diversities of expression of the
immunoglobulin repertoire expressed by the immunized animal.
Typically, if multiple immunizations are given, they will be given
one to two months apart.
[0215] According to the present invention, an "effective amount" of
a therapeutic composition is one which is sufficient to achieve a
desired biological effect. Generally, the dosage needed to provide
an effective amount of the composition will vary depending upon
such factors as the animal's or human's age, condition, sex, and
extent of disease, if any, and other variables which can be
adjusted by one of ordinary skill in the art.
[0216] The antigenic preparations of the invention can be
administered by either single or multiple dosages of an effective
amount. Effective amounts of the compositions of the invention can
vary from 0.01-1,000 .mu.g/ml per dose, more preferably 0.1-500
.mu.g/ml per dose, and most preferably 10-300 .mu.g/ml per
dose.
[0217] Having now generally described the invention, the same will
be more readily understood through reference to the following
example which is provided by way of illustration, and is not
intended to be limiting of the present invention, unless
specified.
EXAMPLES
[0218] 1. Preparation of PCR Primers and Amplification of DNA
[0219] Various fragments of the Borrelia burgdorferi genome, such
as those of Table 1, can be used, in accordance with the present
invention, to prepare PCR primers for a variety of uses. The PCR
primers are preferably at least 15 bases, and more preferably at
least 18 bases in length. When selecting a primer sequence, it is
preferred that the primer pairs have approximately the same G/C
ratio, so that melting temperatures are approximately the same. The
PCR primers and amplified DNA of this Example find use in the
Examples that follow.
[0220] 2. Isolation of a Selected DNA Clone from B. burgdorferi
[0221] Three approaches are used to isolate a B. burgdorferi clone
comprising a polynucleotide of the present invention from any B.
burgdorferi genomic DNA library. The B. burgdorferi strain B31PU
has been deposited as a convienent source for obtaining a B.
burgdorferi strain although a wide varity of strains B. burgdorferi
strains can be used which are known in the art.
[0222] B. burgdorferi genomic DNA is prepared using the following
method. A 20 ml overnight bacterial culture grown in a rich medium
(e.g., Trypticase Soy Broth, Brain Heart Infusion broth or Super
broth), pelleted, ished two times with TES (30 mM Tris-pH 8.0, 25
mM EDTA, 50 mM NaCl), and resuspended in 5 ml high salt TES (2.5M
NaCl). Lysostaphin is added to final concentration of approx 50
ug/ml and the mixture is rotated slowly 1 hour at 37 C to make
protoplast cells. The solution is then placed in incubator (or
place in a shaking water bath) and warmed to 55 C. Five hundred
micro liter of 20% sarcosyl in TES (final concentration 2%) is then
added to lyse the cells. Next, guanidine HCl is added to a final
concentration of 7M (3.69 g in 5.5 ml). The mixture is swirled
slowly at 55 C for 60-90 min (solution should clear). A CsCl
gradient is then set up in SW41 ultra clear tubes using 2.0 ml 5.7M
CsCl and overlaying with 2.85M CsCl. The gradient is carefully
overlayed with the DNA-containing GuHCl solution. The gradient is
spun at 30,000 rpm, 20 C for 24 hr and the lower DNA band is
collected. The volume is increased to 5 ml with TE buffer. The DNA
is then treated with protease K (10 ug/ml) overnight at 37 C, and
precipitated with ethanol. The precipitated DNA is resuspended in a
desired buffer.
[0223] In the first method, a plasmid is directly isolated by
screening a plasmid B. burgdorferi genomic DNA library using a
polynucleotide probe corresponding to a polynucleotide of the
present invention. Particularly, a specific polynucleotide with
30-40 nucleotides is synthesized using an Applied Biosystems DNA
synthesizer according to the sequence reported. The oligonucleotide
is labeled, for instance, with .sup.32P-.gamma.-ATP using T4
polynucleotide kinase and purified according to routine methods.
(See, e.g., Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The
library is transformed into a suitable host, as indicated above
(such as XL-1 Blue (Stratagene)) using techniques known to those of
skill in the art. See, e.g., Sambrook et al. MOLECULAR CLONING: A
LABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel
et al., CURRENT PROTOCALS IN MOLECULAR BIOLOGY (John Wiley and
Sons, N.Y. 1989). The transformants are plated on 1.5% agar plates
(containing the appropriate selection agent, e.g., ampicillin) to a
density of about 150 transformants (colonies) per plate. These
plates are screened using Nylon membranes according to routine
methods for bacterial colony screening. See, e.g., Sambrook et al.
MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor, N.Y.
2nd ed. 1989); Ausubel et al., CURRENT PROTOCALS IN MOLECULAR
BIOLOGY (John Wiley and Sons, N.Y. 1989) or other techniques known
to those of skill in the art.
[0224] Alternatively, two primers of 15-25 nucleotides derived from
the 5' and 3 ends of a polynucleotide of Table 1 are synthesized
and used to amplify the desired DNA by PCR using a B. burgdorferi
genomic DNA prep as a template. PCR is carried out under routine
conditions, for instance, in 25 VI of reaction mixture with 0.5 ug
of the above DNA template. A convenient reaction mixture is 1.5-5
mM MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP, dCTP,
dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
Thirty five cycles of PCR (denaturation at 94.degree. C. for 1 min;
annealing at 55.degree. C. for 1 min; elongation at 72.degree. C.
for 1 min) are performed with a Perkin-Elmer Cetus automated
thermal cycler. The amplified product is analyzed by agarose gel
electrophoresis and the DNA band with expected molecular weight is
excised and purified. The PCR product is verified to be the
selected sequence by subcloning and sequencing the DNA product.
[0225] Finally, overlapping oligos of the DNA sequences of Table 1
can be chemically synthesized and used to generate a nucleotide
sequence of desired length using PCR methods known in the art.
[0226] 3(a). Expression and Purification Borrelia Polypeptides in
E. coli
[0227] The bacterial expression vector pQE60 is used for bacterial
expression of some of the polypeptide fragments of the present
invention. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,
91311). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and
contains a bacterial origin of replication ("ori"), an IPTG
inducible promoter, a ribosome binding site ("RBS"), six codons
encoding histidine residues that allow affinity purification using
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin (QIAGEN,
Inc., supra) and suitable single restriction enzyme cleavage sites.
These elements are arranged such that an inserted DNA fragment
encoding a polypeptide expresses that polypeptide with the six His
residues (i.e., a "6.times.His tag") covalently linked to the
carboxyl terminus of that polypeptide.
[0228] The DNA sequence encoding the desired portion of a B.
burgdorferi protein of the present invention is amplified from B.
burgdorferi genomic DNA using PCR oligonucleotide primers which
anneal to the 5' and 3' sequences coding for the portions of the B.
burgdorferi polynucleotide shown in Table 1. Additional nucleotides
containing restriction sites to facilitate cloning in the pQE60
vector are added to the 5' and 3' sequences, respectively.
[0229] For cloning the mature protein, the 5' primer has a sequence
containing an appropriate restriction site followed by nucleotides
of the amino terminal coding sequence of the desired B. burgdorferi
polynucleotide sequence in Table 1. One of ordinary skill in the
art would appreciate that the point in the protein coding sequence
where the 5' and 3' primers begin may be varied to amplify a DNA
segment encoding any desired portion of the complete protein
shorter or longer than the mature form. The 3' primer has a
sequence containing an appropriate restriction site followed by
nucleotides complementary to the 3' end of the polypeptide coding
sequence of Table 1, excluding a stop codon, with the coding
sequence aligned with the restriction site so as to maintain its
reading frame with that of the six His codons in the pQE60
vector.
[0230] The amplified B. burgdorferi DNA fragment and the vector
pQE60 are digested with restriction enzymes which recognize the
sites in the primers and the digested DNAs are then ligated
together. The B. burgdorferi DNA is inserted into the restricted
pQE60 vector in a manner which places the B. burgdorferi protein
coding region downstream from the IPTG-inducible promoter and
in-frame with an initiating AUG and the six histidine codons.
[0231] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
et al., supra. E. coli strain M15/rep4, containing multiple copies
of the plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), is used in carrying out the
illustrative example described herein. This strain, which is only
one of many that are suitable for expressing a B. burgdorferi
polypeptide, is available commercially (QIAGEN, Inc., supra).
Transformants are identified by their ability to grow on LB agar
plates in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
[0232] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-.beta.-D-thiogalactopyranoside ("IPTG") is then added to
a final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0233] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the B. burgdorferi
polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin column (QIAGEN, Inc., supra). Proteins
with a 6.times.His tag bind to the Ni-NTA resin with high affinity
are purified in a simple one-step procedure (for details see: The
QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the
supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8,
the column is first washed with 10 volumes of 6 M guanidine-HCl, pH
8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and
finally the B. burgdorferi polypeptide is eluted with 6 M
guanidine-HCl, pH 5.
[0234] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein could be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4.degree. C. or frozen at -80.degree. C.
[0235] The polypeptide of the present invention are also prepared
using a non-denaturing protein purification method. For these
polypeptides, the cell pellet from each liter of culture is
resuspended in 25 mls of Lysis Buffer A at 4.degree. C. (Lysis
Buffer A=50 mM Na-phosphate, 300 mM NaCl, 10 mM 2-mercaptoethanol,
10% Glycerol, pH 7.5 with 1 tablet of Complete EDTA-free protease
inhibitor cocktail (Boehringer Mannheim #1873580) per 50 ml of
buffer). Absorbance at 550 nm is approximately 10-20 O.D./ml. The
suspension is then put through three freeze/thaw cycles from
-70.degree. C. (using a ethanol-dry ice bath) up to room
temperature. The cells are lysed via sonication in short 10 sec
bursts over 3 minutes at approximately 80 W while kept on ice. The
sonicated sample is then centrifuged at 15,000 RPM for 30 minutes
at 4.degree. C. The supernatant is passed through a column
containing 1.0 ml of CL-4B resin to pre-clear the sample of any
proteins that may bind to agarose non-specifically, and the
flow-through fraction is collected.
[0236] The pre-cleared flow-through is applied to a
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(Qiagen, Inc., supra). Proteins with a 6.times.His tag bind to the
Ni-NTA resin with high affinity and can be purified in a simple
one-step procedure. Briefly, the supernatant is loaded onto the
column in Lysis Buffer A at 4.degree. C., the column is first
washed with 10 volumes of Lysis Buffer A until the A280 of the
eluate returns to the baseline. Then, the column is washed with 5
volumes of 40 mM Imidazole (92% Lysis Buffer A/8% Buffer B) (Buffer
B=50 mM Na-Phosphate, 300 mM NaCl, 10% Glycerol, 10 mM
2-mercaptoethanol, 500 mM Imidazole, pH of the final buffer should
be 7.5). The protein is eluted off of the column with a series of
increasing Imidazole solutions made by adjusting the ratios of
Lysis Buffer A to Buffer B. Three different concentrations are
used: 3 volumes of 75 mM Imidazole, 3 volumes of 150 mM Imidazole,
5 volumes of 500 mM Imidazole. The fractions containing the
purified protein are analyzed using 8%, 10% or 14% SDS-PAGE
depending on the protein size. The purified protein is then
dialyzed 2.times. against phosphate-buffered saline (PBS) in order
to place it into an easily workable buffer. The purified protein is
stored at 4.degree. C. or frozen at -80.degree..
[0237] The following alternative method may be used to purify B.
burgdorferi expressed in E coli when it is present in the form of
inclusion bodies. Unless otherwise specified, all of the following
steps are conducted at 4-10.degree. C.
[0238] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
[0239] The cells are then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0240] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the B. burgdorferi polypeptide-containing supernatant is
incubated at 4.degree. C. overnight to allow further GuHCl
extraction.
[0241] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0242] To clarify the refolded B. burgdorferi polypeptide solution,
a previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
[0243] Fractions containing the B. burgdorferi polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the B. burgdorferi polypeptide (determined, for
instance, by 16% SDS-PAGE) are then pooled.
[0244] The resultant B. burgdorferi polypeptide exhibits greater
than 95% purity after the above refolding and purification steps.
No major contaminant bands are observed from Commassie blue stained
16% SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 ng/ml according to
LAL assays.
[0245] 3(b). Alternative Expression and Purification Borrelia
Polypeptides in E. coli
[0246] The vector pQE10 is alternatively used to clone and express
some of the polypeptides of the present invention for use in the
soft tissue and systemic infection models discussed below. The
difference being such that an inserted DNA fragment encoding a
polypeptide expresses that polypeptide with the six His residues
(i.e., a "6.times.His tag") covalently linked to the amino terminus
of that polypeptide. The bacterial expression vector pQE10 (QIAGEN,
Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311) was used in this
example. The components of the pQE10 plasmid are arranged such that
the inserted DNA sequence encoding a polypeptide of the present
invention expresses the polypeptide with the six His residues
(i.e., a "6.times.His tag")) covalently linked to the amino
terminus.
[0247] The DNA sequences encoding the desired portions of a
polypeptide of Table 1 were amplified using PCR oligonucleotide
primers from genomic B. burgdorferi DNA. The PCR primers anneal to
the nucleotide sequences encoding the desired amino acid sequence
of a polypeptide of the present invention. Additional nucleotides
containing restriction sites to facilitate cloning in the pQE10
vector were added to the 5' and 3' primer sequences,
respectively.
[0248] For cloning a polypeptide of the present invention, the 5'
and 3' primers were selected to amplify their respective nucleotide
coding sequences. One of ordinary skill in the art would appreciate
that the point in the protein coding sequence where the 5' and 3'
primers begins may be varied to amplify a DNA segment encoding any
desired portion of a polypeptide of the present invention. The 5'
primer was designed so the coding sequence of the 6.times.His tag
is aligned with the restriction site so as to maintain its reading
frame with that of B. burgdorferi polypeptide. The 3' was designed
to include an stop codon. The amplified DNA fragment was then
cloned, and the protein expressed, as described above for the pQE60
plasmid.
[0249] The DNA sequences of Table 1 encoding amino acid sequences
may also be cloned and expressed as fusion proteins by a protocol
similar to that described directly above, wherein the pET-32b(+)
vector (Novagen, 601 Science Drive, Madison, Wis. 53711) is
preferentially used in place of pQE10.
[0250] The above methods are not limited to the polypeptide
fragments actually produced. The above method, like the methods
below, can be used to produce either full length polypeptides or
desired fragments thereof.
[0251] 3(c). Alternative Expression and Purification of Borrelia
Polypeptides in E. coli
[0252] The bacterial expression vector pQE60 is used for bacterial
expression in this example (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). However, in this example, the
polypeptide coding sequence is inserted such that translation of
the six His codons is prevented and, therefore, the polypeptide is
produced with no 6.times.His tag.
[0253] The DNA sequence encoding the desired portion of the B.
burgdorferi amino acid sequence is amplified from an B. burgdorferi
genomic DNA prep the deposited DNA clones using PCR oligonucleotide
primers which anneal to the 5' and 3' nucleotide sequences
corresponding to the desired portion of the B. burgdorferi
polypeptides. Additional nucleotides containing restriction sites
to facilitate cloning in the pQE60 vector are added to the 5' and
3' primer sequences.
[0254] For cloning a B. burgdorferi polypeptides of the present
invention, 5' and 3' primers are selected to amplify their
respective nucleotide coding sequences. One of ordinary skill in
the art would appreciate that the point in the protein coding
sequence where the 5' and 3' primers begin may be varied to amplify
a DNA segment encoding any desired portion of a polypeptide of the
present invention. The 3' and 5' primers contain appropriate
restriction sites followed by nucleotides complementary to the 5'
and 3' ends of the coding sequence respectively. The 3' primer is
additionally designed to include an in-frame stop codon.
[0255] The amplified B. burgdorferi DNA fragments and the vector
pQE60 are digested with restriction enzymes recognizing the sites
in the primers and the digested DNAs are then ligated together.
Insertion of the B. burgdorferi DNA into the restricted pQE60
vector places the B. burgdorferi protein coding region including
its associated stop codon downstream from the IPTG-inducible
promoter and in-frame with an initiating AUG. The associated stop
codon prevents translation of the six histidine codons downstream
of the insertion point.
[0256] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
et al. E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), is used in carrying out the
illustrative example described herein. This strain, which is only
one of many that are suitable for expressing B. burgdorferi
polypeptide, is available commercially (QIAGEN, Inc., supra).
Transformants are identified by their ability to grow on LB plates
in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
[0257] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0258] To purify the B. burgdorferi polypeptide, the cells are then
stirred for 3-4 hours at 4.degree. C. in 6M guanidine-HCl, pH 8.
The cell debris is removed by centrifugation, and the supernatant
containing the B. burgdorferi polypeptide is dialyzed against 50 mM
Na-acetate buffer pH 6, supplemented with 200 mM NaCl.
Alternatively, the protein can be successfully refolded by
dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH
7.4, containing protease inhibitors. After renaturation the protein
can be purified by ion exchange, hydrophobic interaction and size
exclusion chromatography. Alternatively, an affinity chromatography
step such as an antibody column can be used to obtain pure B.
burgdorferi polypeptide. The purified protein is stored at
4.degree. C. or frozen at -80.degree. C.
[0259] The following alternative method may be used to purify B.
burgdorferi polypeptides expressed in E coli when it is present in
the form of inclusion bodies. Unless otherwise specified, all of
the following steps are conducted at 4-10.degree. C.
[0260] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
[0261] The cells ware then lysed by passing the solution through a
microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0262] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the B. burgdorferi polypeptide-containing supernatant is
incubated at 4.degree. C. overnight to allow further GuHCl
extraction.
[0263] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0264] To clarify the refolded B. burgdorferi polypeptide solution,
a previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
[0265] Fractions containing the B. burgdorferi polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the B. burgdorferi polypeptide (determined, for
instance, by 16% SDS-PAGE) are then pooled.
[0266] The resultant B. burgdorferi polypeptide exhibits greater
than 95% purity after the above refolding and purification steps.
No major contaminant bands are observed from Commassie blue stained
16% SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 ng/ml according to
LAL assays.
[0267] 3(d). Cloning and Expression of B. burgdorferi in Other
Bacteria
[0268] B. burgdorferi polypeptides can also be produced in: B.
burgdorferi using the methods of S. Skinner et al., (1988) Mol.
Microbiol. 2: 289-297 or J. I. Moreno (1996) Protein Expr. Purif.
8(3): 332-340; Lactobacillus using the methods of C. Rush et al.,
1997 Appl. Microbiol. Biotechnol. 47(5): 537-542; or in Bacillus
subtilis using the methods Chang et al., U.S. Pat. No.
4,952,508.
[0269] Cloning and Expression in COS Cells
[0270] A B. burgdorferi expression plasmid is made by cloning a
portion of the DNA encoding a B. burgdorferi polypeptide into the
expression vector pDNAI/Amp or pDNAIII (which can be obtained from
Invitrogen, Inc.). The expression vector pDNAI/amp contains: (1) an
E. coli origin of replication effective for propagation in E. coli
and other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a DNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al. 1984 Cell 37: 767. The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pDNAIII contains, in addition, the selectable neomycin
marker.
[0271] A DNA fragment encoding a B. burgdorferi polypeptide is
cloned into the polylinker region of the vector so that recombinant
protein expression is directed by the CMV promoter. The plasmid
construction strategy is as follows. The DNA from a B. burgdorferi
genomic DNA prep is amplified using primers that contain convenient
restriction sites, much as described above for construction of
vectors for expression of B. burgdorferi in E. coli. The 5' primer
contains a Kozak sequence, an AUG start codon, and nucleotides of
the 5' coding region of the B. burgdorferi polypeptide. The 3'
primer, contains nucleotides complementary to the 3' coding
sequence of the B. burgdorferi DNA, a stop codon, and a convenient
restriction site.
[0272] The PCR amplified DNA fragment and the vector, pDNAI/Amp,
are digested with appropriate restriction enzymes and then ligated.
The ligation mixture is transformed into an appropriate E. coli
strain such as SURE.TM. (Stratagene Cloning Systems, La Jolla,
Calif. 92037), and the transformed culture is plated on ampicillin
media plates which then are incubated to allow growth of ampicillin
resistant colonies. Plasmid DNA is isolated from resistant colonies
and examined by restriction analysis or other means for the
presence of the fragment encoding the B. burgdorferi
polypeptide.
[0273] For expression of a recombinant B. burgdorferi polypeptide,
COS cells are transfected with an expression vector, as described
above, using DEAE-dextran, as described, for instance, by Sambrook
et al. (supra). Cells are incubated under conditions for expression
of B. burgdorferi by the vector.
[0274] Expression of the B. burgdorferi-HA fusion protein is
detected by radiolabeling and immunoprecipitation, using methods
described in, for example Harlow et al., supra. To this end, two
days after transfection, the cells are labeled by incubation in
media containing .sup.35S-cysteine for 8 hours. The cells and the
media are collected, and the cells are washed and the lysed with
detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS,
1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et
al. (supra). Proteins are precipitated from the cell lysate and
from the culture media using an HA-specific monoclonal antibody.
The precipitated proteins then are analyzed by SDS-PAGE and
autoradiography. An expression product of the expected size is seen
in the cell lysate, which is not seen in negative controls.
[0275] 5. Cloning and Expression in CHO Cells
[0276] The vector pC4 is used for the expression of B. burgdorferi
polypeptide in this example. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains
the mouse DHFR gene under control of the SV40 early promoter.
Chinese hamster ovary cells or other cells lacking dihydrofolate
activity that are transfected with these plasmids can be selected
by growing the cells in a selective medium (alpha minus MEM, Life
Technologies) supplemented with the chemotherapeutic agent
methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented. See,
e.g., Alt et al., 1978, J. Biol. Chem. 253: 1357-1370; Hamlin et
al., 1990, Biochem. et Biophys. Acta, 1097: 107-143; Page et al.,
1991, Biotechnology 9: 64-68. Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
[0277] Plasmid pC4 contains the strong promoter of the long
terminal repeat (LTR) of the Rouse Sarcoma Virus, for expressing a
polypeptide of interest, Cullen, et al. (1985) Mol. Cell. Biol. 5:
438-447; plus a fragment isolated from the enhancer of the
immediate early gene of human cytomegalovirus (CMV), Boshart, et
al., 1985, Cell 41: 521-530. Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: Bam HI, Xba I, and Asp 718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and
similar systems can be used to express the B. burgdorferi
polypeptide in a regulated way in mammalian cells (Gossen et al.,
1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551. For the
polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well. Stable cell
lines carrying a gene of interest integrated into the chromosomes
can also be selected upon co-transfection with a selectable marker
such as gpt, G418 or hygromycin. It is advantageous to use more
than one selectable marker in the beginning, e.g., G418 plus
methotrexate.
[0278] The plasmid pC4 is digested with the restriction enzymes and
then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel. The DNA sequence encoding the B. burgdorferi
polypeptide is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the desired portion of
the gene. A 5' primer containing a restriction site, a Kozak
sequence, an AUG start codon, and nucleotides of the 5' coding
region of the B. burgdorferi polypeptide is synthesized and used. A
3' primer, containing a restriction site, stop codon, and
nucleotides complementary to the 3' coding sequence of the B.
burgdorferi polypeptides is synthesized and used. The amplified
fragment is digested with the restriction endonucleases and then
purified again on a 1% agarose gel. The isolated fragment and the
dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB 101 or XL-1 Blue cells are then transformed and bacteria
are identified that contain the fragment inserted into plasmid pC4
using, for instance, restriction enzyme analysis.
[0279] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using a
lipid-mediated transfection agent such as Lipofectin.TM. or
LipofectAMINE.TM. (Life Technologies Gaithersburg, Md.). The
plasmid pSV2-neo contains a dominant selectable marker, the neo
gene from Tn5 encoding an enzyme that confers resistance to a group
of antibiotics including G418. The cells are seeded in alpha minus
MEM supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of methotrexate plus 1 mg/ml G418. After about 10-14 days single
clones are trypsinized and then seeded in 6-well petri dishes or 10
ml flasks using different concentrations of methotrexate (50 nM,
100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well
plates containing even higher concentrations of methotrexate (1
.mu.M, 2 .mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is
repeated until clones are obtained which grow at a concentration of
100-200 .mu.M. Expression of the desired gene product is analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase
HPLC analysis.
[0280] 6. Immunization and Detection of Immune Responses
[0281] 6(a). B. burgdorferi Propagation
[0282] B. burgdorferi sensu stricto isolate B31 is propagated in
tightly-closed containers at 34.degree. C. in modified
Barbour-Stoenner-Kelly (BSKII) medium (Barbour, A. G., Yale J.
Biol. Med. 57: 521-525 (1984)) overlaid with a 5% O.sub.2/5%
CO.sub.2/90% N.sub.2 gas mixture. Cell densities of these cultures
are determined by darkfield microscopy at 400.times..
[0283] Immunization of Mice and Challenge with B. burgdorferi. For
active immunizations BALB/cByJ mice (BALB, Jackson Laboratories)
are injected intraperitoneally (i.p.) at week 0 with 20 g of
recombinant borrelial protein, or phosphate-buffered saline (PBS),
emulsified with complete Freund's adjuvant (CFA), given a similar
booster immunization in incomplete Freund's adjuvant (IFA) at week
4, and challenged at week 6. For challenge B. burgdorferi are
diluted in BSKII from exponentially-growing cultures and mice are
injected subcutaneously (s.c.) at the base of the tail with 0.1 ml
of these dilutions (typically 10.sup.3-10.sup.4 borreliae;
approximately 10-100 times the median infectious dose). Borreliae
used for challenge are passaged fewer than six times in vitro. To
assess infection, mice are sacrificed at 14-17 days post-challenge,
and specimens derived from ear, bladder, and tibiotarsal joints are
placed in BSKII plus 1.4% gelatin, 13 g/ml amphotericin B, 1.5 g/ml
phosphomycin, and 15 g/ml rifampicin, and borrelia outgrowth at two
or three weeks is quantified by darkfield microscopy. Batches of
BSKII are qualified for infection testing by confirming that they
supported the growth of 1-5 cells of isolate B31. In some instances
seroconversion for protein P39 reactivity is also used to confirm
infections (see below). Others have previously shown that mice
elicited antibodies to P39 when inoculated with live borreliae by
syringe or tick bite, but not with killed borreliae (Simpson, W.
J., et al., J. Clin. Microbiol. 29: 236-243 (1991)).
[0284] 6(b). Immunoassays
[0285] Several immunoassay formats are used to quantify levels of
borrelia-specific antibodies (ELISA and immunoblot), and to
evaluate the functional properties of these antibodies (growth
inhibition assay). The ELISA and immunoblot assays are also used to
detect and quantify antibodies elicited in response to borrelial
infection that react with specific borrelial antigens. Where
antibodies to certain borrelial antigens are elicited by infection
this is taken as evidence that the borrelial proteins in question
are expressed in vivo. Absence of infection-derived antibodies
(seroconversion) following borrelial challenge is evidence that
infection is prevented or suppressed. The immunoblot assay is also
used to ascertain whether antibodies raised against recombinant
borrelial antigens recognize a protein of similar size in extracts
of whole borreliae. Where the natural protein is of similar, or
identical, size in the immunoblot assay to the recombinant version
of the same protein, this is taken as evidence that the recombinant
protein is the product of a full-length clone of the respective
gene.
[0286] Enzyme-Linked Immunosorbant Assay (ELISA). The ELISA is used
to quantify levels of antibodies reactive with borrelial antigens
elicited in response to immunization with these borrelial antigens.
Wells of 96 well microtiter plates (Immunlon 4, Dynatech,
Chantilly, Va., or equivalent) are coated with antigen by
incubating 50 l of 1 g/ml protein antigen solution in a suitable
buffer, typically 0.1 M sodium carbonate buffer at pH 9.6. After
decanting unbound antigen, additional binding sites are blocked by
incubating 100 l of 3% nonfat milk in wash buffer (PBS, 0.2% Tween
20, pH 7.4). After washing, duplicate serial two-fold dilutions of
sera in PBS, Tween 20, 1% fetal bovine serum, are incubated for 1
hr, removed, wells are washed three times, and incubated with
horseradish peroxidase-conjugated goat anti-mouse IgG. After three
washes, bound antibodies are detected with H.sub.2O.sub.2 and
2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (Schwan, T. G., et
al., Proc. Natl. Acad. Sci. USA 92: 2909-2913 (1985)) (ABTS.RTM.,
Kirkegaard & Perry Labs., Gaithersburg, Md.) and A.sub.405 is
quantified with a Molecular Devices, Corp. (Menlo Park, Calif.)
Vmax.TM. plate reader. IgG levels twice the background level in
serum from naive mice are assigned the minimum titer of 1:100.
[0287] 6(c). In Vitro Growth Inhibition Assay
[0288] Unlike other bacteria, borreliae can be killed by the
binding of specific antibodies to their surface antigens. The
mechanism for this in vitro killing or growth-inhibitory effect is
not known, but can occur in the absence of serum complement, or
other immune effector functions. Antibodies elicited in animals
receiving immunizations with specific borrelial antigens that
result in protection from borrelial challenge usually will directly
kill borreliae in vitro. Thus, the in vitro growth inhibition assay
also has a high predictive value for the protective potency of the
borrelial antibodies, although exceptions, such as antibodies
against OspC which are weak at in vitro growth inhibition, have
been observed. Also, this assay can be used to evaluate the
serologic conservation of epitope binding protective antibodies. A
microwell antibody titration assay (Sadziene, A., et al., J.
Infect. Dis. 167: 165-172 (1993)) is used to evaluate the growth
inhibition (GI) properties of antisera against recombinant
borrelial antigens against the homologous B31 isolate, and against
various strains of borrelia. Briefly, 10.sup.5 borrelia in 100 l
BSKII are added to serial two-fold dilutions of sera in 100 l BSKII
in 96-well plates, and the plates are covered and incubated at
34.degree. C. in a 5% O.sub.2/5% CO.sub.2/90% N.sub.2 gas mixture
for 72 h prior to quantification of borrelia growth by darkfield
microscopy.
[0289] 6(d). Sodiumdodecylsulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) and Immunoblotting
[0290] Using a single well format, total borrelial protein
extracts, recombinant borrelial antigen, or recombinant P39 samples
(2 g of purified protein, or more for total borrelial extracts) are
boiled in SDS/2-ME sample buffer before electrophoresis through 3%
acrylamide stacking gels, and resolving gels of higher acrylamide
concentration, typically 10-15% acrylamide monomer. Gels are
electro-blotted to nitrocellulose membranes and lanes are probed
with dilutions of antibody to be tested for reactivity with
specific borrelial antigens, followed by the appropriate secondary
antibody-enzyme (horseradish peroxidase) conjugate. When it is
desirable to confirm that the protein had transferred following
electro-blotting, membranes are stained with Ponceau S. Immunoblot
signals from bound antibodies are detected on x-ray film as
chemiluminescence using ECL.TM. reagents (Amersham Corp., Arlington
Heights, Ill.).
[0291] 6(e). Detection of Borrelia mRNA Expression
[0292] Northern blot analysis is carried out using methods
described by, among others, Sambrook et al., supra. to detect the
expression of the B. burgdorferi nucleotide sequences of the
present invention in animal tissues. A cDNA probe containing an
entire nucleotide sequence shown in Table 1 is labeled with
.sup.32P using the rediprime.TM. DNA labeling system (Amersham Life
Science), according to manufacturer's instructions. After labeling,
the probe is purified using a CHROMA SPIN-100.TM. column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number
PT1200-1. The purified labeled probe is then used to detect the
expression of Borrelia mRNA in an animal tissue sample.
[0293] Animal tissues, such as blood or spinal fluid, are examined
with the labeled probe using ExpressHyb.TM. hybridization solution
(Clontech) according to manufacturer's protocol number PT1190-1.
Following hybridization and washing, the blots are mounted and
exposed to film at -70 C overnight, and films developed according
to standard procedures.
[0294] The disclosure of all publications (including patents,
patent applications, journal articles, laboratory manuals, books,
or other documents) cited herein are hereby incorporated by
reference in their entireties.
[0295] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention. Functionally
equivalent methods and components are within the scope of the
invention, in addition to those shown and described herein and will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
1TABLE 1 Nucleotide and Amino Acid Sequences. f101.aa (SEQ ID NO:1)
t101.aa (SEQ ID NO:2) f101.nt (SEQ ID NO:3) t101.nt (SEQ ID NO:4)
f11.aa (SEQ ID NO:5) t11.aa (SEQ ID NO:6) f11.nt (SEQ ID NO:7)
t11.nt (SEQ ID NO:8) f12.aa (SEQ ID NO:9) t12.aa (SEQ ID NO:10)
f12.nt (SEQ ID NO:11) t12.nt (SEQ ID NO:12) f129.aa (SEQ ID NO:13)
t129.aa (SEQ ID NO:14) f129.nt (SEQ ID NO:15) t129.nt (SEQ ID
NO:16) f142.aa (SEQ ID NO:17) t142.aa (SEQ ID NO:18) f142.nt (SEQ
ID NO:19) t142.nt (SEQ ID NO:20) f147.aa (SEQ ID NO:21) t147.aa
(SEQ ID NO:22) f147.nt (SEQ ID NO:23) t147.nt (SEQ ID NO:24)
f152.aa (SEQ ID NO:25) t152.aa (SEQ ID NO:26) f152.nt (SEQ ID
NO:27) t152.nt (SEQ ID NO:28) f154.aa (SEQ ID NO:29) t154.aa (SEQ
ID NO:30) f154.nt (SEQ ID NO:31) t154.nt (SEQ ID NO:32) f157.aa
(SEQ ID NO:33) t157.aa (SEQ ID NO:34) f157.nt (SEQ ID NO:35)
t157.nt (SEQ ID NO:36) f17.aa (SEQ ID NO:37) t17.aa (SEQ ID NO:38)
f17.nt (SEQ ID NO:39) t17.nt (SEQ ID NO:40) f170.aa (SEQ ID NO:41)
t170.aa (SEQ ID NO:42) f170.nt (SEQ ID NO:43) t170.nt (SEQ ID
NO:44) f186.aa (SEQ ID NO:45) t186.aa (SEQ ID NO:46) f186.nt (SEQ
ID NO:47) t186.nt (SEQ ID NO:48) f196.aa (SEQ ID NO:49) t196.aa
(SEQ ID NO:50) f196.nt (SEQ ID NO:51) t196.nt (SEQ ID NO:52)
f899.aa (SEQ ID NO:53) t899.aa (SEQ ID NO:54) f899.nt (SEQ ID
NO:55) t899.nt (SEQ ID NO:56) f924.aa (SEQ ID NO:57) t924.aa (SEQ
ID NO:58) f924.nt (SEQ ID NO:59) t924.nt (SEQ ID NO:60) f925.aa
(SEQ ID NO:61) t925.aa (SEQ ID NO:62) f925.nt (SEQ ID NO:63)
t925.nt (SEQ ID NO:64) f929.aa (SEQ ID NO:65) t929.aa (SEQ ID
NO:66) f929.nt (SEQ ID NO:67) t929.nt (SEQ ID NO:68) f933.aa (SEQ
ID NO:69) t933.aa (SEQ ID NO:70) f933.nt (SEQ ID NO:71) t933.nt
(SEQ ID NO:72) f940.aa (SEQ ID NO:73) t940.aa (SEQ ID NO:74)
f940.nt (SEQ ID NO:75) t940.nt (SEQ ID NO:76) f943.aa (SEQ ID
NO:77) t943.aa (SEQ ID NO:78) f943.nt (SEQ ID NO:79) t943.nt (SEQ
ID NO:80) f952.aa (SEQ ID NO:81) t952.aa (SEQ ID NO:82) f952.nt
(SEQ ID NO:83) t952.nt (SEQ ID NO:84) f378.aa (SEQ ID NO:85)
t378.aa (SEQ ID NO:86) f378.nt (SEQ ID NO:87) t378.nt (SEQ ID
NO:88) f4.aa (SEQ ID NO:89) t4.aa (SEQ ID NO:90) f4.nt (SEQ ID
NO:91) t4.nt (SEQ ID NO:92) f43.aa (SEQ ID NO:93) t43.aa (SEQ ID
NO:94) f43.nt (SEQ ID NO:95) t43.nt (SEQ ID NO:96) f50.aa (SEQ ID
NO:97) t50.aa (SEQ ID NO:98) f50.nt (SEQ ID NO:99) t50.nt (SEQ ID
NO:100) f65.aa (SEQ ID NO:101) t65.aa (SEQ ID NO:102) f65.nt (SEQ
ID NO:103) t65.nt (SEQ ID NO:104) f8.aa (SEQ ID NO:105) t8.aa (SEQ
ID NO:106) f8.nt (SEQ ID NO:107) t8.nt (SEQ ID NO:108) f82.aa (SEQ
ID NO:109) t82.aa (SEQ ID NO:110) f82.nt (SEQ ID NO:111) f82.nt
(SEQ ID NO:112) f86.aa (SEQ ID NO:113) t86.aa (SEQ ID NO:114)
f86.nt (SEQ ID NO:115) t86.nt (SEQ ID NO:116) f90.aa (SEQ ID
NO:117) t90.aa (SEQ ID NO:118) f90.nt (SEQ ID NO:119) t90.nt (SEQ
ID NO:120) f469.aa (SEQ ID NO:121) t469.aa (SEQ ID NO:122) f469.nt
(SEQ ID NO:123) t469.nt (SEQ ID NO:124) f477.aa (SEQ ID NO:125)
t477.aa (SEQ ID NO:126) f477.nt (SEQ ID NO:127) t477.nt (SEQ ID
NO:128) f488.aa (SEQ ID NO:129) t488.aa (SEQ ID NO:130) f488.nt
(SEQ ID NO:131) t488.nt (SEQ ID NO:132) f494.aa (SEQ ID NO:133)
t494.aa (SEQ ID NO:134) f494.nt (SEQ ID NO:135) t494.nt (SEQ ID
NO:136) f516.aa (SEQ ID NO:137) t516.aa (SEQ ID NO:138) f516.nt
(SEQ ID NO:139) t516.nt (SEQ ID NO:140) f517.aa (SEQ ID NO:141)
t517.aa (SEQ ID NO:142) f517.nt (SEQ ID NO:143) t517.nt (SEQ ID
NO:144) f519.aa (SEQ ID NO:145) t519.aa (SEQ ID NO:146) f519.nt
(SEQ ID NO:147) t519.nt (SEQ ID NO:148) f520.aa (SEQ ID NO:149)
t520.aa (SEQ ID NO:150) f520.nt (SEQ ID NO:151) t520.nt (SEQ ID
NO:152) f523.aa (SEQ ID NO:153) t523.aa (SEQ ID NO:154) f523.nt
(SEQ ID NO:155) f523.nt (SEQ ID NO:156) f526.aa (SEQ ID NO:157)
t526.aa (SEQ ID NO:158) f526.nt (SEQ ID NO:159) t526.nt (SEQ ID
NO:160) f544.aa (SEQ ID NO:161) t544.aa (SEQ ID NO:162) f544.nt
(SEQ ID NO:163) t544.nt (SEQ ID NO:164) f545.aa (SEQ ID NO:165)
t545.aa (SEQ ID NO:166) f545.nt (SEQ ID NO:167) t545.nt (SEQ ID
NO:168) f577.aa (SEQ ID NO:169) t577.aa (SEQ ID NO:170) f577.nt
(SEQ ID NO:171) t577.nt (SEQ ID NO:172) f584.aa (SEQ ID NO:173)
t584.aa (SEQ ID NO:174) f584.nt (SEQ ID NO:175) t584.nt (SEQ ID
NO:176) f596.aa (SEQ ID NO:177) t596.aa (SEQ ID NO:178) f596.nt
(SEQ ID NO:179) t596.nt (SEQ ID NO:180) f598.aa (SEQ ID NO:181)
t598.aa (SEQ ID NO:182) f598.nt (SEQ ID NO:183) t598.nt (SEQ ID
NO:184) f600.aa (SEQ ID NO:185) t600.aa (SEQ ID NO:186) f600.nt
(SEQ ID NO:187) t600.nt (SEQ ID NO:188) f603.aa (SEQ ID NO:189)
t603.aa (SEQ ID NO:190) f603.nt (SEQ ID NO:191) t603.nt (SEQ ID
NO:192) f607.aa (SEQ ID NO:193) t607.aa (SEQ ID NO:194) f607.nt
(SEQ ID NO:195) t607.nt (SEQ ID NO:196) f611.aa (SEQ ID NO:197)
t611.aa (SEQ ID NO:198) f611.nt (SEQ ID NO:199) t611.nt (SEQ ID
NO:200) f617.aa (SEQ ID NO:201) t617.aa (SEQ ID NO:202) f617.nt
(SEQ ID NO:203) t617.nt (SEQ ID NO:204) f631.aa (SEQ ID NO:205)
t631.aa (SEQ ID NO:206) f631.nt (SEQ ID NO:207) t631.nt (SEQ ID
NO:208) f647.aa (SEQ ID NO:209) t647.aa (SEQ ID NO:210) f647.nt
(SEQ ID NO:211) t647.nt (SEQ ID NO:212) f653.aa (SEQ ID NO:213)
t653.aa (SEQ ID NO:214) f653.nt (SEQ ID NO:215) t653.nt (SEQ ID
NO:216) f664.aa (SEQ ID NO:217) t664.aa (SEQ ID NO:218) f664.nt
(SEQ ID NO:219) t664.nt (SEQ ID NO:220) f680.aa (SEQ ID NO:221)
t680.aa (SEQ ID NO:222) f680.nt (SEQ ID NO:223) t680.nt (SEQ ID
NO:224) f688.aa (SEQ ID NO:225) t688.aa (SEQ ID NO:226) f688.nt
(SEQ ID NO:227) t688.nt (SEQ ID NO:228) f704.aa (SEQ ID NO:229)
t704.aa (SEQ ID NO:230) f704.nt (SEQ ID NO:231) t704.nt (SEQ ID
NO:232) f707.aa (SEQ ID NO:233) t707.aa (SEQ ID NO:234) f707.nt
(SEQ ID NO:235) t707.nt (SEQ ID NO:236) f709.aa (SEQ ID NO:237)
t709.aa (SEQ ID NO:238) f709.nt (SEQ ID NO:239) t709.nt (SEQ ID
NO:240) f730.aa (SEQ ID NO:241) t730.aa (SEQ ID NO:242) f730.nt
(SEQ ID NO:243) t730.nt (SEQ ID NO:244) f197.aa (SEQ ID NO:245)
t197.aa (SEQ ID NO:246) f197.nt (SEQ ID NO:247) t197.nt (SEQ ID
NO:248) f200.aa (SEQ ID NO:249) t200.aa (SEQ ID NO:250) f200.nt
(SEQ ID NO:251) t200.nt (SEQ ID NO:252) f208.aa (SEQ ID NO:253)
t208.aa (SEQ ID NO:254) f208.nt (SEQ ID NO:255) t208.nt (SEQ ID
NO:256) f210.aa (SEQ ID NO:257) t210.aa (SEQ ID NO:258) f210.nt
(SEQ ID NO:259) t210.nt (SEQ ID NO:260) f22.aa (SEQ ID NO:261)
t22.aa (SEQ ID NO:262) f22.nt (SEQ ID NO:263) t22.nt (SEQ ID
NO:264) f221.aa (SEQ ID NO:265) t221.aa (SEQ ID NO:266) f221.nt
(SEQ ID NO:267) t221.nt (SEQ ID NO:268) f253.aa (SEQ ID NO:269)
t253.aa (SEQ ID NO:270) f253.nt (SEQ ID NO:271) t253.nt (SEQ ID
NO:272) f265.aa (SEQ ID NO:273) t265.aa (SEQ ID NO:274) f265.nt
(SEQ ID NO:275) t265.nt (SEQ ID NO:276) f269.aa (SEQ ID NO:277)
t269.aa (SEQ ID NO:278) f269.nt (SEQ ID NO:279) t269.nt (SEQ ID
NO:280) f29.aa (SEQ ID NO:281) t29.aa (SEQ ID NO:282) f29.nt (SEQ
ID NO:283) t29.nt (SEQ ID NO:284) f290.aa (SEQ ID NO:285) t290.aa
(SEQ ID NO:286) f290.nt (SEQ ID NO:287) t290.nt (SEQ ID NO:288)
f291.aa (SEQ ID NO:289) t291.aa (SEQ ID NO:290) f291.nt (SEQ ID
NO:291) t291.nt (SEQ ID NO:292) f296.aa (SEQ ID NO:293) t296.aa
(SEQ ID NO:294) f296.nt (SEQ ID NO:295) t296.nt (SEQ ID NO:296)
f3.aa (SEQ ID NO:297) t3.aa (SEQ ID NO:298) f3.nt (SEQ ID NO:299)
t3.nt (SEQ ID NO:300) f30.aa (SEQ ID NO:301) t30.aa (SEQ ID NO:302)
f30.nt (SEQ ID NO:303) t30.nt (SEQ ID NO:304) f308.aa (SEQ ID
NO:305) t308.aa (SEQ ID NO:306) f308.nt (SEQ ID NO:307) t308.nt
(SEQ ID NO:308) f31.aa (SEQ ID NO:309) t31.aa (SEQ ID NO:310)
f31.nt (SEQ ID NO:311) t31.nt (SEQ ID NO:312) f939.aa (SEQ ID
NO:313) f939.aa (SEQ ID NO:314) f939.nt (SEQ ID NO:315) t939.nt
(SEQ ID NO:316) f739.aa (SEQ ID NO:317) t739.aa (SEQ ID NO:318)
f739.nt (SEQ ID NO:319) t739.nt (SEQ ID NO:320) f742.aa (SEQ ID
NO:321) t742.aa (SEQ ID NO:322) f742.nt (SEQ ID NO:323) t742.nt
(SEQ ID NO:324) f743.aa (SEQ ID NO:325) t743.aa (SEQ ID NO:326)
f743.nt (SEQ ID NO:327) t743.nt (SEQ ID NO:328) f748.aa (SEQ ID
NO:329) t748.aa (SEQ ID NO:330) t748.nt (SEQ ID NO:331) t748.nt
(SEQ ID NO:332) t764.aa (SEQ ID NO:333) f764.aa (SEQ ID NO:334)
f764.nt (SEQ ID NO:335) t764.nt (SEQ ID NO:336) f770.aa (SEQ ID
NO:337) t770.aa (SEQ ID NO:338) f770.nt (SEQ ID NO:339) t770.nt
(SEQ ID NO:340) f790.aa (SEQ ID NO:341) t790.aa (SEQ ID NO:342)
f790.nt (SEQ ID NO:343) t790.nt (SEQ ID NO:344) f792.aa (SEQ ID
NO:345) t792.aa (SEQ ID NO:346) f792.nt (SEQ ID NO:347) t792.nt
(SEQ ID NO:348) f797.aa (SEQ ID NO:349) t797.aa (SEQ ID NO:350)
f797.nt (SEQ ID NO:351) t797.nt (SEQ ID NO:352) f799.aa (SEQ ID
NO:353) t799.aa (SEQ ID NO:354) f799.nt (SEQ ID NO:355) t799.nt
(SEQ ID NO:356) f800.aa (SEQ ID NO:357) t800.aa (SEQ ID NO:358)
f800.nt (SEQ ID NO:359) t800.nt (SEQ ID NO:360) f810.aa (SEQ ID
NO:361) t810.aa (SEQ ID NO:362) f810.nt (SEQ ID NO:363) t810.nt
(SEQ ID NO:364) f814.aa (SEQ ID NO:365) t814.aa (SEQ ID NO:366)
f814.nt (SEQ ID NO:367) t814.nt (SEQ ID NO:368) f818.aa (SEQ ID
NO:369) t818.aa (SEQ ID NO:370) f818.nt (SEQ ID NO:371) t818.nt
(SEQ ID NO:372) f820.aa (SEQ ID NO:373) t820.aa (SEQ ID NO:374)
f820.nt (SEQ ID NO:375) t820.nt (SEQ ID NO:376) f831.aa (SEQ ID
NO:377) t831.aa (SEQ ID NO:378) f831.nt (SEQ ID NO:379) t831.nt
(SEQ ID NO:380) f843.aa (SEQ ID NO:381) t843.aa (SEQ ID NO:382)
f843.nt (SEQ ID NO:383) t843.nt (SEQ ID NO:384) f850.aa (SEQ ID
NO:385) t850.aa (SEQ ID NO:386) f850.nt (SEQ ID NO:387) t850.nt
(SEQ ID NO:388) f853.aa (SEQ ID NO:389) t853.aa (SEQ ID NO:390)
f853.nt (SEQ ID NO:391) t853.nt (SEQ ID NO:392) f859.aa (SEQ ID
NO:393) t859.aa (SEQ ID NO:394) f859.nt (SEQ ID NO:395) t859.nt
(SEQ ID NO:396) f861.aa (SEQ ID NO:397) t861.aa (SEQ ID NO:398)
f861.nt (SEQ ID NO:399) t861.nt (SEQ ID NO:400) f363.aa (SEQ ID
NO:401) t363.aa (SEQ ID NO:402) f363.nt (SEQ ID NO:403) t363.nt
(SEQ ID NO:404) f368.aa (SEQ ID NO:405) t368.aa (SEQ ID NO:406)
f368.nt (SEQ ID NO:407) t368.nt (SEQ ID NO:408) f371.aa (SEQ ID
NO:409) t371.aa (SEQ ID NO:410) f371.nt (SEQ ID NO:411) t371.nt
(SEQ ID NO:412) f502.aa (SEQ ID NO:413) t502.aa (SEQ ID NO:414)
f502.nt (SEQ ID NO:415) t502.nt (SEQ ID NO:416) f527.aa (SEQ ID
NO:417) t527.aa (SEQ ID NO:418) f527.nt (SEQ ID NO:419) t527.nt
(SEQ ID NO:420) f541.aa (SEQ ID NO:421) t541.aa (SEQ ID NO:422)
f541.nt (SEQ ID NO:423) t541.nt (SEQ ID NO:424) f561.aa (SEQ ID
NO:425) t561.aa (SEQ ID NO:426) f561.nt (SEQ ID NO:427)
t561.nt (SEQ ID NO:428) f604.aa (SEQ ID NO:429) t604.aa (SEQ ID
NO:430) f604.nt (SEQ ID NO:431) t604.nt (SEQ ID NO:432) f736.aa
(SEQ ID NO:433) t736.aa (SEQ ID NO:434) f736.nt (SEQ ID NO:435)
t736.nt (SEQ ID NO:436) f752.aa (SEQ ID NO:437) t752.aa (SEQ ID
NO:438) f752.nt (SEQ ID NO:439) t752.nt (SEQ ID NO:440) f798.aa
(SEQ ID NO:441) t798.aa (SEQ ID NO:442) t798.nt (SEQ ID NO:443)
t798.nt (SEQ ID NO:444) f805.aa (SEQ ID NO:445) t805.aa (SEQ ID
NO:446) f805.nt (SEQ ID NO:447) t805.nt (SEQ ID NO:448) f635.aa
(SEQ ID NO:449) t635.aa (SEQ ID NO:450) f635.nt (SEQ ID NO:451)
t635.nt (SEQ ID NO:452) f314.aa (SEQ ID NO:453) t314.aa (SEQ ID
NO:454) f314.nt (SEQ ID NO:455) t314.nt (SEQ ID NO:456) f32.aa (SEQ
ID NO:457) t32.aa (SEQ ID NO:458) f32.nt (SEQ ID NO:459) t32.nt
(SEQ ID NO:460) f320.aa (SEQ ID NO:461) t320.aa (SEQ ID NO:462)
f320.nt (SEQ ID NO:463) t320.nt (SEQ ID NO:464) f342.aa (SEQ ID
NO:465) t342.aa (SEQ ID NO:466) f342.nt (SEQ ID NO:467) t342.nt
(SEQ ID NO:468) f352.aa (SEQ ID NO:469) t352.aa (SEQ ID NO:470)
t352.nt (SEQ ID NO:471) t352.nt (SEQ ID NO:472) f301.aa (SEQ ID
NO:473) t301.aa (SEQ ID NO:474) f301.nt (SEQ ID NO:475) t301.nt
(SEQ ID NO:476) f346.aa (SEQ ID NO:477) t346.aa (SEQ ID NO:478)
f346.nt (SEQ ID NO:479) t346.nt (SEQ ID NO:480) f373.aa (SEQ ID
NO:481) t373.aa (SEQ ID NO:482) f373.nt (SEQ ID NO:483) t373.nt
(SEQ ID NO:484) f384.aa (SEQ ID NO:485) t384.aa (SEQ ID NO:486)
f384.nt (SEQ ID NO:487) t384.nt (SEQ ID NO:488) f860.aa (SEQ ID
NO:489) t860.aa (SEQ ID NO:490) f860.nt (SEQ ID NO:491) t860.nt
(SEQ ID NO:492) f446.aa (SEQ ID NO:493) t446.aa (SEQ ID NO:494)
f446.nt (SEQ ID NO:495) t446.nt (SEQ ID NO:496) f457.aa (SEQ ID
NO:497) t457.aa (SEQ ID NO:498) f457.nt (SEQ ID NO:499) t457.nt
(SEQ ID NO:500) f542.aa (SEQ ID NO:501) t542.aa (SEQ ID NO:502)
f542.nt (SEQ ID NO:503) t542.nt (SEQ ID NO:504) f93.aa (SEQ ID
NO:505) t93.aa (SEQ ID NO:506) f93.nt (SEQ ID NO:507) t93.nt (SEQ
ID NO:508) f105.aa (SEQ ID NO:509) t105.aa (SEQ ID NO:510) f105.nt
(SEQ ID NO:511) t105.nt (SEQ ID NO:512) f150.aa (SEQ ID NO:513)
t150.aa (SEQ ID NO:514) f150.nt (SEQ ID NO:515) t150.nt (SEQ ID
NO:516) f219.aa (SEQ ID NO:517) t219.aa (SEQ ID NO:518) f219.nt
(SEQ ID NO:519) t219.nt (SEQ ID NO:520) f229.aa (SEQ ID NO:521)
t229.aa (SEQ ID NO:522) f229.nt (SEQ ID NO:523) t229.nt (SEQ ID
NO:524) f22.aa (SEQ ID NO:525) t22.aa (SEQ ID NO:526) f22.nt (SEQ
ID NO:527) t22.nt (SEQ ID NO:528) f32.aa (SEQ ID NO:529) t32.aa
(SEQ ID NO:530) f32.nt (SEQ ID NO:531) t32.nt (SEQ ID NO:532)
f186.aa (SEQ ID NO:533) t186.aa (SEQ ID NO:534) f186.nt (SEQ ID
NO:535) t186.nt (SEQ ID NO:536) f216.aa (SEQ ID NO:537) t216.aa
(SEQ ID NO:538) f216.nt (SEQ ID NO:539) t216.nt (SEQ ID NO:540)
f328.aa (SEQ ID NO:541) t328.aa (SEQ ID NO:542) f328.nt (SEQ ID
NO:543) t328.nt (SEQ ID NO:544) t352.aa (SEQ ID NO:545) t352.aa
(SEQ ID NO:546) f352.nt (SEQ ID NO:547) t352.nt (SEQ ID NO:548)
f867.aa (SEQ ID NO:549) t867.aa (SEQ ID NO:550) f867.nt (SEQ ID
NO:551) t867.nt (SEQ ID NO:552) f868.aa (SEQ ID NO:553) t868.aa
(SEQ ID NO:554) f868.nt (SEQ ID NO:555) t868.nt (SEQ ID NO:556)
f872.aa (SEQ ID NO:557) t872.aa (SEQ ID NO:558) f872.nt (SEQ ID
NO:559) t872.nt (SEQ ID NO:560) f874.aa (SEQ ID NO:561) t874.aa
(SEQ ID NO:562) f874.nt (SEQ ID NO:563) t874.nt (SEQ ID NO:564)
f886.aa (SEQ ID NO:565) t886.aa (SEQ ID NO:566) f886.nt (SEQ ID
NO:567) t886.nt (SEQ ID NO:568) f888.aa (SEQ ID NO:569) t888.aa
(SEQ ID NO:570) f888.nt (SEQ ID NO:571) t888.nt (SEQ ID NO:572)
f893.aa (SEQ ID NO:573) t893.aa (SEQ ID NO:574) f893.nt (SEQ ID
NO:575) t893.nt (SEQ ID NO:576) f895.aa (SEQ ID NO:577) t895.aa
(SEQ ID NO:578) f895.nt (SEQ ID NO:579) t895.nt (SEQ ID NO:580)
f605.aa (SEQ ID NO:581) t605.aa (SEQ ID NO:582) f605.nt (SEQ ID
NO:583) t605.nt (SEQ ID NO:584) f606.aa (SEQ ID NO:585) t606.aa
(SEQ ID NO:586) f606.nt (SEQ ID NO:587) t606.nt (SEQ ID NO:588)
f679.aa (SEQ ID NO:589) t679.aa (SEQ ID NO:590) f679.nt (SEQ ID
NO:591) t679.nt (SEQ ID NO:592) f11-12.nt (SEQ ID NO:593) t11-12.nt
(SEQ ID NO:594) f11-12.aa (SEQ ID NO:595) t11-12.aa (SEQ ID NO:596)
f11-4.nt (SEQ ID NO:597) t11-4.nt (SEQ ID NO:598) f11-4.aa (SEQ ID
NO:599) t11-4.aa (SEQ ID NO:600) f112-1.nt (SEQ ID NO:601)
t112-1.nt (SEQ ID NO:602) f112-1.aa (SEQ ID NO:603) t112-1.aa (SEQ
ID NO:604) f14-8.nt (SEQ ID NO:605) t14-8.nt (SEQ ID NO:606)
f14-8.aa (SEQ ID NO:607) t14-8.aa (SEQ ID NO:608) f17-6.nt (SEQ ID
NO:609) t17-6.nt (SEQ ID NO:610) f17-6.aa (SEQ ID NO:611) t17-6.aa
(SEQ ID NO:612) f19-2.nt (SEQ ID NO:613) t19-2.nt (SEQ ID NO:614)
f19-2.aa (SEQ ID NO:615) t19-2.aa (SEQ ID NO:616) f19-4.nt (SEQ ID
NO:617) t19-4.nt (SEQ ID NO:618) f19-4.aa (SEQ ID NO:619) t19-4.aa
(SEQ ID NO:620) f19-6.nt (SEQ ID NO:621) t19-6.nt (SEQ ID NO:622)
f19-6.aa (SEQ ID NO:623) t19-6.aa (SEQ ID NO:624) f21-4.nt (SEQ ID
NO:625) t21-4.nt (SEQ ID NO:626) f21-4.aa (SEQ ID NO:627) t21-4.aa
(SEQ ID NO:628) f24-1.nt (SEQ ID NO:629) t24-1.nt (SEQ ID NO:630)
f24-1.aa (SEQ ID NO:631) t24-1.aa (SEQ ID NO:632) f28-2.nt (SEQ ID
NO:633) t28-2.nt (SEQ ID NO:634) f28-2.aa (SEQ ID NO:635) t28-2.aa
(SEQ ID NO:636) f28-3.nt (SEQ ID NO:637) t28-3.nt (SEQ ID NO:638)
f28-3.aa (SEQ ID NO:639) t28-3.aa (SEQ ID NO:640) f31-2.nt (SEQ ID
NO:641) t31-2.nt (SEQ ID NO:642) f31-2.aa (SEQ ID NO:643) t31-2.aa
(SEQ ID NO:644) f32-4.nt (SEQ ID NO:645) t32-4.nt (SEQ ID NO:646)
f32-4.aa (SEQ ID NO:647) t32-4.aa (SEQ ID NO:648) f4-15.nt (SEQ ID
NO:649) t4-15.nt (SEQ ID NO:650) f4-15.aa (SEQ ID NO:651) t4-15.aa
(SEQ ID NO:652) f4-50.nt (SEQ ID NO:653) t4-50.nt (SEQ ID NO:654)
f4-50.aa (SEQ ID NO:655) t4-50.aa (SEQ ID NO:656) f4-66.nt (SEQ ID
NO:657) t4-66.nt (SEQ ID NO:658) f4-66.aa (SEQ ID NO:659) t4-66.aa
(SEQ ID NO:660) f42-1.nt (SEQ ID NO:661) t42-1.nt (SEQ ID NO:662)
f42-1.aa (SEQ ID NO:663) t42-1.aa (SEQ ID NO:664) f43-3.nt (SEQ ID
NO:665) t43-3.nt (SEQ ID NO:666) f43-3.aa (SEQ ID NO:667) t43-3.aa
(SEQ ID NO:668) f45-2.nt (SEQ ID NO:669) t45-2.nt (SEQ ID NO:670)
f45-2.aa (SEQ ID NO:671) t45-2.aa (SEQ ID NO:672) f47-2.nt (SEQ ID
NO:673) t47-2.nt (SEQ ID NO:674) f47-2.aa (SEQ ID NO:675) t47-2.aa
(SEQ ID NO:676) f49-2.nt (SEQ ID NO:677) t49-2.nt (SEQ ID NO:678)
f49-2.aa (SEQ ID NO:679) t49-2.aa (SEQ ID NO:680) f5-14.nt (SEQ ID
NO:681) t5-14.nt (SEQ ID NO:682) f5-14.aa (SEQ ID NO:683) t5-14.aa
(SEQ ID NO:684) f5-15.nt (SEQ ID NO:685) t5-15.nt (SEQ ID NO:686)
f5-15.aa (SEQ ID NO:687) t5-15.aa (SEQ ID NO:688) f51-2.nt (SEQ ID
NO:689) t51-2.nt (SEQ ID NO:690) f51-2.aa (SEQ ID NO:691) t51-2.aa
(SEQ ID NO:692) f6-21.nt (SEQ ID NO:693) t6-21.nt (SEQ ID NO:694)
f6-21.aa (SEQ ID NO:695) t6-21.aa (SEQ ID NO:696) f6-27.nt (SEQ ID
NO:697) t6-27.nt (SEQ ID NO:698) f6-27.aa (SEQ ID NO:699) t6-27.aa
(SEQ ID NO:700) f6-5.nt (SEQ ID NO:701) t6-5.nt (SEQ ID NO:702)
f6-5.aa (SEQ ID NO:703) t6-5.aa (SEQ ID NO:704) f7-30.nt (SEQ ID
NO:705) t7-30.nt (SEQ ID NO:706) f7-30.aa (SEQ ID NO:707) t7-30.aa
(SEQ ID NO:708) f76-1.nt (SEQ ID NO:709) t76-1.nt (SEQ ID NO:710)
f76-1.aa (SEQ ID NO:711) t76-1.aa (SEQ ID NO:712) f8-10.nt (SEQ ID
NO:713) t8-10.nt (SEQ ID NO:714) f8-10.aa (SEQ ID NO:715) t8-10.aa
(SEQ ID NO:716) f8-14.nt (SEQ ID NO:717) t8-14.nt (SEQ ID NO:718)
f8-14.aa (SEQ ID NO:719) t8-14.aa (SEQ ID NO:720) f01A.nt BB001
(SEQ ID NO:721) t01A.nt BB001 (SEQ ID NO:722) f01A.aa BB001 (SEQ ID
NO:723) t01A.aa BB001 (SEQ ID NO:724) f02A.nt BB002 (SEQ ID NO:725)
t02A.nt BB002 (SEQ ID NO:726) f02A.aa BB002 (SEQ ID NO:727) t02A.aa
BB002 (SEQ ID NO:728) f03A.nt BB006 (SEQ ID NO:729) t03A.nt BB006
(SEQ ID NO:730) f03A.aa BB006 (SEQ ID NO:731) t03A.aa BB006 (SEQ ID
NO:732) f04A.nt BB011 (SEQ ID NO:733) t04A.nt BB011 (SEQ ID NO:734)
f04A.aa BB011 (SEQ ID NO:735) t04A.aa BB011 (SEQ ID NO:736) f05A.nt
BB009 (SEQ ID NO:737) t05A.nt BB009 (SEQ ID NO:738) f05A.aa BB009
(SEQ ID NO:739) t05A.aa BB009 (SEQ ID NO:740) f06A.nt BB014 (SEQ ID
NO:741) t06A.nt BB014 (SEQ ID NO:742) f06A.aa BB014 (SEQ ID NO:743)
t06A.aa BB014 (SEQ ID NO:744) f07A.nt BB023 (SEQ ID NO:745) t07A.nt
BB023 (SEQ ID NO:746) f07A.aa BB023 (SEQ ID NO:747) t07A.aa BB023
(SEQ ID NO:748) f08A.nt BB024 (SEQ ID NO:749) t08A.nt BB024 (SEQ ID
NO:750) f08A.aa BB024 (SEQ ID NO:751) t08A.aa BB024 (SEQ ID NO:752)
f09A.nt BB025 (SEQ ID NO:753) t09A.nt BB025 (SEQ ID NO:754) t09A.aa
BB025 (SEQ ID NO:755) t09A.aa BB025 (SEQ ID NO:756)
[0296]
2TABLE 2 Closest matching sequences between the polypeptides of the
present invention and sequences in GenBank and Derwent databases.
GenSeq BLAST BLAST Query Access No. GenSeq Gene Description Score
P-Value f01A.aa gi.vertline.2690256 (AE000790) antigen, P35,
putative 1523 5.90E-206 [Borrelia burgdorferi] f02A.aa
gi.vertline.2690286 (AE000790) B. burgdorferi predicted 1320
2.10E-174 coding region BBA69 [Borrelia f02A.aa gi.vertline.2690285
(AE000790) B. burgdorferi predicted 278 7.50E-71 coding region
BBA68 [Borrelia f02A.aa gi.vertline.2690105 (AE000789) B.
burgdorferi predicted 151 8.40E-54 coding region BBI38 [Borrelia
f02A.aa gi.vertline.2690092 (AE000789) antigen, P35, putative 151
2.70E-48 [Borrelia burgdorferi] f02A.aa gi.vertline.2690183
(AE000787) antigen, P35, putative 155 4.20E-22 [Borrelia
burgdorferi] f02A.aa gi.vertline.2690106 (AE000789) B. burgdorferi
predicted 154 1.30E-21 coding region BBI39 [Borrelia f03A.aa
gi.vertline.2688051 (AE001127) antigen, S2, putative 1223 7.60E-164
[Borrelia burgdorferi] f03A.aa gi.vertline.1063419 S2 gene product
[Borrelia burgdorferi] 116 3.00E-22 f03A.aa gi.vertline.2690227
(AE000790) antigen, S2 [Borrelia 116 9.70E-22 burgdorferi]
>pir.vertline.D70207.vertline.D70207 f03A.aa gi.vertline.2690128
(AE000788) protein p23 [Borrelia 110 5.70E-19 burgdorferi]
>pir.vertline.C70257.vertline.C70257 f03A.aa gi.vertline.2689956
(AE000785) protein p23 [Borrelia 104 7.90E-15 burgdorferi]
>pir.vertline.D70225.vertline.D70225 f04A.aa gi.vertline.2690078
(AE000784) B. burgdorferi predicted 1873 5.60E-250 coding region
BBH18 [Borrelia f04A.aa gi.vertline.2690192 (AE000787) B.
burgdorferi predicted 167 1.40E-15 coding region BBJ13 [Borrelia
f05A.aa gi.vertline.2687919 (AE001117) B. burgdorferi predicted 696
4.20E-92 coding region BB0028 [Borrelia f06A.aa gi.vertline.2690129
(AE000788) outer membrane protein 884 4.80E-124 [Borrelia
burgdorferi] f06A.aa gi.vertline.2690089 (AE000789) conserved
hypothetical 731 2.20E-118 protein [Borrelia burgdorferi] f06A.aa
gi.vertline.520783 unknown [Borrelia burgdorferi] 337 4.30E-58
>gi.vertline.551742 unknown [Borrelia f07A.aa
gi.vertline.2688608 (AE001168) flagellar filament outer 1668
2.50E-224 layer protein (flaA) [Borrelia f07A.aa
gi.vertline.1575447 FlaA protein [Borrelia burgdorferi] 1645
3.60E-221 >gi.vertline.1019754 orf [Borrelia f07A.aa
gi.vertline.152896 flagellar filament surface antigen 144 1.70E-38
[Spirochaeta aurantia] f07A.aa gi.vertline.155059 endoflagellar
sheath protein 139 3.80E-28 [Treponema pallidum] f07A.aa
gi.vertline.433524 flagellin FlaA1 [Serpulina 119 3.00E-26
hyodysenteriae] >gi.vertline.904393 endoflagellar f07A.aa
pir.vertline.A32814.vertline.A32814 flagellar filament surface
antigen - 116 9.40E-11 Spirochaeta aurantia f08A.aa
gi.vertline.1209837 lipoprotein [Borrelia burgdorferi] 508 2.10E-78
f08A.aa gi.vertline.2121280 (AF000270) lipoprotein [Borrelia 547
4.00E-70 burgdorferi] >gi.vertline.3095109 f08A.aa
gi.vertline.1209873 lipoprotein [Borrelia burgdorferi] 303 3.70E-51
f08A.aa gi.vertline.1209843 lipoprotein [Borrelia burgdorferi] 395
2.20E-49 f08A.aa gi.vertline.1209849 lipoprotein [Borrelia
burgdorferi] 219 2.60E-27 f08A.aa gi.vertline.3095105 (AF046998)
2.9-8 lipoprotein [Borrelia 234 4.30E-27 burgdorferi] f08A.aa
gi.vertline.1209831 lipoprotein [Borrelia burgdorferi] 209 1.10E-22
f08A.aa gi.vertline.3095107 (AF046999) 2.9-9 lipoprotein [Borrelia
200 1.80E-22 burgdorferi] f08A.aa gi.vertline.1209857 lipoprotein
[Borrelia burgdorferi] 200 2.50E-21 f08A.aa
gnl.vertline.PID.vertline.e268244 surface-exposed lipoprotein
[Borrelia 142 1.80E-11 afzelii] f09A.aa gi.vertline.1209843
lipoprotein [Borrelia burgdorferi] 453 8.60E-67 f09A.aa
gi.vertline.2121280 (AF000270) lipoprotein [Borrelia 379 1.00E-56
burgdorferi] >gi.vertline.3095109 f09A.aa gi.vertline.1209873
lipoprotein [Borrelia burgdorferi] 282 1.10E-45 f09A.aa
gi.vertline.1209837 lipoprotein [Borrelia burgdorferi] 357 7.10E-44
f09A.aa gi.vertline.1209849 lipoprotein [Borrelia burgdorferi] 143
1.60E-13 f09A.aa gnl.vertline.PID.vertline.e26824- 4
surface-exposed lipoprotein [Borrelia 111 3.60E-13 afzelii] f09A.aa
gi.vertline.3095105 (AF046998) 2.9-8 lipoprotein [Borrelia 142
5.40E-13 burgdorferi] f101.aa gi.vertline.2688708 (AE001176)
conserved hypothetical 1099 4.50E-152 protein [Borrelia
burgdorferi] f105.aa gi.vertline.2688693 (AE001175) B. burgdorferi
predicted 1276 2.20E-177 coding region BB0758 [Borrelia f11-
gi.vertline.2690139 (AE000788) B. burgdorferi predicted 1473
4.70E-193 12.aa coding region BBK01 [Borrelia f11-
gi.vertline.2690030 (AE000786) B. burgdorferi predicted 1066
1.40E-138 12.aa coding region BBG01 [Borrelia f11-
gi.vertline.2690074 (AE000784) B. burgdorferi predicted 173
6.20E-93 12.aa coding region BBH37 [Borrelia f11-
gi.vertline.2690188 (AE000787) B. burgdorferi predicted 192
2.70E-75 12.aa coding region BBJ08 [Borrelia f11-4.aa
gi.vertline.2690150 (AE000788) B. burgdorferi predicted 1144
2.70E-147 coding region BBK12 [Borrelia f11-4.aa
gi.vertline.2690145 (AE000788) B. burgdorferi predicted 852
5.70E-127 coding region BBK07 [Borrelia f11-4.aa
gi.vertline.2690095 (AE000789) B. burgdorferi predicted 153
1.30E-34 coding region BBI10 [Borrelia f11-4.aa gi.vertline.2690197
(AE000787) B. burgdorferi predicted 115 1.40E-12 coding region
BBJ31 [Borrelia f11-4.aa gi.vertline.2690219 (AE000787) B.
burgdorferi predicted 115 1.40E-12 coding region BBJ45 [Borrelia
f112- gi.vertline.2690054 (AE000784) B. burgdorferi predicted 573
7.00E-75 1.aa coding region BBH06 [Borrelia f12.aa
gi.vertline.2688785 (AE001182) B. burgdorferi predicted 6008 0
coding region BB0838 [Borrelia f129.aa gi.vertline.2688685
(AE001174) B. burgdorferi predicted 987 6.20E-133 coding region
BB0739 [Borrelia f14-8.aa gi.vertline.2689955 (AE000785) antigen,
P35, putative 385 2.70E-75 [Borrelia burgdorferi] f14-8.aa
gi.vertline.2690120 (AE000789) B. burgdorferi predicted 330
2.60E-66 coding region BBI34 [Borrelia f14-8.aa gi.vertline.2690052
(AE000784) antigen, P35, putative 287 4.00E-64 [Borrelia
burgdorferi] f14-8.aa gi.vertline.2690100 (AE000789) B. burgdorferi
predicted 172 1.10E-38 coding region BBI16 [Borrelia f14-8.aa
gi.vertline.2690115 (AE000789) B. burgdorferi predicted 173
1.70E-28 coding region BBI28 [Borrelia f14-8.aa gi.vertline.2690116
(AE000789) B. burgdorferi predicted 163 8.20E-24 coding region
BBI29 [Borrelia f14-8.aa gi.vertline.2690207 (AE000787) B.
burgdorferi predicted 220 1.90E-23 coding region BBJ02 [Borrelia
f14-8.aa gi.vertline.2690099 (AE000789) B. burgdorferi predicted
140 3.60E-12 coding region BBI15 [Borrelia f14-8.aa
gi.vertline.2690125 (AE000788) antigen, P35, putative 111 1.00E-11
[Borrelia burgdorferi] f142.aa gi.vertline.2688655 (AE001172)
glutamate transporter 2233 7.19999999999982e-311 (gltP) [Borrelia
burgdorferi] f142.aa gnl.vertline.PID.vertline.e233874 hypothetical
protein [Bacillus subtilis] 727 2.60E-156
>gnl.vertline.PID.vertline.e1182902 f142.aa
gnl.vertline.PID.vertline.d1016231 Proton/sodium-glutamate symport
762 6.60E-146 protein (Glutamate-aspartate f142.aa
gi.vertline.1574711 proton glutamate symport protein (gltP) 903
2.10E-131 [Haemophilus influenzae] f142.aa gi.vertline.2983758
(AE000735) proton/sodium-glutamate 111 8.40E-36 symport protein
[Aquifex f142.aa gi.vertline.143000 proton glutamate symport
protein 125 1.20E-30 [Bacillus stearothermophilus] f142.aa
gi.vertline.143002 proton glutamate symport protein 125 1.90E-28
[Bacillus caldotenax] f142.aa gnl.vertline.PID.vertline.e- 1183024
proton/sodium-glutamate symport 122 2.20E-25 protein [Bacillus
subtilis] f142.aa gnl.vertline.PID.vertline.d1022697 glutamate
transporter [Caenorhabditis 121 1.80E-22 elegans] f142.aa
gi.vertline.1255318 coded for by C. elegans cDNA cm08h9; 121
2.10E-22 coded for by C. elegans cDNA f142.aa gi.vertline.2388712
(AF017105) amino acid transporter 135 3.60E-22 [Chlamydia psittaci]
f142.aa gi.vertline.2655021 (AF018259) glutamate transporter 5A 125
7.70E-22 [Ambystoma tigrinum] f142.aa
gnl.vertline.PID.vertline.e149542 gluT-R gene product [Clostridium
199 4.60E-21 perfringens] f142.aa gi.vertline.396412 gltP
[Escherichia coli] >gi.vertline.147160 109 7.90E-21
proton-glutamate [Escherichia f147.aa gi.vertline.2688656
(AE001172) NADH oxidase, water- 2245 7.20E-303 forming (nox)
[Borrelia burgdorferi] f147.aa gi.vertline.642030 NADH oxidase
[Serpulina 318 9.20E-105 hyodysenteriae] f147.aa
gi.vertline.2650234 (AE001077) NADH oxidase (noxA-2) 303 2.90E-93
[Archaeoglobus fulgidus] f147.aa gi.vertline.2792490 (AF041467)
coenzyme A disulfide 194 2.60E-90 reductase [Staphylococcus aureus]
f147.aa gi.vertline.2650383 (AE001088) NADH oxidase (noxA-1) 286
3.30E-88 [Archaeoglobus fulgidus] f147.aa
gnl.vertline.PID.vertline.d1009320 H2O-forming NADH Oxidase 369
4.30E-85 [Streptococcus mutans] f147.aa gi.vertline.49023 NADH
peroxidase [Enterococcus 638 3.20E-83 faecalis]
>pir.vertline.S18332.vertline.S18332 NADH f147.aa
gi.vertline.1591361 NADH oxidase (nox) [Methanococcus 535 4.80E-83
jannaschii] >pir.vertline.A64381.vertline.A64381 f147.aa
gi.vertline.2622461 (AE000898) NADH oxidase 303 8.40E-72
[Methanobacterium thermoautotrophicum] f147.aa gi.vertline.47045
NADH oxidase [Enterococcus faecalis] 547 8.80E-71
>pir.vertline.S26965.vertline.S26965 NADH oxidase f147.aa
gi.vertline.2650233 (AE001077) NADH oxidase (noxA-3) 312 2.00E-63
[Archaeoglobus fulgidus] f147.aa gi.vertline.1674132 (AE000044)
Mycoplasma pneumoniae, 175 7.00E-61 NADH oxidase; similar to
f147.aa gi.vertline.1045969 NADH oxidase [Mycoplasma 164 4.10E-51
genitalium] >pir.vertline.D64230.vertline.D64230 NADH f147.aa
gi.vertline.2648692 (AE000975) NADH oxidase (noxA-5) 143 2.00E-40
[Archaeoglobus fulgidus] f147.aa gi.vertline.2983379 (AE000709)
NADH oxidase [Aquifex 162 5.50E-30 aeolicus] f150.aa
gi.vertline.2688659 (AE001172) conserved hypothetical 1319
2.70E-179 protein [Borrelia burgdorferi] f150.aa
gi.vertline.2983887 (AE000743) hypothetical protein 238 1.40E-25
[Aquifex aeolicus] f150.aa gi.vertline.2581796 (AF001974) putative
TrkA 175 5.80E-23 [Thermoanaerobacter ethanolicus] f150.aa
gi.vertline.1377829 unknown [Bacillus subtilis] 212 1.50E-21
>gnl.vertline.PID.vertline.d1007628 orf4 [Bacillus f150.aa
gnl.vertline.PID.vertline.e1185982 similar to hypothetical proteins
181 6.00E-17 [Bacillus subtilis] f150.aa
gnl.vertline.PID.vertline.d1011497 hypothetical protein
[Synechocystis sp.] 128 3.70E-11
>pir.vertline.S75999.vertline.S75999 f152.aa gi.vertline.2688660
(AE001172) K+ transport protein (ntpJ) 2200 2.40000000001213e-313
[Borrelia burgdorferi] f152.aa gi.vertline.2983882 (AE000743) K+
transport protein 239 3.60E-106 homolog [Aquifex aeolicus] f152.aa
gnl.vertline.PID.vertline.e118- 4940 similar to Na+-transporting
ATP 158 6.60E-64 synthase [Bacillus subtilis] f152.aa
gnl.vertline.PID.vertline.e1185983 similar to Na+-transporting ATP
131 3.40E-62 synthase [Bacillus subtilis] f152.aa
gnl.vertline.PID.vertline.d1018749 Na+-ATPase subunit J
[Synechocystis 141 1.70E-55 sp.]
>pir.vertline.S75455.vertline.S75455 f152.aa
gnl.vertline.PID.vertline.d1004799 Na+-ATPase subunit J
[Enterococcus 209 4.00E-45 hirae] f152.aa gi.vertline.2581795
(AF001974) putative TrkG 149 2.20E-29 [Thermoanaerobacter
ethanolicus] f152.aa gi.vertline.1674061 (AE000036) Mycoplasma
pneumoniae, 104 4.00E-28 Na(+) translocating ATPase f152.aa
gi.vertline.1046024 Na+ ATPase subunit J [Mycoplasma 114 2.80E-27
genitalium] >pir.vertline.F64235.vertline.F64235 Na+ f152.aa
gi.vertline.567062 HKT1 [Triticum aestivum] 137 2.00E-17
>pir.vertline.S47582.vertline.S47582 high-affinity potassium
f154.aa gi.vertline.2688664 (AE001172) B. burgdorferi predicted
2456 0 coding region BB0722 [Borrelia f157.aa gi.vertline.2688641
(AE001171) rod shape-determining 2300 0 protein (mreB-2) [Borrelia
f157.aa gi.vertline.143657 endospore forming protein [Bacillus 224
2.60E-61 subtilis] f157.aa gi.vertline.580938 internal open reading
frame (AA 1-290) 224 2.60E-61 [Bacillus subtilis] f157.aa
gi.vertline.2982781 (AE000670) rod shape determining 333 5.40E-61
protein RodA [Aquifex aeolicus] f157.aa gi.vertline.580937 spoVE
gene product (AA 1-366) 224 7.70E-59 [Bacillus subtilis]
>gnl.vertline.PID.vertline.e- 1185111 f157.aa gi.vertline.147695
rod-shape-determining protein 340 6.10E-58 [Escherichia coli]
>gi.vertline.1778551 f157.aa gnl.vertline.PID.vertline.e328589
sfr [Streptomyces coelicolor] 362 6.40E-58 f157.aa
gi.vertline.1572976 rod shape-determining protein (mreB) 307
4.00E-56 [Haemophilus influenzae] f157.aa
gnl.vertline.PID.vertline.e1185075 similar to cell-division protein
[Bacillus 203 2.60E-45 subtilis] f157.aa gi.vertline.1469784
putative cell division protein ftsW 231 6.90E-45 [Enterococcus
hirae] f157.aa gi.vertline.1016213 strong sequence similarity to
FtsW, 206 3.00E-41 RodA, and SpoV-E [Cyanophora f157.aa
gnl.vertline.PID.vertline.d1019002 rod-shape-determining protein
184 1.60E-38 [Synechocystis sp.] f157.aa gi.vertline.146039 cell
division protein [Escherichia coli] 104 8.30E-35
>gi.vertline.40857 FtsW protein f157.aa gi.vertline.1574692 cell
division protein (ftsW) 114 3.30E-33 [Haemophilus influenzae]
f157.aa gi.vertline.1165286 FtsW [Borrelia burgdorferi] 170
6.20E-32 >gi.vertline.2688164 (AE001137) cell division f17-6.aa
gi.vertline.2690100 (AE000789) B. burgdorferi predicted 1250
1.70E-164 coding region BBI16 [Borrelia f17-6.aa
gi.vertline.2690120 (AE000789) B. burgdorferi predicted 142
3.40E-59 coding region BBI34 [Borrelia f17-6.aa gi.vertline.2690115
(AE000789) B. burgdorferi predicted 447 6.70E-56 coding region
BBI28 [Borrelia f17-6.aa gi.vertline.2690052 (AE000784) antigen,
P35, putative 182 1.10E-34 [Borrelia burgdorferi] f17-6.aa
gi.vertline.2689955 (AE000785) antigen, P35, putative 196 6.60E-34
[Borrelia burgdorferi] f17-6.aa gi.vertline.2690114 (AE000789) B.
burgdorferi predicted 176 1.00E-16 coding region BBI27 [Borrelia
f17-6.aa gnl.vertline.PID.vertline.d1012343 gene required for
phosphoylation of 178 2.80E-15 oligosaccharides/has f17-6.aa
gi.vertline.2690207 (AE000787) B. burgdorferi predicted 114
3.50E-13 coding region BBJ02 [Borrelia f17-6.aa
gnl.vertline.PID.vertli- ne.e329895 (AJ000496) cyclic
nucleotide-gated 152 1.10E-11 channel beta subunit f170.aa
gi.vertline.2688652 (AE001171) B. burgdorferi predicted 524
2.60E-73 coding region BB0708 [Borrelia f186.aa gi.vertline.2688622
(AE001169) B. burgdorferi predicted 792 1.80E-105 coding region
BB0689 [Borrelia f186.aa gi.vertline.2688622 (AE001169) B.
burgdorferi predicted 792 1.80E-105 coding region BB0689 [Borrelia
f19-2.aa gi.vertline.2690120 (AE000789) B. burgdorferi predicted
1341 2.70E-177 coding region BBI34 [Borrelia f19-2.aa
gi.vertline.2689955 (AE000785) antigen, P35, putative 347 7.00E-53
[Borrelia burgdorferi] f19-2.aa gi.vertline.2690052 (AE000784)
antigen, P35, putative 254 7.70E-53 [Borrelia burgdorferi] f19-2.aa
gi.vertline.2690100 (AE000789) B. burgdorferi predicted 142
6.60E-50 coding region BBI16 [Borrelia f19-2.aa gi.vertline.2690115
(AE000789) B. burgdorferi predicted 144 7.60E-34 coding region
BBI28 [Borrelia f19-2.aa gi.vertline.2690116 (AE000789) B.
burgdorferi predicted 183 2.20E-21 coding region BBI29 [Borrelia
f19-2.aa gi.vertline.2690207 (AE000787) B. burgdorferi predicted
171 2.00E-16 coding region BBJ02 [Borrelia f19-2.aa
gi.vertline.2690099 (AE000789) B. burgdorferi predicted 166
1.20E-15 coding region BBI15 [Borrelia f19-2.aa gi.vertline.2690125
(AE000788) antigen, P35, putative 122 5.70E-14 [Borrelia
burgdorferi] f19-4.aa gi.vertline.2690116 (AE000789) B. burgdorferi
predicted 1129 1.30E-150 coding region BBI29 [Borrelia f19-4.aa
gi.vertline.2690099 (AE000789) B. burgdorferi predicted 260
3.00E-30 coding region BBI15 [Borrelia f19-4.aa gi.vertline.2689955
(AE000785) antigen, P35, putative 180 1.80E-23 [Borrelia
burgdorferi] f19-4.aa gi.vertline.2690120 (AE000789) B. burgdorferi
predicted 183 1.50E-21 coding region BBI34 [Borrelia f19-4.aa
gi.vertline.2690052 (AE000784) antigen, P35, putative 192 1.20E-19
[Borrelia burgdorferi] f19-4.aa gi.vertline.2690207 (AE000787) B.
burgdorferi predicted 149 8.90E-14 coding region BBJ02 [Borrelia
f19-4.aa gi.vertline.2690098 (AE000789) B. burgdorferi predicted
138 8.00E-12 coding region BBI14 [Borrelia f19-6.aa
gi.vertline.2690115 (AE000789) B. burgdorferi predicted 995
1.20E-131 coding region BBI28 [Borrelia f19-6.aa
gi.vertline.2690100 (AE000789) B. burgdorferi predicted 447
3.00E-55 coding region BBI16 [Borrelia f19-6.aa gi.vertline.2689955
(AE000785) antigen, P35, putative 219 2.00E-36 [Borrelia
burgdorferi] f19-6.aa gi.vertline.2690120 (AE000789) B. burgdorferi
predicted 144 3.50E-34 coding region BBI34 [Borrelia f19-6.aa
gi.vertline.2690052 (AE000784) antigen, P35, putative 130 6.30E-12
[Borrelia burgdorferi] f196.aa gi.vertline.2688620 (AE001169)
methyl-accepting 3093 0 chemotaxis protein (mcp-5) [Borrelia
f196.aa gi.vertline.2688621 (AE001169) methyl-accepting 615
1.90E-83 chemotaxis protein (mcp-4) [Borrelia f196.aa
gi.vertline.496484 tlpC gene product [Bacillus subtilis] 180
6.90E-28 >pir.vertline.I40496.vertline.I40496 methylation
f196.aa gnl.vertline.PID.vertline.d1007002 methyl-accepting
chemotaxis protein 180 4.90E-27 TlpC [Bacillus subtilis] f196.aa
gnl.vertline.PID.vertline.e1173493 methyl-accepting chemotaxis
protein 162 5.10E-25 [Bacillus subtilis] f196.aa gi.vertline.882594
ORF_f506 [Escherichia coli] 204 1.70E-24 >gi.vertline.1789453
(AE000389) aerotaxis f196.aa gi.vertline.148350 tas [Enterobacter
aerogenes] 179 1.80E-24 >pir.vertline.D32302.vertline.D32302
probable aspartate f196.aa gi.vertline.1066850 putative
[Rhodobacter capsulatus] 207 1.80E-24
>pir.vertline.JC4735.vertline.JC4735 f196.aa gi.vertline.154381
chemoreceptor [Salmonella 230 2.00E-24 typhimurium]
>pir.vertline.A47178.vertline.A47178 f196.aa gi.vertline.459690
transmembrane receptor [Bacillus 212 1.40E-23 subtilis]
>gnl.vertline.PID.vertline.e1185997 f196.aa gi.vertline.805015
MCPA protein [Rhodobacter 237 2.10E-23 sphaeroides]
>pir.vertline.S70094.vertline.S54262 f196.aa gi.vertline.40424
mcpA gene product [Caulobacter 238 7.30E-23 crescentus]
>pir.vertline.S23064.vertline.S23064 mcpA f196.aa
gi.vertline.144913 sensory transducer protein [Clostridium 227
8.90E-23 thermocellum] f196.aa gi.vertline.1061063 Trg sensory
transducer protein 211 2.40E-20 [Escherichia coli] f196.aa
gnl.vertline.PID.vertline.d1015762 Methyl-accepting chemotaxis
protein III 211 2.50E-20 (MCP-III) (Ribose and f197.aa
gi.vertline.2688621 (AE001169) methyl-accepting 3724 0 chemotaxis
protein (mcp-4) [Borrelia f197.aa gi.vertline.2688620 (AE001169)
methyl-accepting 615 8.40E-83 chemotaxis protein (mcp-5) [Borrelia
f197.aa gi.vertline.1066850 putative [Rhodobacter capsulatus] 227
9.80E-27 >pir.vertline.JC4735.ve- rtline.JC4735 f197.aa
gi.vertline.882594 ORF_f506 [Escherichia coli] 217 1.00E-26
>gi.vertline.1789453 (AE000389) aerotaxis f197.aa
gi.vertline.154381 chemoreceptor [Salmonella 239 2.80E-25
typhimurium] >pir.vertline.A47178.vertline.A47178 f197.aa
gi.vertline.496484 tlpC gene product [Bacillus subtilis] 202
5.10E-25 >pir.vertline.I40496.vertline.I40496 methylation
f197.aa gnl.vertline.PID.vertline.d1007002 methyl-accepting
chemotaxis protein 202 5.10E-25 TlpC [Bacillus subtilis] f197.aa
gi.vertline.2564665 (AF022807) putative methyl accepting 212
7.20E-24 chemotaxis protein [Rhizobium f197.aa gi.vertline.459691
transmembrane receptor [Bacillus 215 1.10E-23 subtilis]
>gnl.vertline.PID.vertline.e1185996 f197.aa gi.vertline.43218
serine chemoreceptor [Escherichia coli] 236 2.80E-23
>bbs.vertline.127562 serine f197.aa gi.vertline.537197 CG Site
No. 63; alternate gene name 236 2.90E-23 cheD [Escherichia coli]
f197.aa gi.vertline.148077 methyl-accepting chemotaxis protein I
236 2.90E-23 [Escherichia coli] >gi.vertline.2367378 f197.aa
gnl.vertline.PID.vertline.d1009948 transducer [Pseudomonas
aeruginosa] 178 4.20E-23 f197.aa gi.vertline.148349 tse
[Enterobacter aerogenes] 234 5.50E-23 >pir.vertline.C32302.ve-
rtline.C32302 serine transducer f197.aa gi.vertline.2626835
chemotactic transducer [Pseudomonas 177 5.70E-23 aeruginosa]
f200.aa gi.vertline.2688600 (AE001168) ribose/galactose ABC 1887
5.10E-266 transporter, permease protein f200.aa
gnl.vertline.PID.vertline.e311453 unknown [Bacillus subtilis] 283
1.50E-63 >gnl.vertline.PID.vertline.e1184234 similar to f200.aa
gi.vertline.2649711 (AE001042) ribose ABC transporter, 202 1.10E-47
permease protein (rbsC-1) f200.aa gi.vertline.2130609 (AF000308)
putative polytopic protein 119 2.10E-27 [Mycoplasma fermentans]
f200.aa gnl.vertline.PID.vertline.e3- 11493 unknown [Bacillus
subtilis] 112 1.10E-18 >gnl.vertline.PID.vertline.e1184235
similar to f200.aa gi.vertline.950073 membrane forming protein 161
5.60E-16 [Mycoplasma capricolum]
>pir.vertline.S77790.vertline.S77790 f200.aa gi.vertline.2688599
(AE001168) ribose/galactose ABC 108 2.00E-14 transporter, permease
protein f208.aa gi.vertline.2688610 (AE001168) B. burgdorferi
predicted 1726 6.70E-244 coding region BB0674 [Borrelia f21-4.aa
gi.vertline.1197833 Bbk2.11 [Borrelia burgdorferi] 474 3.00E-70
>pir.vertline.S70531.vertline.S70531 bbk2.11 protein f21-4.aa
gi.vertline.2627267 ErpL [Borrelia burgdorferi] 477 6.30E-69
f21-4.aa gi.vertline.1707281 putative outer membrane protein 503
6.60E-66 [Borrelia burgdorferi] f21-4.aa gi.vertline.896042 OspF
[Borrelia burgdorferi] 503 6.60E-66 >pir.vertline.S70532.vert-
line.S70532 outer surface protein f21-4.aa gi.vertline.1707287
putative outer membrane protein 489 3.00E-60 [Borrelia burgdorferi]
f21-4.aa gi.vertline.1707290 putative outer surface protein
[Borrelia 342 3.20E-49 burgdorferi] f21-4.aa gi.vertline.1663633
ErpK [Borrelia burgdorferi] 268 1.70E-48 f21-4.aa
gi.vertline.466482 outer surface protein F [Borrelia 321 3.80E-38
burgdorferi] >pir.vertline.I40287.vertline.I40287 f21-4.aa
gi.vertline.896038 BbK2.10 precursor [Borrelia 121 3.90E-34
burgdorferi] >pir.vertline.S70534.vertline.S70534 bbK2.10
f21-4.aa gi.vertline.896040 BbK2.10 precursor [Borrelia 118
2.30E-33 burgdorferi] >pir.vertline.S70533.vertline.S7053- 3
bbK2.10 f21-4.aa gi.vertline.1051120 outer surface protein G
[Borrelia 107 3.30E-33 burgdorferi] >gi.vertline.1373118 ErpG
f21-4.aa gi.vertline.2444428 (AF020657) ErpX protein [Borrelia 118
6.00E-14 burgdorferi] f210.aa gi.vertline.2688603 (AE001168)
conserved hypothetical 867 2.60E-116 protein [Borrelia burgdorferi]
f210.aa gi.vertline.2688604 (AE001168) chemotaxis response 733
1.40E-97 regulator (cheY-3) [Borrelia f210.aa gi.vertline.1408274
CheY [Borrelia burgdorferi] 720 9.00E-96 f210.aa
gi.vertline.1765976 chemotaxis protein CheY [Treponema 405 6.60E-52
pallidum] f210.aa gi.vertline.142682 chemotactic response protein
[Bacillus 184 8.00E-30 subtilis]
>gnl.vertline.PID.vertline.e1185224 f210.aa gi.vertline.940149
CheY [Thermotoga maritima] 171 1.50E-27 f210.aa gi.vertline.2649557
(AE001031) chemotaxis response 168 1.50E-26 regulator (cheY)
[Archaeoglobus f210.aa gi.vertline.620085 cheY gene product
[Listeria 183 3.00E-26 monocytogenes] f210.aa
gnl.vertline.PID.vertline.e249646 YneI [Bacillus subtilis]
>gi.vertline.870926 166 4.00E-24 response regulator f210.aa
gi.vertline.149620 ORF2 [Leptospira borgpetersenii] 121 4.70E-22
>sp.vertline.P24086.vertline.YLB3_LEPIN HYPOTHETICAL f210.aa
gi.vertline.1408275 orfX; putative OrfX protein [Borrelia 208
9.20E-22 burgdorferi] f210.aa gi.vertline.994802 cheY gene product
[Halobacterium 139 8.90E-18 salinarium]
>pir.vertline.S58645.vertline.S58645 CheY f210.aa
gi.vertline.143598 spo0F [Bacillus subtilis] >gi.vertline.143601
113 4.70E-11 Spo0F protein [Bacillus f216.aa gi.vertline.2688586
(AE001167) conserved hypothetical 804 1.20E-109 protein [Borrelia
burgdorferi] f216.aa gi.vertline.1575446 orfA [Borrelia
burgdorferi] 472 1.10E-91 f219.aa gi.vertline.2688594 (AE001167) B.
burgdorferi predicted 1122 3.10E-148 coding region BB0664 [Borrelia
f22.aa gi.vertline.2688779 (AE001181) B. burgdorferi predicted 1400
4.90E-188 coding region BB0832 [Borrelia f22.aa gi.vertline.2688779
(AE001181) B. burgdorferi predicted 1400 4.90E-188 coding region
BB0832 [Borrelia f221.aa gi.vertline.2688596 (AE001167) B.
burgdorferi predicted 692 2.60E-93 coding region BB0662 [Borrelia
f229.aa gi.vertline.2688591 (AE001167) oxygen-independent 863
7.80E-120 coproporphyrinogen III oxidase, f24-1.aa
gi.vertline.2039285 putative vls recombination cassette Vls6 924
1.80E-114 [Borrelia burgdorferi] f24-1.aa gi.vertline.2039284
putative vls recombination cassette Vls5 867 6.30E-107 [Borrelia
burgdorferi] f24-1.aa gi.vertline.2039287 putative vls
recombination cassette Vls8 824 1.50E-104 [Borrelia burgdorferi]
f24-1.aa gi.vertline.2039289 putative vls recombination cassette
829 7.50E-102 Vls10 [Borrelia burgdorferi] f24-1.aa
gi.vertline.2039320 vmp-like sequence protein VlsE 644 1.10E-98
[Borrelia burgdorferi] f24-1.aa gi.vertline.2039288 putative vls
recombination cassette Vls9 783 8.20E-96 [Borrelia burgdorferi]
f24-1.aa gi.vertline.2039330 vmp-like sequence protein VlsE 742
6.30E-95 [Borrelia burgdorferi] f24-1.aa gi.vertline.2039336
vmp-like sequence protein VlsE 509 1.50E-92 [Borrelia burgdorferi]
f24-1.aa gi.vertline.2039286 putative vls recombination cassette
Vls7 754 6.60E-92 [Borrelia burgdorferi] f24-1.aa
gi.vertline.2039324 vmp-like sequence protein VlsE 488 8.10E-86
[Borrelia burgdorferi] f24-1.aa gi.vertline.2039316 vmp-like
sequence protein VlsE 531 1.70E-85 [Borrelia burgdorferi] f24-1.aa
gi.vertline.2039312 vmp-like sequence protein VlsE 531 1.20E-83
[Borrelia burgdorferi] f24-1.aa gi.vertline.2039326 vmp-like
sequence protein VlsE 476 2.00E-82 [Borrelia burgdorferi] f24-1.aa
gi.vertline.2039332 vmp-like sequence protein VlsE 474 5.10E-82
[Borrelia burgdorferi] f24-1.aa gi.vertline.2039328 vmp-like
sequence protein VlsE 420 3.50E-59 [Borrelia burgdorferi] f253.aa
gi.vertline.2688567 (AE001165) Na+/H+ antiporter (nhaC- 2247 0 1)
[Borrelia burgdorferi] f253.aa gi.vertline.2688566 (AE001165)
Na+/H+ antiporter (nhaC- 609 6.40E-155 2) [Borrelia burgdorferi]
f253.aa gi.vertline.2209268 Na+/H+ antiporter [Bacillus firmus] 158
9.40E-15 >pir.vertline.A41594.vertline.A41594 f253.aa
gi.vertline.1574661 Na+/H+ antiporter (nhaC) 143 4.20E-14
[Haemophilus influenzae] f253.aa gnl.vertline.PID.vertline.e11856-
25 similar to Na+/H+ antiporter [Bacillus 137 1.20E-11 subtilis]
f253.aa gnl.vertline.PID.vertline.e324972 hypothetical protein
[Bacillus subtilis] 133 2.00E-11 >gnl.vertline.PID.vertline.e-
1182969 f265.aa gi.vertline.2688555 (AE001164) conserved
hypothetical 1196 9.90E-161 protein [Borrelia burgdorferi] f269.aa
gi.vertline.2688560 (AE001164) B. burgdorferi predicted 1654
5.50E-226 coding region BB0624 [Borrelia f28-2.aa
gi.vertline.2690174 (AE000788) B. burgdorferi predicted 1683
2.80E-222 coding region BBK47 [Borrelia f28-2.aa
gi.vertline.2690161 (AE000788) B. burgdorferi predicted 1068
2.20E-163 coding region BBK49 [Borrelia f28-3.aa
gi.vertline.2690138 (AE000788) immunogenic protein P37, 281
6.00E-48 putative [Borrelia burgdorferi] f28-3.aa
gi.vertline.2690127 (AE000788) immunogenic protein P37 209 3.20E-28
[Borrelia burgdorferi] f28-3.aa gi.vertline.2459605 immunogenic
protein P37 [Borrelia 208 4.50E-28 burgdorferi] f28-3.aa
gi.vertline.2690137 (AE000788) immunogenic protein P37, 172
5.50E-17 putative [Borrelia burgdorferi] f29.aa gi.vertline.2688764
(AE001180) B. burgdorferi predicted 869 8.20E-116 coding region
BB0826 [Borrelia f290.aa gi.vertline.2688537 (AE001162) serine-type
D-Ala-D-Ala 2046 1.50E-281 carboxypeptidase (dacA) f290.aa
gi.vertline.143439 DD-carboxypeptidase [Bacillus subtilis] 161
6.60E-36 >pir.vertline.B42708.vertline.B42708 f290.aa
gnl.vertline.PID.vertline.e1185617 D-alanyl-D-alanine
carboxypeptidase 161 6.60E-36 (penicilin binding f290.aa
gnl.vertline.PID.vertline.d1016562 Probable penicillin-binding
protein. 131 3.30E-28 [Escherichia coli] f290.aa
sp.vertline.P37604.vertline.DACD_SALTY PENICILLIN-BINDING PROTEIN
6B 135 9.10E-28 PRECURSOR f290.aa gi.vertline.1572974
penicillin-binding protein 5 (dacA) 145 3.00E-27 [Haemophilus
influenzae] f290.aa gi.vertline.580849 D-alanine carboxypeptidase
[Bacillus 170 4.10E-27 stearothermophilus] f290.aa
gi.vertline.1778549 penicillin-binding protein 5 152 3.20E-26
[Escherichia coli] >gi.vertline.41212 precursor f290.aa
gi.vertline.142820 penicillin-binding protein 5 [Bacillus 137
4.60E-26 subtilis] f290.aa gi.vertline.410134 penicillin-binding
protein [Bacillus 137 4.60E-26 subtilis] >gnl.vertline.PID.ve-
rtline.e1185588 f290.aa gi.vertline.41218 precursor [Escherichia
coli] 136 1.30E-25 f290.aa gnl.vertline.PID.vertline.d1015262
Penicillin-binding protein 6 precursor 136 1.30E-25
(D-alanyl-D-alanine f290.aa gi.vertline.1864022 pencillin binding
protein 4 155 5.10E-22 [Staphylococcus aureus] f290.aa
gnl.vertline.PID.vertline.e154145 penicillin binding protein 4 155
5.10E-22 [Staphylococcus aureus] f290.aa
gnl.vertline.PID.vertline.e264682 penicillin-binding protein 4 155
5.10E-22 [Staphylococcus aureus] f291.aa gi.vertline.2688538
(AE001162) L-lactate permease (lctP) 2473 0 [Borrelia burgdorferi]
f291.aa gnl.vertline.PID.vertline.e274704 lactate premease
[Streptococcus iniae] 586 1.20E-132 f291.aa gi.vertline.882504
ORF_f560 [Escherichia coli] 345 3.60E-95 >gi.vertline.1789347
(AE000380) f560; This 560 aa f291.aa gi.vertline.2313225 (AE000535)
L-lactate permease (lctP) 359 1.10E-94 [Helicobacter pylori]
f291.aa gi.vertline.2313224 (AE000535) L-lactate permease (lctP)
348 2.90E-93 [Helicobacter pylori] f291.aa gi.vertline.404693
L-lactate permease [Escherichia coli] 331 7.20E-82
>gi.vertline.466741 aug is 3rd start f291.aa
gnl.vertline.PID.vertline.e313006 hypothetical protein [Bacillus
subtilis] 330 9.00E-80 >gnl.vertline.PID.vertline.e1186107
f291.aa gnl.vertline.PID.vertline.d1022632 lactate permease
[Bacillus subtilis] 300 1.70E-61 f291.aa
gnl.vertline.PID.vertline.e1182258 L-lactate permease [Bacillus
subtilis] 300 1.10E-60 >pir.vertline.F69649.vertline.F69649
f291.aa gnl.vertline.PID.vertline.d1009575 homologue of L-lactate
permease of E. coli 265 6.40E-56 [Bacillus f291.aa
gi.vertline.2649804
(AE001049) L-lactate permease (lctP) 170 1.50E-47 [Archaeoglobus
fulgidus] f291.aa gnl.vertline.PID.vertline.e283914 L-lactate
permease [Sulfolobus 163 2.60E-44 solfataricus] f291.aa
gi.vertline.1574148 L-lactate permease (lctP) [Haemophilus 173
6.00E-35 influenzae] f296.aa gi.vertline.2688517 (AE001161)
chaperonin, putative 1276 4.40E-177 [Borrelia burgdorferi] f296.aa
gi.vertline.840643 mucZ gene product [Coxiella burnetii] 101
7.90E-12 >pir.vertline.I40852.vertline.I40852 mucZ f3.aa
gi.vertline.2688797 (AE001183) B. burgdorferi predicted 1604
1.40E-211 coding region BB0844 [Borrelia f30.aa gi.vertline.2688765
(AE001180) B. burgdorferi predicted 1343 2.00E-181 coding region
BB0825 [Borrelia f301.aa gi.vertline.2688521 (AE001161)
methyl-accepting 2756 0 chemotaxis protein (mcp-3) [Borrelia
f301.aa gi.vertline.1805311 methyl-accepting chemotaxis protein B
211 7.00E-20 [Treponema denticola] f301.aa gi.vertline.2688522
(AE001161) methyl-accepting 189 2.80E-18 chemotaxis protein (mcp-2)
[Borrelia f301.aa gi.vertline.2367665 (AF016689) Mcp-2 [Treponema
189 3.50E-17 pallidum] f301.aa gi.vertline.2352917 (AF012922)
methyl-accepting 187 5.70E-17 chemotaxis protein [Treponema f301.aa
gi.vertline.1354776 MCP-1 [Treponema pallidum] 189 5.90E-17 f301.aa
gi.vertline.2619023 (AF027868) YoaH [Bacillus subtilis] 184
2.80E-16 >gnl.vertline.PID.vertline.e1185333 similar to f301.aa
gi.vertline.1654421 transducer HtB protein [Halobacterium 177
2.20E-15 salinarum] f301.aa gi.vertline.415694 chemoreceptor
[Desulfovibrio vulgaris] 163 3.50E-15 >pir.vertline.G36943.ve-
rtline.G36943 f301.aa gi.vertline.459691 transmembrane receptor
[Bacillus 163 4.90E-15 subtilis] >gnl.vertline.PID.vertline.e-
1185996 f301.aa gi.vertline.2104730 ORF2 [Desulfurococcus sp. ] 173
5.80E-15 f301.aa gi.vertline.2914132 methyl accepting chemotaxis
homolog 170 1.10E-14 [Treponema denticola] f301.aa
gi.vertline.459689 transmembrane receptor [Bacillus 164 1.30E-14
subtilis] >gnl.vertline.PID.vertline.e1185998 f301.aa
gi.vertline.496484 tlpC gene product [Bacillus subtilis] 170
3.80E-14 >pir.vertline.I40496.vertline.I40496 methylation
f301.aa gi.vertline.2313163 (AE000530) methyl-accepting 170
6.30E-14 chemotaxis transducer (tlpC) f308.aa gi.vertline.2688527
(AE001161) B. burgdorferi predicted 1227 1.70E-176 coding region
BB0592 [Borrelia f31-2.aa gi.vertline.2690202 (AE000787) B.
burgdorferi predicted 1771 7.20E-235 coding region BBJ36 [Borrelia
f31-2.aa gi.vertline.2690200 (AE000787) B. burgdorferi predicted
423 4.60E-88 coding region BBJ34 [Borrelia f31.aa
gi.vertline.2688766 (AE001180) B. burgdorferi predicted 957
7.80E-133 coding region BB0824 [Borrelia f314.aa
gi.vertline.2688509 (AE001160) pfs protein (pfs-2) 1329 7.40E-180
[Borrelia burgdorferi] f314.aa gi.vertline.2690087 (AE000789) pfs
protein (pfs) [Borrelia 335 1.50E-77 burgdorferi] f314.aa
gi.vertline.2688288 (AE001143) pfs protein (pfs-1) 266 1.00E-65
[Borrelia burgdorferi] f314.aa gi.vertline.2738591 (AF012886) Pfs
[Buchnera aphidicola] 115 1.70E-52 f314.aa gi.vertline.1552737
similar to purine nucleoside 133 6.90E-52 phosphorylase (deoD)
[Escherichia f314.aa gnl.vertline.PID.vertline.e1183957 similar to
purine nucleoside 157 1.20E-49 phosphorylase [Bacillus f314.aa
gi.vertline.147158 pfs [Escherichia coli] >gi.vertline.457107
ORF 133 2.50E-42 [Escherichia coli] {(SUB 9-219} f314.aa
gi.vertline.1574146 pfs protein (pfs) [Haemophilus 110 2.70E-37
influenzae] >pir.vertline.C64169.vertline.C64169 pfs f314.aa
gi.vertline.2267164 (AF009177) pfs protein homolog 118 3.30E-23
[Helicobacter pylori] f314.aa gi.vertline.2313168 (AE000530) pfs
protein (pfs) 115 1.00E-22 [Helicobacter pylori] f314.aa
gi.vertline.1777939 Pfs [Treponema pallidum] 102 1.90E-20 f314.aa
gi.vertline.2689970 (AE000785) B. burgdorferi predicted 191
1.50E-19 coding region BBE07 [Borrelia f314.aa
gnl.vertline.PID.vertlin- e.e249405 unknown [Mycobacterium
tuberculosis] 105 7.60E-16
>sp.vertline.Q10889.vertline.Y05A_MYCTU f32-4.aa
gi.vertline.2690221 (AE000787) B. burgdorferi predicted 1192
4.00E-163 coding region BBJ47 [Borrelia f32-4.aa
gi.vertline.2689979 (AE000785) B. burgdorferi predicted 103
4.10E-11 coding region BBE16 [Borrelia f32.aa gi.vertline.2688767
(AE001180) B. burgdorferi predicted 623 1.80E-81 coding region
BB0823 [Borrelia f32.aa gi.vertline.2688767 (AE001180) B.
burgdorferi predicted 623 1.80E-81 coding region BB0823 [Borrelia
f320.aa gi.vertline.2688497 (AE001159) carboxypeptidase, putative
1373 6.40E-186 [Borrelia burgdorferi] f320.aa gi.vertline.2529473
(AF006665) YokZ [Bacillus subtilis] 136 9.80E-28 f320.aa
gi.vertline.2415396 (AF015775) carboxypeptidase [Bacillus 136
1.90E-27 subtilis] >gnl.vertline.PID.vertline.e1185433 f320.aa
gi.vertline.1209528 D,D-carboxypeptidase [Enterococcus 148 3.30E-16
faecalis] >sp.vertline.Q47746.vertline.VANY_ENTFA f320.aa
gi.vertline.155044 vanY [Transposon Tn1546] >gi.vertline.149126
142 1.60E-13 D,D-carboxypeptidase [Plasmid f328.aa
gi.vertline.2688502 (AE001159) CTP synthase (pyrG) 869 6.10E-119
[Borrelia burgdorferi] f328.aa gi.vertline.1591801 CTP synthase
(pyrG) [Methanococcus 325 6.20E-59 jannaschii]
>pir.vertline.E64446.vertline.E64446 f328.aa gi.vertline.2650385
(AE001088) CTP synthase (pyrG) 304 4.20E-54 [Archaeoglobus
fulgidus] f328.aa gi.vertline.1399854 CTP synthetase [Synechococcus
313 3.30E-52 PCC7942] >sp.vertline.Q54775.vertline.PYRG_SYNP7
CTP f328.aa gnl.vertline.PID.vertline.d1019032 CTP synthetase
[Synechocystis sp.] 295 1.80E-50
>pir.vertline.S75840.vertline.S75840 CTP f328.aa
gi.vertline.143597 CTP synthetase [Bacillus subtilis] 274 1.60E-49
>gi.vertline.853762 CTP synthase [Bacillus f328.aa
gi.vertline.2983754 (AE000735) CTP synthetase [Aquifex 271 1.50E-46
aeolicus] f328.aa gi.vertline.1574630 CTP synthetase (pyrG)
[Haemophilus 234 1.90E-44 influenzae]
>pir.vertline.F64181.vertline.F64181 f328.aa gi.vertline.413755
CTP synthetase [Spiroplasma citri] 231 3.00E-44
>sp.vertline.P52200.vertline.PYRG_SPICI CTP f328.aa
gi.vertline.2621483 (AE000826) CTP synthase 257 2.80E-40
[Methanobacterium thermoautotrophicum] f328.aa gi.vertline.950067
CTP synthase [Mycoplasma 220 4.10E-39 capricolum]
>pir.vertline.S77767.vertline.S77767 CTP synthase f328.aa
gi.vertline.904007 cytidine triphosphate synthetase 219 2.00E-38
precursor [Giardia intestinalis] f328.aa gi.vertline.147478 CTP
synthetase (EC 6.3.4.2) 217 2.90E-38 [Escherichia coli] f328.aa
gi.vertline.882674 CTP synthetase [Escherichia coli] 214 7.70E-38
>gi.vertline.1789142 (AE000361) CTP f328.aa gi.vertline.38688
CTP synthase [Azospirillum brasilense] 132 3.20E-37
>pir.vertline.I39496.v- ertline.S25101 CTP f342.aa
gi.vertline.2688495 (AE001158) B. burgdorferi predicted 944
5.30E-130 coding region BB0563 [Borrelia f346.aa
gi.vertline.1272356 phosphotransferase enzyme II [Borrelia 828
1.10E-108 burgdorferi] >gi.vertline.2688474 f346.aa
gi.vertline.145603 PTS enzyme III glc [Escherichia coli] 385
8.80E-53 >gi.vertline.145605 PTS enzyme III glc f346.aa
gi.vertline.1314675 glucose-specific component IIA of the 385
9.30E-53 PTS system [Escherichia coli] f346.aa gi.vertline.47658
III(Glc) (crr) (AA 1-169) [Salmonella 382 2.30E-52 typhimurium]
f346.aa gi.vertline.1574566 glucose phosphotransferase enzyme III-
397 8.70E-50 glc (crr) [Haemophilus f346.aa gi.vertline.43819 nagE
gene product [Klebsiella 349 2.80E-41 pneumoniae]
>pir.vertline.S18607.ver- tline.S18607 f346.aa
gi.vertline.146913 N-acetylglucosamine transport protein 334
3.20E-39 [Escherichia coli] f346.aa gi.vertline.1072418 glcA
[Staphylococcus carnosus] 317 7.20E-37
>pir.vertline.S46952.vertline.S46952 f346.aa gi.vertline.1072419
glcB [Staphylococcus carnosus] 315 1.40E-36
>pir.vertline.S63606.vertline.S46953 f346.aa gi.vertline.1146177
phosphotransferase system glucose- 295 7.30E-36 specific enzyme II
[Bacillus f346.aa gi.vertline.529001 PTS glucose-specific permease
294 8.80E-36 [Bacillus stearothermophilus] f346.aa
gnl.vertline.PID.vertline.e1182187 alternate gene name: yzfA;
similar to 293 1.40E-33 phosphotransferase f346.aa
gi.vertline.580912 enzyme III-glucose [Bacillus subtilis] 257
1.20E-30 f346.aa gi.vertline.602681 phosphocarrier protein (enzyme
IIA) 243 1.00E-28 [Mycoplasma capricolum] f346.aa
gi.vertline.1432153 cellobiose-specific PTS permease 257 1.20E-28
[Klebsiella oxytoca] f352.aa gi.vertline.2688482 (AE001157) B.
burgdorferi predicted 2547 0 coding region BB0553 [Borrelia f352.aa
gi.vertline.2688482 (AE001157) B. burgdorferi predicted 1005
1.30E-132 coding region BB0553 [Borrelia f363.aa
gi.vertline.2688468 (AE001156) B. burgdorferi predicted 1109
5.40E-153 coding region BB0543 [Borrelia f368.aa
gi.vertline.2688450 (AE001155) conserved hypothetical 1133
4.10E-157 integral membrane protein f368.aa gi.vertline.1787004
(AE000181) o234; This 234 aa ORF is 417 1.40E-67 26 pct identical
(15 gaps) to f368.aa gi.vertline.2314055 (AE000601) conserved
hypothetical 129 3.50E-16 integral membrane protein f368.aa
gnl.vertline.PID.vertline.e128- 9272 S1R [Cowpox virus] 135
1.80E-14 f368.aa gnl.vertline.PID.vertline.d1003176 24 K membrane
protein [Pseudomonas 108 9.00E-13 aeruginosa] f368.aa
gi.vertline.41284 put. 23.5-kd protein [Escherichia coli] 101
1.00E-11 >gi.vertline.1787205 (AE000199) f371.aa
gi.vertline.2688452 (AE001155) conserved hypothetical 1066
3.60E-143 protein [Borrelia burgdorferi] f371.aa
gi.vertline.2196997 Orf256 [Treponema pallidum] 154 1.10E-15
f373.aa gi.vertline.2688453 (AE001155) zinc protease, putative 3663
0 [Borrelia burgdorferi] f373.aa gi.vertline.1574200 hypothetical
[Haemophilus influenzae] 295 2.70E-67
>pir.vertline.E64171.vertline.E64171 f373.aa gi.vertline.1787770
(AE000246) f931; residues 5-650 are 289 1.10E-57 99 pct identical
to YDDC_ECOLI f373.aa gi.vertline.535004 cds106 gene product
[Escherichia coli] 289 3.20E-57 f373.aa gi.vertline.799369
metalloendopeptidase [Pisum sativum] 148 7.10E-28 f373.aa
gi.vertline.2827039 (AF008444) chloroplast processing 150 1.70E-26
enzyme [Arabidopsis thaliana] f373.aa gi.vertline.2983709
(AE000732) processing protease 136 4.30E-24 [Aquifex aeolicus]
f373.aa gi.vertline.2314155 (AE000609) protease (pqqE) 115 5.30E-23
[Helicobacter pylori] >pir.vertline.D64646.vertline.D64646
f378.aa gi.vertline.2688458 (AE001155) B. burgdorferi predicted
1030 1.30E-136 coding region BB0531 [Borrelia f384.aa
gi.vertline.2688435 (AE001154) inositol monophosphatase 1470
3.80E-201 [Borrelia burgdorferi] f4-15.aa gi.vertline.2690238
(AE000790) surface lipoprotein P27 1400 1.50E-185 [Borrelia
burgdorferi] f4-15.aa gi.vertline.144008 P27 [Borrelia burgdorferi]
462 2.40E-96 >pir.vertline.S34995.vertline.S34995 surface
lipoprotein f4-50.aa gi.vertline.2690243 (AE000790) decorin binding
protein B 900 6.30E-117 (dbpB) [Borrelia burgdorferi] f4-50.aa
gi.vertline.2062381 decorin binding protein B [Borrelia 897
1.60E-116 burgdorferi] f4-50.aa gi.vertline.2809217 (AF042796)
putative decorin-binding 887 3.60E-115 protein precursor [Borrelia
f4-50.aa gi.vertline.2809218 (AF042796) decorin-binding protein 172
2.00E-33 precursor [Borrelia burgdorferi] f4-50.aa
gi.vertline.2690249 (AE000790) decorin binding protein A 176
9.50E-33 (dbpA) [Borrelia burgdorferi] f4-50.aa gi.vertline.2062379
decorin binding protein A [Borrelia 177 6.10E-32 burgdorferi]
f4-66.aa gi.vertline.2690229 (AE000790) chpAI protein, putative 807
1.60E-107 [Borrelia burgdorferi] f4.aa gi.vertline.2688787
(AE001183) conserved hypothetical 2408 0 integral membrane protein
f4.aa gi.vertline.2697115 (AF008219) unknown [Borrelia afzelii]
1138 1.90E-305 f4.aa gi.vertline.1573583 H. influenzae predicted
coding region 337 2.10E-109 HI0594 [Haemophilus f4.aa
gi.vertline.1788636 (AE000319) o513; This 513 aa ORF is 327
9.10E-80 31 pct identical (30 gaps) to f4.aa
gnl.vertline.PID.vertline.d1009571 homologue of hypothetical
protein 357 5.40E-69 HI10594 of H. influenzae f42-1.aa
gi.vertline.2689993 (AE000794) conserved hypothetical 495 2.70E-62
protein [Borrelia burgdorferi] f42-1.aa gi.vertline.2689934
(AE000793) conserved hypothetical 312 1.00E-37 protein [Borrelia
burgdorferi] f43-3.aa gi.vertline.1209843 lipoprotein [Borrelia
burgdorferi] 546 1.50E-69 f43-3.aa gi.vertline.2121280 (AF000270)
lipoprotein [Borrelia 442 1.80E-55 burgdorferi]
>gi.vertline.3095109 f43-3.aa gi.vertline.1209837 lipoprotein
[Borrelia burgdorferi] 365 3.10E-55 f43-3.aa gi.vertline.1209873
lipoprotein [Borrelia burgdorferi] 269 5.30E-32 f43-3.aa
gi.vertline.1209849 lipoprotein [Borrelia burgdorferi] 141 1.70E-13
f43-3.aa gi.vertline.3095105 (AF046998) 2.9-8 lipoprotein [Borrelia
140 9.60E-13 burgdorferi] f43-3.aa gi.vertline.3095107 (AF046999)
2.9-9 lipoprotein [Borrelia 132 1.40E-11 burgdorferi] f43.aa
gi.vertline.2688752 (AE001179) B. burgdorferi predicted 2337
6.60000000084856e-315 coding region BB0811 [Borrelia f446.aa
gi.vertline.2688383 (AE001151) B. burgdorferi predicted 920
7.20E-124 coding region BB0464 [Borrelia f45-2.aa
gi.vertline.1699017 ErpB2 [Borrelia burgdorferi] 364 7.50E-78
>gi.vertline.1373133 ErpB [Borrelia f45-2.aa gi.vertline.2627270
ErpJ [Borrelia burgdorferi] 364 2.50E-77 f45-2.aa
gi.vertline.2627268 ErpM [Borrelia burgdorferi] 452 9.70E-60
f45-2.aa gi.vertline.1373144 ErpD [Borrelia burgdorferi] 316
1.60E-58 f45-2.aa gi.vertline.2444428 (AF020657) ErpX protein
[Borrelia 380 2.80E-55 burgdorferi] f45-2.aa gi.vertline.1051120
outer surface protein G [Borrelia 213 7.10E-35 burgdorferi]
>gi.vertline.1373118 ErpG f45-2.aa gi.vertline.1663633 ErpK
[Borrelia burgdorferi] 152 1.60E-21 f45-2.aa
gnl.vertline.PID.vertline.e329895 (AJ000496) cyclic
nucleotide-gated 198 2.80E-16 channel beta subunit f45-2.aa
gi.vertline.466482 outer surface protein F [Borrelia 111 5.70E-14
burgdorferi] >pir.vertline.I40287.vertline.I40287 f45-2.aa
gi.vertline.2246532 ORF 73, contains large complex repeat 174
5.90E-14 CR 73 [Kaposi's f45-2.aa gi.vertline.160299 glutamic
acid-rich protein [Plasmodium 169 1.00E-13 falciparum] f45-2.aa
gi.vertline.1707287 putative outer membrane protein 101 2.20E-13
[Borrelia burgdorferi] f45-2.aa gi.vertline.1633572 Herpesvirus
saimiri ORF73 homolog 175 4.10E-13 [Kaposi's sarcoma-associated
f45-2.aa gnl.vertline.PID.vertline.- d1012343 gene required for
phosphoylation of 166 5.60E-13 oligosaccharides/has f45-2.aa
gi.vertline.2690100 (AE000789) B. burgdorferi predicted 161
2.70E-12 coding region BBI16 [Borrelia f457.aa gi.vertline.2688369
(AE001150) B. burgdorferi predicted 1021 6.20E-139 coding region
BB0456 [Borrelia f469.aa gi.vertline.2688368 (AE001150) Na+/H+
antiporter (napA) 1544 1.10E-211 [Borrelia burgdorferi] f47-2.aa
gi.vertline.1209849 lipoprotein [Borrelia burgdorferi] 742 2.30E-97
f47-2.aa gi.vertline.1209857 lipoprotein [Borrelia burgdorferi] 407
7.80E-86 f47-2.aa gi.vertline.1209831 lipoprotein [Borrelia
burgdorferi] 393 5.00E-82 f47-2.aa
gnl.vertline.PID.vertline.e268245 surface-exposed lipoprotein
[Borrelia 321 2.60E-73 burgdorferi] f47-2.aa gi.vertline.1209874
lipoprotein [Borrelia burgdorferi] 348 1.10E-64 f47-2.aa
gnl.vertline.PID.vertline.e268239 surface-exposed lipoprotein
[Borrelia 333 1.40E-57 garinii]
f47-2.aa gnl.vertline.PID.vertline.e268244 surface-exposed
lipoprotein [Borrelia 292 9.60E-44 afzelii] f47-2.aa
gi.vertline.3095107 (AF046999) 2.9-9 lipoprotein [Borrelia 328
3.80E-40 burgdorferi] f47-2.aa gnl.vertline.PID.vertline.e268242
surface-exposed lipoprotein [Borrelia 320 1.70E-39 garinii]
f47-2.aa gi.vertline.1209837 lipoprotein [Borrelia burgdorferi] 210
4.80E-29 f47-2.aa gi.vertline.2121280 (AF000270) lipoprotein
[Borrelia 205 1.10E-27 burgdorferi] >gi.vertline.3095109
f47-2.aa gi.vertline.3095105 (AF046998) 2.9-8 lipoprotein [Borrelia
217 6.30E-25 burgdorferi] f47-2.aa gi.vertline.1209873 lipoprotein
[Borrelia burgdorferi] 113 2.40E-11 f477.aa gi.vertline.2688350
(AE001149) fructose-bisphosphate 1506 3.60E-202 aldolase (fba)
[Borrelia f477.aa gi.vertline.882454 fructose 1,6-bisphosphate
aldolase 651 1.10E-131 [Escherichia coli] >gi.vertline.41423
f477.aa gi.vertline.2708661 (AF037440) fructose 1,6-bisphosphate
593 1.40E-124 aldolase [Edwardsiella f477.aa gi.vertline.1573507
fructose-bisphosphate aldolase (fba) 560 8.50E-120 [Haemophilus
influenzae] f477.aa gi.vertline.671841 fructose 1,6-bisphosphate
aldolase 856 3.80E-113 [Campylobacter jejuni] f477.aa
gnl.vertline.PID.vertline.d10047- 56 fructose 1,6-bisphosphate
aldolase 749 1.70E-98 [Schizosaccharomyces f477.aa
gi.vertline.433637 yeast fructose-bisphate-aldolase 459 1.20E-92
[Saccharomyces cerevisiae] >gi.vertline.3696 f477.aa
gnl.vertline.PID.vertline- .e190134 fructose-1,6-bisphosphate
aldolase 701 6.30E-92 [Euglena gracilis] f477.aa
gi.vertline.1334980 fructose 1,6 bisphosphate-aldolase 647 1.50E-84
[Neurospora crassa] f477.aa gi.vertline.40495 fructose-bisphosphate
aldolase 204 6.80E-37 [Corynebacterium glutamicum] f477.aa
gnl.vertline.PID.vertlin- e.e315480 Fba [Mycobacterium
tuberculosis] 207 1.50E-35 f477.aa gi.vertline.1045692
fructose-bisphosphate aldolase 108 2.10E-23 [Mycoplasma genitalium]
f477.aa gnl.vertline.PID.vertline.d1003809 hypothetical protein
[Bacillus subtilis] 102 2.70E-15
>gnl.vertline.PID.vertline.e1184692 f488.aa gi.vertline.2688338
(AE001148) DNA gyrase, subunit A 3222 0 (gyrA) [Borrelia
burgdorferi] f488.aa gi.vertline.1790876 DNA gyrase subunit A
[Clostridium 822 1.80E-171 acetobutylicum] f488.aa
gi.vertline.2650163 (AE001072) DNA gyrase, subunit A 483 1.10E-162
(gyrA) [Archaeoglobus fulgidus] f488.aa gi.vertline.40019 ORF 821
(aa 1-821) [Bacillus subtilis] 836 6.10E-159
>gnl.vertline.PID.vertline.d1005785 A subunit of f488.aa
gi.vertline.459929 gyrase A subunit [Pseudomonas 418 7.00E-155
aeruginosa] >sp.vertline.P48372.vertline.GYRA_PSEAE DNA f488.aa
gi.vertline.144206 DNA gyrase A [Campylobacter jejuni] 508
7.50E-154 >pir.vertline.A48902.vertline.A48902 DNA gyrase
f488.aa gi.vertline.466275 gyrase A [Mycobacterium tuberculosis]
395 3.50E-152 >sp.vertline.Q07702.vertline.GYRA_MYCTU DNA
f488.aa gnl.vertline.PID.vertline.e266924 GyrA [Mycobacterium
tuberculosis] 395 2.00E-151 f488.aa gi.vertline.43485 DNA gyrase A
subunit [Haloferax] 275 6.10E-151 >pir.vertline.S30571.vertli-
ne.S30571 DNA topoisomerase f488.aa
gnl.vertline.PID.vertline.d1025098 (AB010081) A subunit of DNA
gyrase 549 1.20E-150 [Bacillus sp.] f488.aa
gnl.vertline.PID.vertlin- e.e214031 DNA gyrase subunit A
[Mycobacterium 388 5.90E-150 smegmatis] f488.aa gi.vertline.2731385
DNA gyrase [Serratia marcescens] 378 6.00E-148 f488.aa
gnl.vertline.PID.vertline.e13703- 8 DNA topoisomerase
(ATP-hydrolysing) 388 7.30E-147 [Mycobacterium smegmatis] f488.aa
gi.vertline.41634 gyrA gene product (AA 1-875) 383 2.40E-146
[Escherichia coli] >gi.vertline.41636 DNA gyrase f488.aa
gi.vertline.497648 DNA gyrase subunit A [Mycoplasma 514 5.20E-146
genitalium] f49-2.aa gi.vertline.2039282 putative vls recombination
cassette Vls3 943 2.30E-120 [Borrelia burgdorferi] f49-2.aa
gi.vertline.2547241 vmp-like sequence protein VlsE 434 4.10E-106
[Borrelia burgdorferi] f49-2.aa gi.vertline.2039324 vmp-like
sequence protein VlsE 458 3.00E-104 [Borrelia burgdorferi] f49-2.aa
gi.vertline.2039281 putative vls recombination cassette Vls2 793
1.80E-100 [Borrelia burgdorferi] f49-2.aa gi.vertline.2039283
putative vls recombination cassette Vls4 729 4.60E-92 [Borrelia
burgdorferi] f49-2.aa gi.vertline.2039308 vmp-like sequence protein
VlsE 652 1.40E-88 [Borrelia burgdorferi] f49-2.aa
gi.vertline.2039288 putative vls recombination cassette Vls9 352
1.80E-88 [Borrelia burgdorferi] f49-2.aa gi.vertline.2039332
vmp-like sequence protein VlsE 550 4.40E-88 [Borrelia burgdorferi]
f49-2.aa gi.vertline.2039328 vmp-like sequence protein VlsE 629
1.50E-85 [Borrelia burgdorferi] f49-2.aa gi.vertline.2039336
vmp-like sequence protein VlsE 460 1.40E-82 [Borrelia burgdorferi]
f49-2.aa gi.vertline.2039318 vmp-like sequence protein VlsE 367
6.20E-82 [Borrelia burgdorferi] f49-2.aa gi.vertline.2039320
vmp-like sequence protein VlsE 449 1.80E-77 [Borrelia burgdorferi]
f49-2.aa gi.vertline.2483796 VlsE1 [Borrelia burgdorferi] 497
8.20E-76 f49-2.aa gi.vertline.2039326 vmp-like sequence protein
VlsE 427 2.50E-64 [Borrelia burgdorferi] f49-2.aa
gi.vertline.2039291 putative vls recombination cassette 409
1.30E-47 Vls13 [Borrelia burgdorferi] f494.aa gi.vertline.2688346
(AE001148) B. burgdorferi predicted 547 8.20E-74 coding region
BB0428 [Borrelia f5-14.aa gi.vertline.2627268 ErpM [Borrelia
burgdorferi] 1836 2.60E-236 f5-14.aa gi.vertline.1373144 ErpD
[Borrelia burgdorferi] 543 4.40E-87 f5-14.aa gi.vertline.2627270
ErpJ [Borrelia burgdorferi] 503 4.30E-83 f5-14.aa
gi.vertline.1699017 ErpB2 [Borrelia burgdorferi] 503 2.60E-82
>gi.vertline.1373133 ErpB [Borrelia f5-14.aa gi.vertline.2444428
(AF020657) ErpX protein [Borrelia 399 9.30E-57 burgdorferi]
f5-14.aa gnl.vertline.PID.vertline.e329895 (AJ000496) cyclic
nucleotide-gated 228 1.50E-20 channel beta subunit f5-14.aa
gnl.vertline.PID.vertline.d1012343 gene required for phosphoylation
of 203 8.70E-18 oligosaccharides/has f5-14.aa gi.vertline.2246532
ORF 73, contains large complex repeat 197 3.30E-17 CR 73 [Kaposi's
f5-14.aa gi.vertline.1633572 Herpesvirus saimiri ORF73 homolog 192
1.20E-16 [Kaposi's sarcoma-associated f5-14.aa gi.vertline.3068583
(AF000580) Rep-like [Dictyostelium 197 3.60E-16 discoideum]
f5-14.aa gi.vertline.2690100 (AE000789) B. burgdorferi predicted
183 2.90E-15 coding region BBI16 [Borrelia f5-14.aa
gi.vertline.1825739 No definition line found 168 1.60E-14
[Caenorhabditis elegans] f5-14.aa gi.vertline.3044185 (AF056936)
mature parasite-infected 166 2.00E-14 erythrocyte surface antigen
f5-14.aa gnl.vertline.PID.vertline.e349084 E02A10.2 [Caenorhabditis
elegans] 176 2.30E-14 f5-14.aa gi.vertline.1051120 outer surface
protein G [Borrelia 157 3.30E-12 burgdorferi]
>gi.vertline.1373118 ErpG f5-15.aa gi.vertline.2627267 ErpL
[Borrelia burgdorferi] 1152 4.40E-147 f5-15.aa gi.vertline.1197833
Bbk2.11 [Borrelia burgdorferi] 856 3.30E-108
>pir.vertline.S70531.vertline.S705- 31 bbk2.11 protein f5-15.aa
gi.vertline.896042 OspF [Borrelia burgdorferi] 325 1.00E-72
>pir.vertline.S70532.vertline.S7053- 2 outer surface protein
f5-15.aa gi.vertline.1707281 putative Outer membrane protein 323
1.80E-72 [Borrelia burgdorferi] f5-15.aa gi.vertline.1707287
putative outer membrane protein 322 6.60E-70 [Borrelia burgdorferi]
f5-15.aa gi.vertline.466482 outer surface protein F [Borrelia 448
6.80E-68 burgdorferi] >pir.vertline.I40287.vertline.I40287
f5-15.aa gi.vertline.1707290 putative outer surface protein
[Borrelia 290 1.90E-52 burgdorferi] f5-15.aa gi.vertline.1663633
ErpK [Borrelia burgdorferi] 172 8.70E-43 f5-15.aa
gi.vertline.896038 BbK2.10 precursor [Borrelia 153 1.10E-42
burgdorferi] >pir.vertline.S70534.vertline.S70534 bbK2.10
f5-15.aa gi.vertline.896040 BbK2.10 precursor [Borrelia 124
4.30E-39 burgdorferi] >pir.vertline.S70533.vertline.S70533
bbK2.10 f5-15.aa gi.vertline.1051120 outer surface protein G
[Borrelia 105 3.10E-23 burgdorferi] >gi.vertline.1373118 ErpG
f5-15.aa gi.vertline.1373144 ErpD [Borrelia burgdorferi] 103
1.10E-14 f50.aa gi.vertline.2688754 (AE001179) B. burgdorferi
predicted 2651 0 coding region BB0806 [Borrelia f502.aa
gi.vertline.2688313 (AE001146) sensory transduction 7570 0
histidine kinase, putative f502.aa gnl.vertline.PID.vertline.d1025-
877 (AB006363) homologue of histidine 296 3.80E-58 kinase [Candida
albicans] f502.aa gi.vertline.1354473 Os-1p [Neurospora crassa] 275
3.30E-57 f502.aa gi.vertline.1679757 two-component histidine kinase
CHK-1 382 4.20E-57 [Glomerella cingulata] f502.aa
gi.vertline.1262208 Nik-1 [Neurospora crassa]
>gi.vertline.1262210 273 6.30E-57 Nik-1 [Neurospora crassa]
f502.aa gi.vertline.2460283 (AF024654) hybrid histidine kinase 273
3.90E-55 DHKB [Dictyostelium discoideum] f502.aa
gnl.vertline.PID.vertline.d1017789 sensory transduction histidine
kinase 288 8.50E-54 [Synechocystis sp. ] f502.aa
gi.vertline.2623815 (AF030352) two component sensor 252 4.00E-52
[Pseudomonas aeruginosa] f502.aa gi.vertline.939724 putative sensor
kinase; regulatory 252 1.80E-50 protein for production of f502.aa
gi.vertline.151329 regulatory protein [Pseudomonas 248 1.20E-49
syringae] >sp.vertline.P48027.vertline.LEMA_PSESY f502.aa
pir.vertline.B41863.vertline.B41863 two-component regulatory
protein lemA - 248 1.30E-49 Pseudomonas syringae f502.aa
gnl.vertline.PID.vertline.d1018725 sensory transduction histidine
kinase 252 2.10E-49 [Synechocytis sp. ] f502.aa
gnl.vertline.PID.vertline.d1002185 sensor-regulator protein
[Escherichia 262 6.20E-49 coli] >gi.vertline.1789149 f502.aa
gi.vertline.463195 pectate lyase [Pseudomonas viridiflava] 247
7.50E-49 f502.aa gnl.vertline.PID.vertline.d1018731 sensory
transduction histidine kinase 244 1.00E-48 [Synechocystis sp. ]
f51-2.aa gi.vertline.2444428 (AF020657) ErpX protein [Borrelia 1755
2.20E-227 burgdorferi] f51-2.aa gi.vertline.2627268 ErpM [Borrelia
burgdorferi] 399 3.20E-57 f51-2.aa gi.vertline.1373144 ErpD
[Borrelia burgdorferi] 282 2.20E-50 f51-2.aa gi.vertline.2627270
ErpJ [Borrelia burgdorferi] 271 6.00E-34 f51-2.aa
gi.vertline.1699017 ErpB2 [Borrelia burgdorferi] 271 2.50E-33
>gi.vertline.1373133 ErpB [Borrelia f51-2.aa gi.vertline.1051120
outer surface protein G [Borrelia 109 3.70E-22 burgdoferi]
>gi.vertline.1373118 ErpG f51-2.aa
gnl.vertline.PID.vertline.d1012343 gene required for phosphoylation
of 203 5.40E-18 oligosaccharides/has f51-2.aa gi.vertline.1707287
putative outer membrane protein 111 7.50E-18 [Borrelia burgdoferi]
f51-2.aa gi.vertline.896042 OspF [Borrelia burgdorferi] 111
2.10E-17 >pir.vertline.S70532.vertline.S7053- 2 outer surface
protein f51-2.aa gi.vertline.1707281 putative outer membrane
protein 111 7.50E-17 [Borrelia burgdorferi] f51-2.aa
gnl.vertline.PID.vertline.e329895 (AJ000496) cyclic
nucleotide-gated 198 1.60E-16 channel beta subunit f51-2.aa
gi.vertline.2246532 ORF 73, contains large complex repeat 176
2.30E-14 CR 73 [Kaposi's f51-2.aa gnl.vertline.PID.vertli-
ne.e349084 E02A10.2 [Caenorhabditis elegans] 170 2.10E-13 f51-2.aa
gi.vertline.160299 glutamic acid-rich protein [Plasmodium 157
7.30E-12 falciparum] f516.aa gi.vertline.2688326 (AE001146) B.
burgdorferi predicted 1096 2.00E-150 coding region BB0409 [Borrelia
f517.aa gi.vertline.2688320 (AE001146) PTS system, fructose- 1637
2.30E-228 specific IIABC component (fruA-1) f517.aa
gnl.vertline.PID.vertline.e1183221 similar to fructose
phosphotransferase 256 4.00E-88 system enzyme II f517.aa
gi.vertline.396296 similar to phosphotransferase system 305
9.10E-86 enzyme II [Escherichia coli] f517.aa gi.vertline.405893
fructose-specific IIBC component 224 4.30E-84 [Escherichia coli]
>gi.vertline.450372 f517.aa gi.vertline.151932 fructose enzyme
II [Rhodobacter 222 4.70E-79 capsulatus] >gi.vertline.46021
fructose f517.aa gi.vertline.1573422 fructose-permease IIBC
component 225 6.90E-69 (fruA) [Haemophilus influenzae] f517.aa
gi.vertline.2688554 (AE001164) PTS system, fructose- 236 8.20E-66
specific IIABC component (fruA-2) f517.aa
gnl.vertline.PID.vertline.e1185030 phosphotransferase system (PTS)
195 2.80E-65 fructose-specific enzyme IIBC f517.aa
gi.vertline.155369 PTS enzyme-II fructose [Xanthomonas 187 8.10E-62
campestris] >pir.vertline.B40944.vertline.B40944 f517.aa
gi.vertline.305003 similar to fructose-specific 145 1.90E-39
phosphotransferase enzyme II f517.aa gnl.vertline.PID.vertline.d10-
11544 HrsA [Escherichia coli] >gi.vertline.1786951 148 2.80E-39
(AE000176) f517.aa gi.vertline.1813488 phosphotransferase enzyme II
[Bacillus 226 3.90E-39 firmus] f517.aa gi.vertline.757734 fruA gene
product [Bacillus 177 2.50E-36 amyloliquefaciens]
>pir.vertline.S59965.vertline.S59965 f517.aa
gnl.vertline.PID.vertline.d1016984 PTS SYSTEM, FRUCTOSE-SPECIFIC
173 1.10E-34 IIBC COMPONENT (EIIBC-FRU) f517.aa gi.vertline.1673731
(AE000010) Mycoplasma pneumoniae, 143 9.00E-33 fructose-permease
IIBC component; f519.aa gi.vertline.2688327 (AE001146) B.
burgdorferi predicted 1060 5.70E-145 coding region BB0406 [Borrelia
f519.aa gi.vertline.2688328 (AE001146) B. burgdorferi predicted 261
1.20E-47 coding region BB0405 [Borrelia f520.aa gi.vertline.2688328
(AE001146) B. burgdorferi predicted 1022 3.90E-138 coding region
BB0405 [Borrelia f520.aa gi.vertline.2688327 (AE001146) B.
burgdorferi predicted 261 4.00E-47 coding region BB0406 [Borrelia
f523.aa gi.vertline.2688300 (AE001145) glutamate transporter, 2007
9.90E-284 putative [Borrelia burgdorferi] f526.aa
gi.vertline.2688309 (AE001145) B. burgdorferi predicted 1087
1.60E-145 coding region BB0399 [Borrelia f527.aa
gi.vertline.2688310 (AE001145) B. burgdorferi predicted 1814
7.60E-249 coding region BB0398 [Borrelia f541.aa gi.vertline.508421
antigen P39 [Borrelia burgdorferi] 1706 5.40E-230
>gi.vertline.2688281 (AE001143) basic f541.aa
gi.vertline.1753225 BmpA protein [Borrelia burgdorferi] 1698
6.80E-229 f541.aa gnl.vertline.PID.vertline.e117- 2833 bmpA(p39,
ORF1) [Borrelia 1695 1.70E-228 burgdorferi] f541.aa
gnl.vertline.PID.vertline.e1172835 membrane protein A [Borrelia
1642 3.40E-221 burgdorferi] >gi.vertline.516592 membrane f541.aa
gnl.vertline.PID.vertline.e1172834 membrane protein A [Borrelia
1638 1.20E-220 burgdorferi] f541.aa
gnl.vertline.PID.vertline.e1172828 bmpA(p39, ORF1) [Borrelia 1551
1.00E-208 burgdorferi] f541.aa gnl.vertline.PID.vertline.- e1172829
membrane protein A [Borrelia afzelii] 1502 5.60E-202 f541.aa
gnl.vertline.PID.vertline.e1172831 membrane protein A [Borrelia
afzelii] 1499 1.40E-201 f541.aa gnl.vertline.PID.vertline.e1172837
membrane protein A [Borrelia garinii] 1496 3.70E-201 f541.aa
gnl.vertline.PID.vertline.e1172830 membrane protein A [Borrelia
afzelii] 1493 9.60E-201 f541.aa gnl.vertline.PID.vertline.e1172838
membrane protein A [Borrelia garinii] 1488 4.60E-200 f541.aa
gnl.vertline.PID.vertline.e237214 membrane protein A [Borrelia
garinii] 1216 1.20E-162 f541.aa gnl.vertline.PID.vertline.e237209
membrane protein A [Borrelia garinii] 1211 5.90E-162 f541.aa
gnl.vertline.PID.vertline.e237236 membrane protein A [Borrelia
garinii] 1098 2.00E-146 f541.aa gi.vertline.2688282 (AE001143)
basic membrane protein B 518 1.20E-123 (bmpB) [Borrelia
burgdorferi] f542.aa gi.vertline.508422 [Borrelia burgdorferi
immunodominant 711 1.70E-95 antigen P39 gene, complete f542.aa
gi.vertline.2688282 (AE001143) basic membrane protein B 711
1.70E-95
(bmpB) [Borrelia burgdorferi] f542.aa gi.vertline.551744 membrane
lipoprotein [Borrelia 708 8.60E-95 burgdorferi] f542.aa
gnl.vertline.PID.vertline.e1172836 bmpB(p39, ORF2) [Borrelia 699
8.20E-94 burgdorferi] f542.aa gnl.vertline.PID.vertline.e- 1172832
bmpB(p39, ORF2) [Borrelia afzelii] 634 1.00E-84 f542.aa
gnl.vertline.PID.vertline.e1172839 bmpB(p39, ORF2) [Borrelia
garinii] 613 9.20E-82 f542.aa gnl.vertline.PID.vertline.e237209
membrane protein A [Borrelia garinii] 153 1.70E-32 f542.aa
gnl.vertline.PID.vertline.e1172828 bmpA(p39, ORF1) [Borrelia 144
3.80E-32 burgdorferi] f542.aa gnl.vertline.PID.vertline.e237214
membrane protein A [Borrelia garinii] 153 2.00E-31 f542.aa
gi.vertline.1753225 BmpA protein [Borrelia burgdorferi] 155
2.80E-31 f542.aa gnl.vertline.PID.vertline.e1172833 bmpA(p39, ORF1)
[Borrelia 155 2.80E-31 burgdorferi] f542.aa gi.vertline.508421
antigen P39 [Borrelia burgdorferi] 155 2.80E-31
>gi.vertline.2688281 (AE001143) basic f542.aa
gnl.vertline.PID.vertline.e1172837 membrane protein A [Borrelia
garinii] 156 1.00E-30 f542.aa gnl.vertline.PID.vertline.e1172829
membrane protein A [Borrelia afzelii] 144 1.90E-30 f542.aa
gnl.vertline.PID.vertline.e1172830 membrane protein A [Borrelia
afzelii] 144 2.70E-30 f544.aa gi.vertline.2688284 (AE001143) Mg2+
transport protein 860 4.20E-119 (mgtE) [Borrelia burgdorferi]
f544.aa gi.vertline.1753228 MgtE [Borrelia burgdorferi] 855
2.20E-118 f544.aa gi.vertline.619724 MgtE [Bacillus firmus] 176
3.70E-37 >pir.vertline.I40201.vertline.I40201 mgtE protein -
Bacillus f544.aa gi.vertline.780282 extended ORF of mgtE gene; 182
1.30E-34 transcription from this start point is f544.aa
gnl.vertline.PID.vertline.e315479 unknown [Mycobacterium
tuberculosis] 183 4.50E-31 f544.aa
gnl.vertline.PID.vertline.d1018132 Mg2+ transporter [Synechocystis
sp. ] 165 4.60E-31 >pir.vertline.S77552.vertline.S77552 Mg2+
f544.aa gnl.vertline.PID.vertline.e1181529 (AJ002571) YkoK
[Bacillus subtilis] 142 2.30E-30
>gnl.vertline.PID.vertline.e1183350 similar f544.aa
gi.vertline.2621701 (AE000843) Mg2+ transporter 142 3.20E-21
[Methanobacterium thermoautotrophicum] f545.aa gi.vertline.2688284
(AE001143) Mg2+ transport protein 860 4.20E-119 (mgtE) [Borrelia
burgdorferi] f545.aa gi.vertline.1753228 MgtE [Borrelia
burgdorferi] 855 2.20E-118 f545.aa gi.vertline.619724 MgtE
[Bacillus firmus] 176 3.70E-37 >pir.vertline.I40201.vert-
line.I40201 mgtE protein - Bacillus f545.aa gi.vertline.780282
extended ORF of mgtE gene; 182 1.30E-34 transcription from this
start point is f545.aa gnl.vertline.PID.vertline.e315479 unknown
[Mycobacterium tuberculosis] 183 4.50E-31 f545.aa
gnl.vertline.PID.vertline.d1018132 Mg2+ transporter [Synechocystis
sp. ] 165 4.60E-31 >pir.vertline.S77552.vertline.S77552 Mg2+
f545.aa gnl.vertline.PID.vertline.e1181529 (AJ002571) YkoK
[Bacillus subtilis] 142 2.30E-30
>gnl.vertline.PID.vertline.e1183350 similar f545.aa
gi.vertline.2621701 (AE000843) Mg2+ transporter 142 3.20E-21
[Methanobacterium thermoautotrophicum] f561.aa gi.vertline.49245
lipoprotein [Borrelia burgdorferi] 1000 1.30E-132
>gi.vertline.2688271 (AE001142) lipoprotein f561.aa
gi.vertline.495738 P22 [Borrelia burgdorferi] 982 3.70E-130 f577.aa
gi.vertline.2688261 (AE001141) B. burgdorferi predicted 1930
4.00E-264 coding region BB0352 [Borrelia f584.aa
gi.vertline.2688246 (AE001140) B. burgdorferi predicted 1094
4.10E-147 coding region BB0346 [Borrelia f596.aa
gi.vertline.2688241 (AE001140) P26 [Borrelia burgdorferi] 1322
1.20E-180 >pir.vertline.G70141.vertline.G70141 P26 f596.aa
gi.vertline.2281465 (AF000366) P26 [Borrelia burgdorferi] 1010
5.90E-137 >gi.vertline.2281465 (AF000366) P26 f598.aa
gi.vertline.2281462 (AF000366) oligopeptide permease 652 1.20E-85
homolog D [Borrelia burgdorferi] f598.aa gi.vertline.143607
sporulation protein [Bacillus subtilis] 372 1.20E-45 f598.aa
gnl.vertline.PID.vertline.e1183166 oligopeptide ABC transporter
(ATP- 372 1.20E-45 binding protein) [Bacillus f598.aa
gi.vertline.1574676 oligopeptide transport ATP-binding 344 6.70E-42
protein (oppD) [Haemophilus f598.aa gi.vertline.677943 AppD
[Bacillus subtilis] 344 8.00E-42 >gnl.vertline.PID.vertline.e-
1183156 oligopeptide ABC f598.aa gi.vertline.1787051 (AE000185)
o612; 48 pct identical (33 346 2.50E-41 gaps) to 525 residues from
f598.aa gi.vertline.47346 AmiE protein [Streptococcus 338 1.10E-40
pneumoniae] >pir.vertline.S11152.vertline.S11152 amiE f598.aa
gi.vertline.47805 Opp D (AA1-335) [Salmonella 332 5.70E-40
typhimurium] >sp.vertline.P04285.vertline.O- PPD_SALTY f598.aa
pir.vertline.A03413.vertline.QREBOT oligopeptide transport protein
oppD - 332 5.70E-40 Salmonella typhimurium f598.aa
gi.vertline.1787499 (AE000223) oligopeptide transport 332 5.90E-40
ATP-binding protein OppD f598.aa gnl.vertline.PID.vertline.d1015494
Oligopeptide transport ATP-binding 332 5.90E-40 protein OppD.
[Escherichia f598.aa gi.vertline.495177 ATP binding protein
[Lactococcus 331 8.40E-40 lactis]
>sp.vertline.P50980.vertline.OPPD_LACLC f598.aa
gnl.vertline.PID.vertline.e187587 oligopeptidepermease
[Streptococcus 331 1.10E-39 pyogenes] f598.aa gi.vertline.308850
ATP binding protein [Lactococcus 329 1.60E-39 lactis]
>pir.vertline.A53290.vertline.A53290 f598.aa gi.vertline.2313399
(AE000548) dipeptide ABC transporter, 322 2.30E-39 ATP-binding
protein (dppD) f6-21.aa gi.vertline.2281468 (AF000948) OppAIV
[Borrelia 565 4.30E-73 burgdorferi] >gi.vertline.2689891
(AE000792) f6-21.aa gi.vertline.2253286 (AF005657) plasminogen
binding 315 1.20E-37 protein [Borrelia burgdorferi] f6-21.aa
gi.vertline.2688228 (AE001139) oligopeptide ABC 314 1.60E-37
transporter, periplasmic f6-21.aa gi.vertline.2809544 (AF043071)
oligopeptide permease 314 1.60E-37 periplasmic binding protein
f6-21.aa gi.vertline.2281457 (AF000366) oligopeptide permease 314
1.60E-37 homolog AI [Borrelia burgdorferi] f6-21.aa
gi.vertline.2688227 (AE001139) oligopeptide ABC 290 3.90E-34
transporter, periplasmic f6-21.aa gi.vertline.2281458 (AF000366)
oligopeptide permease 290 3.90E-34 homolog AII [Borrelia
burgdorferi] f6-21.aa gi.vertline.2281455 (AF000365) oligopeptide
permease 279 9.90E-34 homolog AV [Borrelia burgdorferi] f6-21.aa
gi.vertline.2690261 (AE000790) oligopeptide ABC 282 5.30E-33
transporter, periplasmic f6-21.aa gi.vertline.1616644 P30 [Borrelia
burgdorferi] 271 6.70E-32 f6-21.aa gi.vertline.2688226 (AE001139)
oligopeptide ABC 268 5.00E-31 transporter, periplasmic f6-21.aa
gi.vertline.2281459 (AF000366) oligopeptide permease 268 5.00E-31
homolog AIII [Borrelia f6-21.aa gi.vertline.2809546 (AF043071)
oligopeptide permease 268 5.00E-31 periplasmic binding protein
f6-21.aa bbs.vertline.161785 60 kda antigen [Borrelia coriaceae,
255 2.90E-30 C053, ATCC 4338, Peptide, 514 f6-21.aa
gi.vertline.2983834 (AE000740) transporter (extracellular 154
3.50E-14 solute binding protein family f6-27.aa gi.vertline.2689911
(AE000792) B. burgdorferi predicted 1773 7.30E-240 coding region
BBB09 [Borrelia f6-5.aa gi.vertline.2689905 (AE000792) B.
burgdorferi predicted 932 7.50E-126 coding region BBB27 [Borrelia
f600.aa gi.vertline.2281461 (AF000366) oligopeptide permease 731
1.40E-100 homolog C [Borrelia burgdorferi] f600.aa
gi.vertline.2688244 (AE001140) oligopeptide ABC 731 1.40E-100
transporter, permease protein (oppC-1) f600.aa gi.vertline.143606
sporulation protein [Bacillus subtilis] 372 5.00E-48
>pir.vertline.C38447.vertline.C38447 f600.aa gi.vertline.40007
OppC gene product [Bacillus subtilis] 372 5.00E-48
>gnl.vertline.PID.vertline.e1183165 oligopeptide f600.aa
gi.vertline.1574677 oligopeptide transport system permease 372
7.30E-48 protein (oppC)C [Haemophilus f600.aa gi.vertline.47804 Opp
C (AA1-301) [Salmonella 366 4.20E-47 typhimurium]
>pir.vertline.C29333.vertline.QREBOC f600.aa
gnl.vertline.PID.vertline.d1015493 Oligopeptide transport system
permease 366 4.20E-47 protein OppC. f600.aa
gnl.vertline.PID.vertline.e1181495 (AJ002571) DppC [Bacillus
subtilis] 267 1.70E-42 >gnl.vertline.PID.vertline.e1183314
f600.aa gi.vertline.1732315 transport system permease homolog 335
5.30E-42 [Listeria monocytogenes] f600.aa gi.vertline.580851 dciAC
[Bacillus subtilis] 258 1.50E-40
>sp.vertline.P26904.vertline.DPPC_BACSU DIPEPTIDE TRANSPORT
f600.aa gnl.vertline.PID.vertline.d1011164 oligopeptide transport
system permease 240 2.50E-39 protein [Synechocystis f600.aa
gi.vertline.677947 AppC [Bacillus subtilis] 236 2.80E-37
>gnl.vertline.PID.vertline.e1183160 oligopeptide ABC f600.aa
gi.vertline.1813497 dipeptide transporter protein dppC 281 1.20E-35
[Bacillus firmus] f600.aa sp.vertline.Q10623.vertline.Y021_MYCT- U
PUTATIVE PEPTIDE TRANSPORT 290 1.50E-35 PERMEASE PROTEIN CY373.01C.
f600.aa gi.vertline.1532201 BldKA [Streptomyces coelicolor] 291
1.60E-35 f603.aa gi.vertline.2281460 (AF000366) oligopeptide
permease 1522 5.80E-214 homolog B [Borrelia burgdorferi] f603.aa
gi.vertline.1574678 dipeptide transport system permease 392
1.30E-100 protein (dppB) [Haemophilus f603.aa
gnl.vertline.PID.vertline.e1183164 oligopeptide ABC transporter 374
3.40E-96 (permease) [Bacillus subtilis] f603.aa gi.vertline.580897
OppB gene product [Bacillus subtilis] 373 6.60E-96
>pir.vertline.S15231.vertline.B38447 f603.aa gi.vertline.47803
Opp B (AA1-306) [Salmonella 371 6.70E-96 typhimurium]
>pir.vertline.B29333.vertline.QREBOB f603.aa gi.vertline.1787497
(AE000223) oligopeptide transport 364 3.50E-95 system permease
protein OppB f603.aa gnl.vertline.PID.vertline.d- 1015492
Oligopeptide transport system permease 357 3.50E-94 protein OppB.
f603.aa gi.vertline.580850 dciAB [Bacillus subtilis] 350 9.10E-90
>gnl.vertline.PID.vertline.e1181494 (AJ002571) DppB f603.aa
gi.vertline.551726 sporulation protein [Bacillus subtilis] 374
2.40E-87 >gi.vertline.143605 sporulation f603.aa
gi.vertline.349226 transmembrane protein [Escherichia 293 9.60E-79
coli] >gi.vertline.466682 dppB f603.aa gi.vertline.1787053
(AE000185) o306; This 306 aa ORF is 284 3.80E-77 46 pct identical
(32 gaps) to f603.aa gi.vertline.972895 DppB [Haemophilus
influenzae] 301 2.50E-76 >gi.vertline.1574114 dipeptide
transport system f603.aa gi.vertline.2182646 (AE000098) Y4tP
[Rhizobium sp. 294 9.10E-74 NGR234] >sp.vertline.Q53191.-
vertline.Y4TP_RHISN f603.aa gi.vertline.2983140 (AE000692)
transporter (OppBC 169 2.30E-73 family) [Aquifex aeolicus] f603.aa
gi.vertline.677946 AppB [Bacillus subtilis] 218 8.70E-73
>gnl.vertline.PID.vertline.e1183159 oligopeptide ABC f604.aa
gi.vertline.2281459 (AF000366) oligopeptide permease 2818 0 homolog
AIII [Borrelia f604.aa gi.vertline.2809546 (AF043071) oligopeptide
permease 2818 0 periplasmic binding protein f604.aa
gi.vertline.2688226 (AE001139) oligopeptide ABC 2823 0 transporter,
periplasmic f604.aa gi.vertline.2688227 (AE001139) oligopeptide ABC
1738 1.40E-234 transporter, periplasmic f604.aa gi.vertline.2281458
(AF000366) oligopeptide permease 1731 1.30E-233 homolog AII
[Borrelia burgdorferi] f604.aa gi.vertline.2281468 (AF000948)
OppAIV [Borrelia 1675 3.60E-229 burgdorferi]
>gi.vertline.2689891 (AE000792) f604.aa gi.vertline.2688228
(AE001139) oligopeptide ABC 718 1.60E-204 transporter, periplasmic
f604.aa gi.vertline.2809544 (AF043071) oligopeptide permease 718
3.00E-204 periplasmic binding protein f604.aa gi.vertline.2253286
(AF005657) plasminogen binding 718 4.10E-204 protein [Borrelia
burgdorferi] f604.aa gi.vertline.2281457 (AF000366) oligopeptide
permease 714 2.00E-203 homolog AI [Borrelia burgdorferi] f604.aa
bbs.vertline.161785 60 kda antigen [Borrelia coriaceae, 704
1.20E-190 C053, ATCC 4338, Peptide, 514 f604.aa gi.vertline.2281455
(AF000365) oligopeptide permease 1402 1.80E-188 homolog AV
[Borrelia burgdorferi] f604.aa gi.vertline.2690261 (AE000790)
oligopeptide ABC 1400 3.40E-188 transporter, periplasmic f604.aa
gi.vertline.1616644 P30 [Borrelia burgdorferi] 858 4.90E-117
f604.aa gi.vertline.47802 Opp A (AA1-542) [Salmonella 296 9.00E-114
typhimurium] >gi.vertline.47808 precursor f606.aa
gi.vertline.2281458 (AF000366) oligopeptide permease 2762 0 homolog
AII [Borrelia burgdorferi] f606.aa gi.vertline.2688227 (AE001139)
oligopeptide ABC 2774 0 transporter, periplasmic f606.aa
gi.vertline.2281468 (AF000948) OppAIV [Borrelia 1817 6.50E-245
burgdorferi] >gi.vertline.2689891 (AE000792) f606.aa
gi.vertline.2809546 (AF043071) oligopeptide permease 1739 3.10E-234
periplasmic binding protein f606.aa gi.vertline.2688226 (AE001139)
oligopeptide ABC 1738 4.20E-234 transporter, periplasmic f606.aa
gi.vertline.2281459 (AF000366) oligopeptide permease 1733 2.00E-233
homolog AIII [Borrelia f606.aa bbs.vertline.161785 60 kda antigen
[Borrelia coriaceae, 762 1.70E-202 C053, ATCC 4338, Peptide, 514
f606.aa gi.vertline.2281455 (AF000365) oligopeptide permease 1456
1.80E-195 homolog AV [Borrelia burgdorferi] f606.aa
gi.vertline.2690261 (AE000790) oligopeptide ABC 1454 3.30E-195
transporter, periplasmic f606.aa gi.vertline.2253286 (AF005657)
plasminogen binding 751 2.00E-192 protein [Borrelia burgdorferi]
f606.aa gi.vertline.2688228 (AE001139) oligopeptide ABC 751
2.70E-192 transporter, periplasmic f606.aa gi.vertline.2809544
(AF043071) oligopeptide permease 751 6.90E-192 periplasmic binding
protein f606.aa gi.vertline.2281457 (AF000366) oligopeptide
permease 748 2.40E-191 homolog AI [Borrelia burgdorferi] f606.aa
gi.vertline.1616644 P30 [Borrelia burgdorferi] 1220 7.30E-163
f606.aa gi.vertline.47802 Opp A (AA1-542) [Salmonella 285 7.80E-106
typhimurium] >gi.vertline.47808 precursor f607.aa
gi.vertline.2281457 (AF000366) oligopeptide permease 2694 0 homolog
AI [Borrelia burgdorferi] f607.aa gi.vertline.2253286 (AF005657)
plasminogen binding 2706 0 protein [Borrelia burgdorferi] f607.aa
gi.vertline.2809544 (AF043071) oligopeptide permease 2708 0
periplasmic binding protein f607.aa gi.vertline.2688228 (AE001139)
oligopeptide ABC 2714 0 transporter, periplasmic f607.aa
bbs.vertline.161785 60 kda antigen [Borrelia coriaceae, 1272
3.80E-242 C053, ATCC 4338, Peptide, 514 f607.aa gi.vertline.2809546
(AF043071) oligopeptide permease 718 1.40E-204 periplasmic binding
protein f607.aa gi.vertline.2688226 (AE001139) oligopeptide ABC 718
3.60E-204 transporter, periplasmic f607.aa gi.vertline.2281459
(AF000366) oligopeptide permease 713 1.70E-203 homolog AIII
[Borrelia f607.aa gi.vertline.2688227 (AE001139) oligopeptide ABC
751 2.40E-192 transporter, periplasmic f607.aa gi.vertline.2281458
(AF000366) oligopeptide permease 751 4.50E-192 homolog AII
[Borrelia burgdorferi] f607.aa gi.vertline.2281468 (AF000948)
OppAIV [Borrelia 806 8.40E-189 burgdorferi] >gi.vertline.2689891
(AE000792) f607.aa gi.vertline.2690261 (AE000790) oligopeptide ABC
601 1.20E-144 transporter, periplasmic f607.aa gi.vertline.2281455
(AF000365) oligopeptide permease 600 1.60E-144 homolog AV [Borrelia
burgdorferi] f607.aa gi.vertline.1616644 P30 [Borrelia burgdorferi]
709 5.40E-103 f607.aa gi.vertline.47802 Opp A (AA1-542) [Salmonella
261 8.50E-69 typhimurium] >gi.vertline.47808 precursor f611.aa
gi.vertline.2688231 (AE001139) B. burgdorferi predicted 1907
1.10E-261 coding region BB0325 [Borrelia f617.aa
gi.vertline.2688213 (AE001138) conserved hypothetical 1574
2.70E-226 integral
membrane protein f617.aa gi.vertline.2649711 (AE001042) ribose ABC
transporter, 109 7.00E-12 permease protein (rbsC-1) f631.aa
gi.vertline.1165286 FtsW [Borrelia burgdorferi] 1820 4.00E-259
>gi.vertline.2688164 (AE001137) cell division f631.aa
gnl.vertline.PID.vertline.e229592 membrane protein [Borrelia 1815
2.10E-258 burgdorferi] >gnl.vertline.PID.vertline.e228289 ftsW
f631.aa gi.vertline.146039 cell division protein [Escherichia coli]
362 1.30E-60 >gi.vertline.40857 FtsW protein f631.aa
gi.vertline.580938 internal open reading frame (AA 1-290) 407
4.90E-55 [Bacillus subtilis] f631.aa
gnl.vertline.PID.vertline.e315953 FtsW [Mycobacterium tuberculosis]
412 5.40E-55 >sp.vertline.O06223.vertline.FTWH_MYCTU f631.aa
gi.vertline.580937 spoVE gene product (AA 1-366) 410 2.90E-53
[Bacillus subtilis] >gnl.vertline.PID.vertline.e1185111 f631.aa
gi.vertline.143657 endospore forming protein [Bacillus 405 1.20E-52
subtilis] f631.aa gnl.vertline.PID.vertline.d101- 9002
rod-shape-determining protein 358 3.10E-51 [Synechocystis sp. ]
f631.aa gnl.vertline.PID.vertline.e1287793 (AL022602) cell divisin
protein FtsW 396 6.70E-51 [Mycobacterium leprae] f631.aa
gi.vertline.1016213 strong sequence similarity to FtsW, 349
1.00E-50 RodA, and SpoV-E [Cyanophora f631.aa gi.vertline.1574692
cell division protein (ftsW) 304 4.20E-50 [Haemophilus influenzae]
f631.aa gnl.vertline.PID.vertline.e118507- 5 similar to
cell-division protein [Bacillus 281 1.80E-46 subtilis] f631.aa
gi.vertline.1469784 putative cell division protein ftsW 247
1.60E-38 [Enterococcus hirae] f631.aa gi.vertline.1572976 rod
shape-determining protein (mreB) 196 1.20E-37 [Haemophilus
influenzae] f631.aa gi.vertline.147695 rod-shape-determining
protein 194 5.00E-35 [Escherichia coli] >gi.vertline.1778551
f635.aa gi.vertline.1165282 orf7; Method: conceptual translation
1166 1.00E-156 supplied by author [Borrelia f635.aa
gi.vertline.1448949 ORF 224; The predicted gene product 621
2.80E-125 showed weak homology with the f647.aa gi.vertline.2688180
(AE001137) flagellar protein (flbB) 1032 1.00E-140 [Borrelia
burgdorferi] f647.aa gi.vertline.1196323 putative [Borrelia
burgdorferi] 1031 1.50E-140 f647.aa gi.vertline.1165270 orf19;
Method: conceptual translation 1019 7.10E-139 supplied by author
[Borrelia f647.aa gi.vertline.2108242 22.5 K protein [Treponema
pallidum] 200 4.70E-24 f65.aa gi.vertline.2688737 (AE001178) B.
burgdorferi predicted 1095 8.10E-148 coding region BB0792 [Borrelia
f653.aa gi.vertline.1165265 MotB [Borrelia burgdorferi] 1220
1.70E-164 >gi.vertline.1185054 flagellar motor apparatus f653.aa
gi.vertline.1399286 MotB [Treponema phagedenis] 168 5.80E-57
f653.aa gi.vertline.2196896 MotB [Treponema pallidum] 179 1.30E-49
f664.aa gi.vertline.1185062 flagellar export protein [Borrelia 1430
1.90E-199 burgdorferi] f664.aa gi.vertline.1165257 FlhB [Borrelia
burgdorferi] 1430 1.90E-199 >gi.vertline.2688194 (AE001137)
flagellar f664.aa gi.vertline.1216382 FlhB' [Treponema pallidum]
272 5.30E-64 >pir.vertline.PC4115.vertline.PC4115 flagellar
protein f664.aa gi.vertline.395390 flagellar biosynthetic protein
[Bacillus 433 1.30E-61 subtilis] f664.aa
gnl.vertline.PID.vertline.e1185229 flagella-associated protein
[Bacillus 433 1.30E-61 subtilis] f664.aa gi.vertline.1147737 third
gene in fliQ operon; membrane 353 1.70E-46 protein homolog
[Caulobacter f664.aa gi.vertline.2313898 (AE000589) flagellar
biosynthetic 203 1.20E-44 protein (flhB) [Helicobacter f664.aa
gi.vertline.2984250 (AE000768) flagellar biosynthetic 319 3.00E-44
protein FlhB [Aquifex aeolicus] f664.aa gi.vertline.2459702 FlhB
[Agrobacterium tumefaciens] 347 6.20E-44 f664.aa gi.vertline.793892
flhB [Yersinia enterocolitica] 330 1.30E-39
>pir.vertline.S54213.vertline.S54213 flhB protein - f664.aa
gnl.vertline.PID.vertline.d1016420 Flagellar biosynthetic protein
FlhB. 325 2.20E-39 [Escherichia coli] f664.aa gi.vertline.475126
yscU [Yersinia pseudotuberculosis] 309 9.80E-38
>gi.vertline.2996233 (AF053946) Yop f664.aa gi.vertline.497216
YscU [Yersinia enterocolitica] 308 1.40E-37 f664.aa
gnl.vertline.PID.vertline.d1007477 flagellar protein FlhB
[Salmonella 312 2.10E-37 typhimurium] f664.aa
gnl.vertline.PID.vertline.e283684 secretion system apparatus, SsaU
312 8.20E-37 [Salmonella typhimurium] f679.aa gi.vertline.2688158
(AE001136) B. burgdorferi predicted 3714 0 coding region BB0259
[Borrelia f679.aa gnl.vertline.PID.vertline.d- 1011473 soluble
lytic transglycosylase 180 1.10E-25 [Synechocystis sp. ] f679.aa
gnl.vertline.PID.vertline.e1183177 similar to lytic
transglycosylase 108 2.10E-22 [Bacillus subtilis] f679.aa
gi.vertline.2984090 (AE000756) hypothetical protein 111 9.30E-17
[Aquifex aeolicus] f680.aa gi.vertline.2688153 (AE001136)
bacitracin resistance 769 3.90E-109 protein (bacA) [Borrelia
f680.aa gnl.vertline.PID.vertline.e1185- 988 similar to bacitracin
resistance protein 174 7.30E-18 (undecaprenol f680.aa
gi.vertline.2622542 (AE000905) bacitracin resistance 116 3.30E-16
protein [Methanobacterium f680.aa gi.vertline.2984378 (AE000777)
undecaprenol kinase 152 3.90E-15 [Aquifex aeolicus] f680.aa
gi.vertline.882579 CG Site No. 29739 [Escherichia coli] 139
2.60E-12 >gi.vertline.1789437 (AE000387) f688.aa
gi.vertline.2688146 (AE001135) conserved hypothetical 2497 0
integral membrane protein f688.aa gi.vertline.2649351 (AE001019)
conserved hypothetical 110 3.70E-18 protein [Archaeoglobus
fulgidus] f688.aa gi.vertline.1592186 M. jannaschii predicted
coding region 174 1.10E-16 MJ1562 [Methanococcus f7-30.aa
gi.vertline.2690009 (AE000786) conserved hypothetical 682 1.90E-90
protein [Borrelia burgdorferi] f704.aa gi.vertline.2688137
(AE001134) glycerol uptake facilitator 1307 4.70E-181 (glpF)
[Borrelia f704.aa gi.vertline.142997 glycerol uptake facilitator
[Bacillus 191 1.50E-50 subtilis]
>gnl.vertline.PID.vertline.e1182917 f704.aa gi.vertline.521003
C01G6.1 [Caenorhabditis elegans] 152 1.60E-50 f704.aa
gi.vertline.529582 water channel protein [Rattus 142 5.80E-50
norvegicus] >pir.vertline.I59266.vertline.I59266 water f704.aa
dbjl.vertline.AB000507_1 (AB000507) aquaporin 7 [Rattus 155
1.30E-49 norvegicus] f704.aa pir.vertline.A57119.vertline.A57119
aquaporin 3 - human 149 4.20E-44 f704.aa gi.vertline.1109920 coded
for by C. elegans cDNA 168 9.30E-44 cm16b11; strong similarity to
MIP f704.aa gnl.vertline.PID.vertline.d1019987 (AB001325) aquaporin
3 [Homo 148 5.30E-43 sapiens] >sp.vertline.Q92482.v-
ertline.AQP3_HUMAN f704.aa gnl.vertline.PID.vertline.d1025786
(AB008775) aquaporin 9 [Homo 144 1.40E-42 sapiens] f704.aa
gi.vertline.146188 glycerol diffusion facilitator 146 1.30E-40
[Escherichia coli] >gi.vertline.305030 CG Site f704.aa
gi.vertline.1065485 strong similarity to the MIP family of 179
1.40E-39 transmembrane channel f704.aa
sp.vertline.P31140.vertline.G- LPF_SHIFL GLYCEROL UPTAKE 146
3.30E-39 FACILITATOR PROTEIN. f704.aa gi.vertline.2587035
(AF026270) PduF [Salmonella 168 7.30E-39 typhimurium]
>sp.vertline.P37451.vertline.PDUF_SALTY f704.aa
gi.vertline.1399489 glycerol dfiffusion facilitator 154 7.90E-39
[Pseudomonas aeruginosa] f704.aa gi.vertline.2649144 (AE001005)
glycerol uptake facilitator, 150 1.30E-38 MIP channel (glpF)
f707.aa gi.vertline.2688143 (AE001134) B. burgdorferi predicted
1300 3.90E-176 coding region BB0238 [Borrelia f709.aa
gi.vertline.2688131 (AE001133) B. burgdorferi predicted 3437 0
coding region BB0236 [Borrelia f730.aa gi.vertline.2688111
(AE001132) gufA protein [Borrelia 1376 3.00E-192 burgdorferi]
>pir.vertline.C70127.vertline.C70127 f730.aa gi.vertline.1707057
coded for by C. elegans cDNA 235 2.80E-83 CEESS55F; coded for by C.
elegans cDNA f730.aa gi.vertline.2621542 (AE000831) conserved
protein 259 1.10E-74 [Methanobacterium thermoautotrophicum] f730.aa
gnl.vertline.PID.vertline.e183440 gufA gene product [Myxococcus 175
2.30E-35 xanthus] >gi.vertline.49253 orfX gene f730.aa
gi.vertline.2984109 (AE000757) hypothetical protein 171 7.00E-28
[Aquifex aeolicus] f736.aa gi.vertline.2688115 (AE001132) phosphate
ABC 1403 2.10E-186 transporter, periplasmic phosphate- binding
f736.aa gi.vertline.2622858 (AE000929) phosphate-binding protein
151 4.40E-30 PstS [Methanobacterium f736.aa gi.vertline.2622859
(AE000929) phosphate-binding protein 145 2.80E-24 PstS homolog
[Methanobacterium f736.aa gnl.vertline.PID.vertline.d1010224 ORF108
[Bacillus subtilis] 120 1.20E-11
>gnl.vertline.PID.vertline.e1185766 alternate gene f739.aa
gi.vertline.2688119 (AE001132) B. burgdorferi predicted 1139
1.10E-156 coding region BB0213 [Borrelia f742.aa
gi.vertline.2688100 (AE001131) surface-located membrane 5654 0
protein 1 (lmp1) [Borrelia f742.aa gi.vertline.2621120 (AE000799)
O-linked GlcNAc 200 9.30E-22 transferase [Methanobacterium f742.aa
gi.vertline.2621106 (AE000798) O-linked GlcNAc 180 5.80E-17
transferase [Methanobacterium f742.aa pir.vertline.E69190.vert-
line.E69190 conserved hypothetical protein MTH68 - 154 1.60E-14
Methanobacterium f742.aa gi.vertline.1591608 transformation
sensitive protein 109 9.90E-14 [Methanococcus jannaschii] f742.aa
gi.vertline.1589778 SPINDLY [Arabidopsis thaliana] 101 1.40E-13
f742.aa gi.vertline.2984175 (AE000762) hypothetical protein 132
7.30E-13 [Aquifex aeolicus] f742.aa gi.vertline.3037137 (AF056198)
Hsp70/Hsp90 organizing 105 5.40E-11 protein homolog [Drosophila
f743.aa gi.vertline.2688104 (AE001131) B. burgdorferi predicted
1299 1.70E-174 coding region BB0209 [Borrelia f748.aa
gi.vertline.2688089 (AE001130) Lambda CII stability- 1615 5.10E-220
governing protein (hflC) [Borrelia f748.aa gi.vertline.436158
putative integral membrane protease 191 4.80E-35 required for high
frequency f748.aa gi.vertline.1573107 Lambda CII
stability-governing protein 193 4.90E-33 (hflC) [Haemophilus
f748.aa gi.vertline.507735 HflC [Vibrio parahaemolyticus] 212
6.10E-26 >sp.vertline.P40606.vertline.HFLC_VIBPA HFLC PROTEIN
f752.aa gi.vertline.2688092 (AE001130) 2585 0 f752.aa
gi.vertline.2984050 (AE000754) UDP-MurNac-tripeptide 202 9.10E-74
synthetase [Aquifex aeolicus] f752.aa gi.vertline.40162 murE gene
product [Bacillus subtilis] 157 6.40E-70
>gnl.vertline.PID.vertline.e1185108 f752.aa
gnl.vertline.PID.vertline.d1011466 UDP-MurNac-tripeptide synthetase
166 5.20E-57 [Synechocystis sp. ] f752.aa
gnl.vertline.PID.vertline.e307808 UDP-MurNAc-tripeptide synthetase
108 2.30E-51 [Rickettsia prowazekii] f752.aa gi.vertline.1574688
UDP-MurNac-tripeptide synthetase 166 3.20E-50 (murE) [Haemophilus
influenzae] f752.aa gnl.vertline.PID.vertline- .e1287797 (AL022602)
udp-n- 183 3.20E-50 acetylmuramoylalanyl-d-g- lutamate f752.aa
gnl.vertline.PID.vertline.e316022 MurE [Mycobacterium tuberculosis]
181 4.10E-46 f752.aa gi.vertline.581032 UDP-MurNac-tripeptide
synthetase 175 1.30E-41 (MurE) [Escherichia coli] f752.aa
gi.vertline.2177098 UDP-MurNAc-Dipeptide: meso- 172 3.70E-41
diaminopimelate ligase [Escherichia f752.aa gi.vertline.2314673
(AE000648) UDP-MurNac-tripeptide 137 9.80E-41 synthetase (murE)
[Helicobacter f752.aa gi.vertline.840843 UDP-N-acetylmuramoylalany-
l-D- 135 1.70E-20 glutamate-2,6-diaminopimelate ligase f76-1.aa
gi.vertline.1209837 lipoprotein [Borrelia burgdorferi] 395 2.80E-49
f76-1.aa gi.vertline.1209873 lipoprotein [Borrelia burgdorferi] 250
7.00E-37 f76-1.aa gi.vertline.1209843 lipoprotein [Borrelia
burgdorferi] 267 7.30E-32 f76-1.aa gi.vertline.2121280 (AF000270)
lipoprotein [Borrelia 258 1.20E-30 burgdorferi]
>gi.vertline.3095109 f76-1.aa gnl.vertline.PID.vertline.e268244
surface-exposed lipoprotein [Borrelia 116 2.40E-18 afzelii]
f76-1.aa gi.vertline.1209849 lipoprotein [Borrelia burgdorferi] 146
8.30E-17 f76-1.aa gi.vertline.3095105 (AF046998) 2.9-8 lipoprotein
[Borrelia 148 5.80E-14 burgdorferi] f76-1.aa gi.vertline.3095107
(AF046999) 2.9-9 lipoprotein [Borrelia 127 7.20E-11 burgdorferi]
f764.aa gi.vertline.2688084 (AE001129) B. burgdorferi predicted
1218 1.20E-164 coding region BB0193 [Borrelia f770.aa
gi.vertline.2688077 (AE001129) conserved hypothetical 646 7.60E-87
protein [Borrelia burgdorferi] f790.aa gi.vertline.2688065
(AE001128) outer membrane protein 2013 2.50E-271 (tpn50) [Borrelia
burgdorferi] f790.aa gi.vertline.458015 TpN50 precursor [Treponema
pallidum] 134 4.30E-33 f790.aa
sp.vertline.P38369.vertline.TP50_TREPA OUTER MEMBRANE PROTEIN 134
4.30E-33 TPN50 PRECURSOR. f790.aa gi.vertline.532658 antigen
[Treponema pallidum] 139 4.30E-31
>pir.vertline.S61867.vertline.S61867 antigen tpp57 - f792.aa
gi.vertline.2688052 (AE001127) B. burgdorferi predicted 3185 0
coding region BB0165 [Borrelia f797.aa gi.vertline.2688056
(AE001127) B. burgdorferi predicted 1116 5.30E-148 coding region
BB0159 [Borrelia f798.aa gi.vertline.2688051 (AE001127) antigen,
S2, putative 1223 9.70E-164 [Borrelia burgdorferi] f798.aa
gi.vertline.1063419 S2 gene product [Borrelia burgdorferi] 116
4.70E-23 f798.aa gi.vertline.2690227 (AE000790) antigen, S2
[Borrelia 116 1.50E-22 burgdorferi] >pir.vertline.D70207.vert-
line.D70207 f798.aa gi.vertline.2690128 (AE000788) protein p23
[Borrelia 110 1.40E-19 burgdorferi] >pir.vertline.C70257.vert-
line.C70257 f798.aa gi.vertline.2689956 (AE000785) protein p23
[Borrelia 104 2.70E-15 burgdorferi] >pir.vertline.D70225.vert-
line.D70225 f799.aa gi.vertline.2688043 (AE001126) B. burgdorferi
predicted 632 1.40E-83 coding region BB0156 [Borrelia f8-10.aa
gi.vertline.2690052 (AE000784) antigen, P35, putative 1241
1.10E-167 [Borrelia burgdorferi] f8-10.aa gi.vertline.2689955
(AE000785) antigen, P35, putative 298 1.70E-57 [Borrelia
burgdorferi] f8-10.aa gi.vertline.2690120 (AE000789) B. burgdorferi
predicted 254 3.80E-54 coding region BBI34 [Borrelia f8-10.aa
gi.vertline.2690100 (AE000789) B. burgdorferi predicted 182
2.90E-31 coding region BBI16 [Borrelia f8-10.aa gi.vertline.2690207
(AE000787) B. burgdorferi predicted 196 1.50E-20 coding region
BBJ02 [Borrelia f8-10.aa gi.vertline.2690116 (AE000789) B.
burgdorferi predicted 192 5.50E-20 coding region BBI29 [Borrelia
f8-10.aa gi.vertline.2690125 (AE000788) antigen, P35, putative 129
5.80E-14 [Borrelia burgdorferi] f8-10.aa gi.vertline.2690206
(AE000787) B. burgdorferi predicted 103 1.10E-13 coding region
BBJ01 [Borrelia f8-10.aa gi.vertline.2690099 (AE000789) B.
burgdorferi predicted 142 8.50E-13 coding region BBI15 [Borrelia
f8-10.aa gi.vertline.2690115 (AE000789) B. burgdorferi predicted
130 3.30E-12 coding region BBI28 [Borrelia f8-14.aa
gi.vertline.2690074 (AE000784) B. burgdorferi predicted 1560
2.60E-206 coding region BBH37 [Borrelia f8-14.aa
gi.vertline.2690188 (AE000787) B. burgdorferi predicted 599
3.50E-123 coding region BBJ08 [Borrelia f8-14.aa
gi.vertline.2690030 (AE000786) B. burgdorferi predicted 337
4.40E-106 coding region BBG01 [Borrelia f8-14.aa
gi.vertline.2690139 (AE000788) B. burgdorferi predicted 173
8.00E-91 coding region BBK01 [Borrelia f8.aa gi.vertline.2688783
(AE001182) B. burgdorferi predicted 2765 0 coding region BB0840
[Borrelia f8.aa gi.vertline.2697112 (AF008219) unknown [Borrelia
afzelii] 1494 2.80E-205 f800.aa gi.vertline.2688044 (AE001126) B.
burgdorferi predicted 1936 1.00E-262 coding region BB0155 [Borrelia
f805.aa gi.vertline.2688039 (AE001126) N-acetylglucosamine-6- 641
6.30E-85 phosphate deacetylase (nagA) f810.aa gi.vertline.2688024
(AE001125) glycine
betaine, L-proline 1527 4.20E-207 ABC transporter, f810.aa
gi.vertline.984805 glycine betaine-binding protein 179 6.80E-21
precursor [Bacillus subtilis] f810.aa gi.vertline.1850605 ProX
[Streptococcus mutans] 181 2.30E-18 f814.aa
pir.vertline.D70117.vertline.D70117 acriflavine resistance protein
(acrB) 5105 0 homolog - Lyme disease f814.aa gi.vertline.2688027
(AE001125) acriflavine resistance 5111 0 protein (acrB) [Borrelia
f814.aa gi.vertline.2983346 (AE000707) cation efflux 325 4.80E-119
(AcrB/AcrD/AcrF family) [Aquifex aeolicus] f814.aa
gi.vertline.2313726 (AE000574) acriflavine resistance 327 4.50E-111
protein (acrB) [Helicobacter f814.aa gi.vertline.3068786 (AF059041)
RND pump protein 297 1.70E-110 [Helicobacter pylori] f814.aa
gnl.vertline.PID.vertline.e1182651 similar to acriflavin resistance
protein 257 8.90E-100 [Bacillus subtilis] f814.aa
gi.vertline.1573914 acriflavine resistance protein (acrB) 294
2.10E-97 [Haemophilus influenzae] f814.aa
gnl.vertline.PID.vertline.e256815 mexF [Pseudomonas aeruginosa] 300
2.00E-88 f814.aa gnl.vertline.PID.vertline.d1019295 cation efflux
system protein CzcA 198 1.30E-87 [Synechocystis sp. ] f814.aa
gnl.vertline.PID.vertline.e285274 membrane-bound cation-proton- 283
2.20E-87 antiporter [Ralstonia eutropha] f814.aa gi.vertline.438854
envD homologue; ORFB 290 6.50E-87 [Pseudomonas aeruginosa]
>pir.vertline.S39630.vertline.S39630 f814.aa
gnl.vertline.PID.vertline.d1011721 CzcA [Alcaligenes sp. ] 275
8.20E-87 >pir.vertline.JC4700.vertline.JC4700 cadmium, zinc,
f814.aa gi.vertline.2314107 (AE000605) cation efflux system 266
2.30E-86 protein (czcA) [Helicobacter f814.aa
pir.vertline.A33830.vertline.A33830 cation efflux system membrane
protein 275 3.10E-86 czcA - Alcaligenes f814.aa
gnl.vertline.PID.vertline.d1017073 envD gene product homolog 283
8.30E-86 [Escherichia coli] >gi.vertline.1788814 f818.aa
gi.vertline.2688032 (AE001125) B. burgdorferi predicted 664
3.00E-87 coding region BB0139 [Borrelia f82.aa gi.vertline.2688729
(AE001177) B. burgdorferi predicted 991 2.20E-132 coding region
BB0776 [Borrelia f820.aa gi.vertline.2688029 (AE001125)
penicillin-binding protein 3171 0 (pbp-1) [Borrelia f820.aa
gi.vertline.580936 SpoVD [Bacillus subtilis] 149 3.00E-49
>gnl.vertline.PID.vertline.e1185107 penicillin-binding f820.aa
gi.vertline.150283 penicillin-binding protein 2 [Neisseria 154
6.90E-43 meningitidis] f820.aa gnl.vertline.PID.vertline.e1287798
(AL022602) penicillin binding protein 2 182 4.20E-42 [Mycobacterium
f820.aa gi.vertline.509190 penicillin-binding protein 2 [Neisseria
158 1.70E-41 meningitidis] f820.aa gi.vertline.509118
penicillin-binding protein 2 [Neisseria 151 7.10E-41 meningitidis]
f820.aa gi.vertline.840842 penicillin-binding protein 3 177
1.20E-40 [Pseudomonas aeruginosa] f820.aa gi.vertline.509065
penicillin-binding protein 2 [Neisseria 152 1.40E-40 meningitidis]
f820.aa gi.vertline.509043 penicillin-binding protein 2 [Neisseria
150 2.70E-40 meningitidis] f820.aa gi.vertline.509159
penicillin-binding protein 2 [Neisseria 147 2.80E-40 meningitidis]
f820.aa gi.vertline.509120 penicillin-binding protein 2 [Neisseria
155 1.60E-39 meningitidis] f820.aa gi.vertline.509157
penicillin-binding protein 2 [Neisseria 155 1.60E-39 meningitidis]
f820.aa gi.vertline.509126 penicillin-binding protein 2 [Neisseria
158 1.70E-39 meningitidis] f820.aa gi.vertline.45178
penicillin-binding protein 2 (AA 1- 155 2.30E-38 581) [Neisseria
meningitidis] f820.aa gi.vertline.150279 penicillin binding protein
2 [Neisseria 154 8.70E-38 gonorrhoeae] f831.aa gi.vertline.2688018
(AE001124) B. burgdorferi predicted 994 1.20E-133 coding region
BB0126 [Borrelia f843.aa gi.vertline.2688014 (AE001124) PTS system,
maltose and 2590 0 glucose-specific IIABC component f843.aa
gi.vertline.2688579 (AE001166) PTS system, glucose- 594 1.80E-129
specific IIBC component (ptsG) f843.aa gi.vertline.1072418 glcA
[Staphylococcus carnosus] 283 1.00E-72
>pir.vertline.S46952.vertline.S46952 f843.aa gi.vertline.1072419
glcB [Staphylococcus carnosus] 248 1.00E-66
>pir.vertline.S63606.vertline.S46953 f843.aa
dbj.parallel.D86417_11 YflF [Bacillus subtilis] 215 7.90E-65
>gnl.vertline.PID.vertline.e1182760 similar to f843.aa
gi.vertline.2197104 (AF003742) MalX homolog 182 8.90E-64
[Escherichia coli] f843.aa gi.vertline.43819 nagE gene product
[Klebsiella 264 8.50E-63 pneumoniae] >pir.vertline.S18607.ver-
tline.S18607 f843.aa gi.vertline.146913 N-acetylglucosamine
transport protein 256 1.10E-62 [Escherichia coli] f843.aa
gi.vertline.39956 IIGlc [Bacillus subtilis] 315 5.20E-62
>gnl.vertline.PID.vertline.e1184979 phosphotransferase system
f843.aa dbj.parallel.D87820_1 NagE [Vibrio cholerae non-O1] 263
3.80E-61 >pir.vertline.JC5651.vertline.JC5651 f843.aa
gi.vertline.2689888 (AE000792) PTS system, maltose and 198 1.10E-60
glucose-specific IIABC component f843.aa gi.vertline.397363 enzyme
II-glc [Salmonella 227 1.20E-58 typhimurium]
>pir.vertline.S36620.vertline.S36620 f843.aa gi.vertline.147393
glucose-specific enzyme II of 226 3.90E-57 phosphotransferase
system [Escherichia f843.aa gnl.vertline.PID.vertline.e1182187
alternate gene name: yzfA; similar to 180 9.00E-56
phosphotransferase f843.aa gi.vertline.1732194 PTS permease for
glucose [Vibrio 349 4.30E-50 furnissii] f850.aa gi.vertline.2687999
(AE001123) B. burgdorferi predicted 2374 0 coding region BB0110
[Borrelia f853.aa gi.vertline.2687994 (AE001123) basic membrane
protein 1672 2.20E-224 [Borrelia burgdorferi] f853.aa
gi.vertline.155055 basic membrane protein precursor 130 3.60E-24
[Treponema pallidum] f859.aa gi.vertline.2688002 (AE001123) B.
burgdorferi predicted 888 1.80E-115 coding region BB0102 [Borrelia
f86.aa gi.vertline.2688725 (AE001177) flagellar P-ring protein 1647
1.50E-217 (flgI) [Borrelia burgdorferi] f86.aa gi.vertline.2920802
(AF019213) FlgI [Vibrio cholerae] 143 3.50E-14 f86.aa
gi.vertline.405550 flagellar P-ring protein [Pseudomonas 102
3.70E-13 putida] >sp.vertline.Q52082.vertline.FLGI_PSEPU f86.aa
gi.vertline.144241 flagellin [Caulobacter crescentus] 110 6.70E-13
>pir.vertline.A41891.vertline.A41891 basal body f860.aa
gi.vertline.2687998 (AE001123) asparaginyl-tRNA 1110 2.40E-149
synthetase (asnS) [Borrelia f860.aa gi.vertline.1574761
asparaginyl-tRNA synthetase (asnS) 634 1.30E-83 [Haemophilus
influenzae] f860.aa gi.vertline.147935 asparaginyl-tRNA synthetase
(asnS) 622 6.10E-82 [Escherichia coli] >gi.vertline.41000
f860.aa gnl.vertline.PID.vertline.e1202698 (AJ222644)
asparaginyl-tRNA 404 2.40E-80 synthetase [Arabidopsis thaliana]
f860.aa gnl.vertline.PID.vertline.d1011495 asparaginyl-tRNA
synthetase 618 4.50E-80 [Synechocystis sp. ] f860.aa
gi.vertline.530408 Asn-tRNA synthetase [Mycoplasma 439 1.60E-65
capricolum] >pir.vertline.S77842.vertline.S77842 f860.aa
gi.vertline.1045792 asparaginyl-tRNA synthetase 365 2.20E-62
[Mycoplasma genitalium] f860.aa gi.vertline.1674281 (AE000057)
Mycoplasma pneumoniae, 338 3.10E-61 asparaginyl-tRNA synthetase;
f860.aa gnl.vertline.PID.vertline.e1202700 (AJ222645)
asparaginyl-tRNA 364 3.90E-59 synthetase [Arabidopsis thaliana]
f860.aa gnl.vertline.PID.vertline.e264488 YCR024c, len: 492
[Saccharomyces 150 3.90E-47 cerevisiae] >pir.vertline.S19435.-
vertline.S19435 f860.aa gnl.vertline.PID.vertline.e254305
asparaginyl-tRNA synthetase 370 1.70E-46 [Salmonella typhi] f860.aa
gnl.vertline.PID.vertline.e188505 asparagine-tRNA ligase
[Lactobacillus 224 1.30E-44 delbrueckii] f860.aa
pir.vertline.S71072.vertline.S71072 asparagine-tRNA ligase (EC
6.1.1.22) 224 1.30E-44 asnS1 - Lactobacillus f860.aa
gnl.vertline.PID.vertline.e188572 asparagine-tRNA ligase
[Lactobacillus 224 2.40E-44 delbrueckii] f860.aa
gi.vertline.1146247 asparaginyl-tRNA synthetase [Bacillus 234
6.10E-44 subtilis] >gnl.vertline.PID.vertline.e1183681 f861.aa
gi.vertline.2687975 (AE001122) glutamate racemase (murI) 1354
2.90E-186 [Borrelia burgdorferi] f861.aa gi.vertline.396314
glutamate synthase [Escherichia coli] 168 1.20E-16
>gi.vertline.290428 glutamate synthase f861.aa
gnl.vertline.PID.vertline.e1165353 glutamate racemase [Bacillus
subtilis] 120 1.80E-13 >gnl.vertline.PID.vertline.e1184088
f861.aa pir.vertline.JC5587.vertline.JC5587 glutamate racemase (EC
5.1.1.3) - 122 1.80E-13 Bacillus pumilus f861.aa
sp.vertline.P52973.vert- line.MURI_HAEIN PROBABLE GLUTAMATE 114
8.10E-13 RACEMASE (EC 5.1.1.3). f867.aa gi.vertline.2687979
(AE001122) V-type ATPase, subunit A 2826 0 (atpA) [Borrelia
burgdorferi] f867.aa pir.vertline.JC5532.vertline.JC5532
vacuolar-type ATPase (EC 3.-.-.-) A 594 2.20E-162 chain
-Desulfurococcus f867.aa gi.vertline.2104726 V-ATPase A subunit
[Desulfurococcus 594 3.10E-162 sp. SY] f867.aa gi.vertline.2605627
ATPase alpha subunit [Thermococcus 592 7.10E-161 sp.] f867.aa
gnl.vertline.PID.vertline.d1003475 Na+-ATPase alpha subunit 601
1.60E-153 [Enterococcus hirae] f867.aa gi.vertline.1590955
H+-transporting ATP synthase, subunit 585 6.00E-147 A (atpA)
[Methanococcus f867.aa gi.vertline.496904 membrane ATPase
[Haloferax volcanii] 728 6.00E-147 >pir.vertline.S55895.vertl-
ine.S45144 f867.aa gi.vertline.152927 ATPase alpha subunit
[Sulfolobus 548 5.00E-163 acidocaldarius]
>pir.vertline.A28652.vertline.A28652 f867.aa gi.vertline.2649416
(AE001023) H+-transporting ATP 748 2.00E-146 synthase, subunit A
(atpA) f867.aa gi.vertline.2622052 (AE000869) ATP synthase, subunit
A 607 9.40E-146 [Methanobacterium f867.aa gi.vertline.168926
vacuolar ATPase vma-1 [Neurospora 302 9.00E-145 crassa]
>pir.vertline.A30799.vertline.PXNCV7 f867.aa gi.vertline.149820
ATPase alpha subunit [Methanosarcina 743 1.40E-143 barkeri]
>pir.vertline.A34283.vertline.A34283 f867.aa gi.vertline.160736
vacuolar ATPase [Plasmodium 305 9.40E-140 falciparum]
>pir.vertline.A48582.vertline.A48582 vacuolar f867.aa
gnl.vertline.PID.vertline.d1009732 adenosine triphosphatase A
subunit 307 9.00E-137 [Acetabularia acetabulum] f867.aa
gi.vertline.49048 ATPase alpha-subunit [Thermus 684 4.80E-136
aquaticus thermophilus] f868.aa gi.vertline.2687980 (AE001122)
V-type ATPase, subunit B 2205 1.80E-298 (atpB) [Borrelia
burgdorferi] f868.aa gi.vertline.1590954 H+-transporting ATP
synthase, subunit 156 2.00E-114 B (atpB) [Methanococcus f868.aa
gi.vertline.2605628 ATPase beta subunit [Thermococcus 151 3.30E-108
sp.] f868.aa gi.vertline.2104727 V-ATPase B subunit
[Desulfurococcus 151 1.10E-107 sp. SY] f868.aa gi.vertline.43641
ATP synthase subunit [Halobacterium 150 1.80E-107 salinarium]
>pir.vertline.S14733- .vertline.S14733 f868.aa
gi.vertline.149821 ATPase beta subunit [Methanosarcina 172
1.00E-105 barkeri] >pir.vertline.B34283.v- ertline.B34283
f868.aa gnl.vertline.PID.vertline.d1003476 Na+-ATPase beta subunit
151 1.40E-105 [Enterococcus hirae] f868.aa gi.vertline.2649415
(AE001023) H+-transporting ATP 151 1.70E-103 synthase, subunit B
(atpB) f868.aa gi.vertline.496905 membrane ATPase [Haloferax
volcanii] 153 5.80E-103 >pir.vertline.S55896.vertline.S45145
f868.aa gi.vertline.1199639 A1AO H+ ATPase, subunit B 173 2.20E-102
[Methanosarcina mazeii] f868.aa gi.vertline.2622051 (AE000869) ATP
synthase, subunit B 155 1.00E-101 [Methanobacterium f868.aa
gnl.vertline.PID.vertline.d1009734 adenosine triphosphatase B
subunit 159 1.30E-101 [Acetabularia acetabulum] f868.aa
gi.vertline.1086645 Similar to vacuolar ATP synthase 163 1.30E-101
(strong). [Caenorhabditis elegans] f868.aa gi.vertline.459198
vacuolar H+- ATPase subunit B 164 4.60E-101 [Gossypium hirsutum]
f868.aa gi.vertline.167108 vacuolar ATPase B subunit [Hordeum 164
4.60E-101 vulgare] >sp.vertline.Q40078.vertline.VAT1_HORVU
f872.aa gi.vertline.2687986 (AE001122) B. burgdorferi predicted
1684 1.60E-230 coding region BB0089 [Borrelia f874.aa
gi.vertline.2687965 (AE001121) L-lactate dehydrogenase 1603
2.80E-217 (ldh) [Borrelia burgdorferi] f874.aa gi.vertline.39758
L-lactate dehydrogenase [Bacillus 520 3.10E-109
psychrosaccharolyticus] f874.aa pir.vertline.S08183.vertline.S0818-
3 L-lactate dehydrogenase (EC 1.1.1.27) 515 4.30E-109 X - Bacillus
f874.aa pir.vertline.A25805.vertline.A25805 L-lactate dehydrogenase
(EC 1.1.1.27) - 520 1.00E-107 Bacillus subtilis f874.aa
gi.vertline.143136 L-lactate dehydrogenase [Bacillus 430 5.20E-107
megaterium] >pir.vertline.S00133.vertline.DEBSLM f874.aa
gi.vertline.143138 lactate dehydrogenase (EC 1.1.1.27) 514
6.60E-107 [Bacillus stearothermophilus] f874.aa
gnl.vertline.PID.vertline.d1009574 L-lactate dehydrogenase
[Bacillus 512 8.90E-107 subtilis]
>gnl.vertline.PID.vertline.e1182257 f874.aa gi.vertline.143134
lactate dehydrogenase (EC 1.1.1.27) 516 1.70E-106 [Bacillus
caldotenax] f874.aa gi.vertline.143132 lactate dehydrogenase (AC
1.1.1.27) 506 2.30E-106 [Bacillus caldolyticus] f874.aa
gi.vertline.412392 NAD-dependent dehydrogenase 508 4.40E-106
[unidentified] f874.aa gi.vertline.143130 L-lactate dehydrogenase
[Bacillus 510 1.10E-105 caldotenax]
>pir.vertline.S00019.vertline.S00019 f874.aa gi.vertline.642256
L-lactate dehydrogenase [Pediococcus 560 1.70E-91 acidilactici]
f874.aa gi.vertline.847956 L-lactate dehydrogenase [Lactobacillus
381 2.30E-91 sake] >sp.vertline.P50934.vertline.LDH_LACSK
f874.aa gi.vertline.581305 L-lactate dehydrogenase [Lactobacillus
547 2.30E-91 plantarum] >pir.vertline.A36957.vertline.A36957
f874.aa gi.vertline.149575 L(+)-lactate dehydrogenase 386 3.20E-91
[Lactobacillus casei] f886.aa gi.vertline.2687958 (AE001120) B.
burgdorferi predicted 1792 9.50E-237 coding region BB0077 [Borrelia
f888.aa gi.vertline.2687959 (AE001120) B. burgdorferi predicted
2351 3.59999944710933e-318 coding region BB0075 [Borrelia f893.aa
gi.vertline.2687962 (AE001120) B. burgdorferi predicted 2514 0
coding region BB0071 [Borrelia f895.aa gi.vertline.2687954
(AE001120) conserved hypothetical 747 3.60E-100 protein [Borrelia
burgdorferi] f895.aa gnl.vertline.PID.vertlin- e.e1184285 similar
to hypothetical proteins 103 2.50E-35 [Bacillus subtilis] f899.aa
gi.vertline.2687946 (AE001119) B. burgdorferi predicted 1161
4.30E-158 coding region BB0066 [Borrelia f924.aa
gi.vertline.2687934 (AE001118) B. burgdorferi predicted 692
3.90E-93 coding region BB0044 [Borrelia f925.aa gi.vertline.2687935
(AE001118) B. burgdorferi predicted 1771 7.50E-242 coding region
BB0043 [Borrelia f929.aa gi.vertline.2687916 (AE001117) B.
burgdorferi predicted 2589 0 coding region BB0038 [Borrelia f93.aa
gi.vertline.2688703 (AE001176) pyridoxal kinase (pdxK) 1334
6.60E-181 [Borrelia burgdorferi] f933.aa gi.vertline.2687917
(AE001117) B. burgdorferi predicted 902 1.90E-122 coding region
BB0034 [Borrelia f933.aa gi.vertline.2690091 (AE000789) conserved
hypothetical 136 3.10E-37 protein [Borrelia burgdorferi] f933.aa
gi.vertline.2690225 (AE000790) conserved hypothetical 149 4.50E-37
protein [Borrelia burgdorferi] f933.aa gi.vertline.2690045
(AE000784) conserved hypothetical 126 5.70E-28 protein [Borrelia
burgdorferi] f933.aa gi.vertline.2239281 No definition line found
[Borrelia 148 2.40E-14 burgdorferi] f939.aa gi.vertline.2687919
(AE001117) B. burgdorferi predicted 1796 7.50E-241 coding region
BB0028 [Borrelia f940.aa gi.vertline.2687920 (AE001117) B.
burgdorferi predicted 1109 1.20E-152 coding region
BB0027 [Borrelia f943.aa gi.vertline.2687905 (AE001116) B.
burgdorferi predicted 2001 5.00E-273 coding region BB0024 [Borrelia
f943.aa gi.vertline.411592 L-sorbosone dehydrogenase 175 2.30E-15
[unidentified] f943.aa gnl.vertline.PID.vertline- .d1006418
L-sorbosone dehydrogenase 173 4.40E-15 [Acetobacter liquefaciens]
f952.aa gi.vertline.2687880 (AE001115) glpE protein (glpE) 628
2.90E-84 [Borrelia burgdorferi] f07A.aa R33279 43 kD endoflagellum
sheath protein. 120 6.10E-25 f142.aa R95044 Apoptosis participating
protein. 103 4.70E-18 f147.aa W18209 Staphylococcus aureus Coenzyme
A 194 4.80E-91 disulphide reductase (CoADR). f147.aa W06425
Water-forming NADH oxidase. 369 8.00E-86 f147.aa R32089 Benzene
dioxygenase polypeptide V. 104 4.70E-11 f147.aa R66733 Aromatic
dihydrodiol/catechol 105 9.00E-11 deoxygenase #5. f152.aa R81549
High affinity potassium uptake 137 3.70E-18 transporter HKT1.
f157.aa W15192 Staphylococcus aureus cell surface 239 3.40E-37
protein. f17-6.aa W30763 Mannose-1-phosphate transferase 178
5.20E-16 protein MNN4. f17-6.aa W03627 Human follicle stimulating
hormone 145 1.30E-11 GPR N-terminal sequence. f17-6.aa W03626 Human
thyrotropin GPR N-terminal 144 1.90E-11 sequence. f17-6.aa W21591
Antibiotic potentiating peptide #3. 141 5.10E-11 f196.aa W05196
Helicobacter pylori 50 kDa protective 183 2.70E-18 antigen G3.8.
f196.aa W20916 H. pylori inner membrane protein 180 3.60E-17
14gp12015orf12. f196.aa W20287 H. pylori inner membrane protein,
169 6.50E-15 24132293.aa. f196.aa W20769 H. pylori inner membrane
protein, 169 1.40E-14 07ee20513orf28. f196.aa W20767 H. pylori
cytoplasmic protein, 140 6.10E-14 07ee20513orf1. f197.aa W20769 H.
pylori inner membrane protein, 190 2.30E-19 07ee20513orf28. f197.aa
W20287 H. pylori inner membrane protein, 190 2.00E-18 24132293.aa.
f197.aa W05196 Helicobacter pylori 50 kDa protective 179 4.00E-16
antigen G3.8. f197.aa W20916 H. pylori inner membrane protein 182
6.30E-16 14gp12015orf12. f197.aa W20767 H. pylori cytoplasmic
protein, 150 1.10E-12 07ee20513orf1. f21-4.aa R69629 B. burgdorferi
OspF operon. 321 7.00E-39 f21-4.aa R89476 B. burgdorferi OspG
lipoprotein. 107 6.10E-34 f24-1.aa W22676 Borrelia variable major
protein (VMP)- 412 4.60E-72 like protein VIsE. f291.aa W20152 H.
pylori transporter protein, 336 1.70E-41 1464715.aa. f291.aa W24682
Helicobacter pylori transporter protein 234 8.20E-27 4882763.aa.
f291.aa W20528 H. pylori cell envelope transporter 234 8.20E-27
protein 4882763.aa. f291.aa W20592 H. pylori transporter protein,
168 7.60E-17 01ce11513orf21. f301.aa W20287 H. pylori inner
membrane protein, 158 1.60E-13 24132293.aa. f301.aa W20916 H.
pylori inner membrane protein 158 1.90E-13 14gp12015orf12. f301.aa
W20769 H. pylori inner membrane protein, 158 2.40E-13
07ee20513orf28. f301.aa W05196 Helicobacter pylori 50 kDa
protective 157 2.80E-13 antigen G3.8. f301.aa W20767 H. pylori
cytoplasmic protein, 138 4.30E-11 07ee20513orf1. f320.aa R24300
Glycopeptide resistance protein VanY 142 2.90E-14 from E. faecium.
f328.aa R15642 CTP synthetase. 274 3.00E-50 f328.aa W20778 H.
pylori cytoplasmic protein, 122 1.90E-34 07ge20415orf6. f352.aa
W03626 Human thyrotropin GPR N-terminal 153 4.70E-12 sequence.
f352.aa W21591 Antibiotic potentiating peptide #3. 152 6.60E-12
f352.aa W03627 Human follicle stimulating hormone 145 5.30E-11 GPR
N-terminal sequence. f4-50.aa W07187 B. garinii IP90 decorin
binding protein. 305 1.30E-41 f4-50.aa W07186 B. afzelii strain
pGau decorin binding 161 1.60E-34 protein. f4-50.aa W07185 B.
burgdorferi HB-19 decorin binding 173 2.80E-34 protein. f4-50.aa
W07183 B. burgdorferi B31 decorin binding 176 1.80E-33 protein.
f4-50.aa W07190 B. burgdorferi JD1 decorin binding 177 1.80E-33
protein. f4-50.aa W07182 B. burgdorferi 297 decorin binding 177
1.10E-32 protein. f4-50.aa W07189 B. burgdorferi LP7 decorin
binding 177 1.10E-32 protein. f4-50.aa W07188 B. burgdorferi LP4
decorin binding 177 3.90E-32 protein. f4-50.aa W07184 B.
burgdorferi Sh.2.82 decorin binding 177 1.30E-31 protein. f45-2.aa
R89476 B. burgdorferi OspG lipoprotein. 213 1.30E-35 f45-2.aa
R70491 Leucocytozoan protozoa structural 206 2.10E-20 protein
epitope. f45-2.aa W03626 Human thyrotropin GPR N-terminal 211
6.10E-20 sequence. f45-2.aa W03627 Human follicle stimulating
hormone 202 8.90E-19 GPR N-terminal sequence. f45-2.aa R69629 B.
burgdorferi OspF operon. 111 1.10E-14 f45-2.aa W30763
Mannose-1-phosphate transferase 166 1.00E-13 protein MNN4. f45-2.aa
R97866 Chicken leucocytozoan immunogenic 154 7.10E-12 protein for
use in vaccines. f488.aa W15078 M. leprae gyrA precursor. 390
2.70E-143 f488.aa R88733 S. aureus mutant grlA protein. 698
6.70E-122 f488.aa R88731 S. aureus topoisomerase IV grlA 698
6.70E-122 subunit. f49-2.aa W22676 Borrelia variable major protein
(VMP)- 497 2.70E-75 like protein VlsE. f5-14.aa W03626 Human
thyrotropin GPR N-terminal 234 6.60E-23 sequence. f5-14.aa W03627
Human follicle stimulating hormone 231 1.40E-22 GPR N-terminal
sequence. f5-14.aa R70491 Leucocytozoan protozoa structural 221
1.00E-20 protein epitope. f5-14.aa W30763 Mannose-1-phosphate
transferase 203 1.60E-18 protein MNN4. f5-14.aa R97866 Chicken
leucocytozoan immunogenic 187 2.10E-15 protein for use in vaccines.
f5-14.aa W21591 Antibiotic potentiating peptide #3. 176 4.60E-15
f5-14.aa R69629 B. burgdorferi OspF operon. 106 3.50E-13 f5-14.aa
R89476 B. burgdorferi OspG lipoprotein. 157 6.20E-13 f5-14.aa
W26536 Trypanosoma cruzi antigen. 143 5.00E-11 f5-15.aa R69629 B.
burgdorferi OspF operon. 448 1.30E-68 f5-15.aa R89476 B.
burgdorferi OspG lipoprotein. 105 5.80E-24 f502.aa R69852 Ethylene
response (ETR) mutant 191 1.90E-35 protein etr1-3. f502.aa R69849
Ethylene response (ETR) gene product. 191 2.70E-35 f502.aa R69853
Ethylene response (ETR) mutant 191 2.70E-35 protein etr1-4. f502.aa
R69850 Ethylene response (ETR) mutant 191 3.60E-35 protein etr1-1.
f502.aa R69851 Ethylene response (ETR) mutant 191 3.60E-35 protein
etr1-2. f502.aa R74632 QETR ethylene response (ETR) protein 190
5.20E-26 from Arabidopsis thaliana. f502.aa R74629 Tomato ethylene
response (TETR) 171 6.50E-23 protein. f502.aa R74633 Nr (never
ripe) tomato ethylene 171 6.50E-23 response (ETR) protein. f502.aa
R74630 Tomato TGETR1 ethylene response 123 1.20E-19 protein.
f51-2.aa W03626 Human thyrotropin GPR N-terminal 235 2.90E-23
sequence. f51-2.aa R89476 B. burgdorferi OspG lipoprotein. 109
6.90E-23 f51-2.aa W03627 Human follicle stimulating hormone 228
2.20E-22 GPR N-terminal sequence. f51-2.aa W30763
Mannose-1-phosphate transferase 203 1.00E-18 protein MNN4. f51-2.aa
R70491 Leucocytozoan protozoa structural 191 7.50E-18 protein
epitope. f51-2.aa R97866 Chicken leucocytozoan immunogenic 183
4.80E-16 protein for use in vaccines. f51-2.aa W21591 Antibiotic
potentiating peptide #3. 159 6.20E-13 f51-2.aa R68838 Plasmodium
falciparum ABRA gene 142 1.10E-12 protein. f51-2.aa R27530
Plasmodium falciparum blood and liver 142 2.80E-12 stage ABRA
antigen. f51-2.aa W31186 Human p160 polypeptide 160.2. 148 2.30E-11
f51-2.aa W31185 Human p160 polypeptide 160.1. 148 2.40E-11 f517.aa
W24296 Staphylococcus aureus Gene #1 237 6.80E-30 polypeptide
sequence 2. f541.aa R31013 P39-alpha. 1253 3.80E-229 f541.aa R33280
P39-beta. 504 1.90E-117 f542.aa R33280 P39-beta. 711 3.20E-96
f542.aa R31013 P39-alpha. 101 7.90E-16 f561.aa R69631 B.
burgdorferi T5 protein. 982 6.90E-131 f598.aa W20289 H. pylori
transporter protein, 264 9.90E-33 24218968.aa. f598.aa W20640 H.
pylori transporter protein, 264 1.00E-30 02ce11022orf8. f598.aa
W20101 H. pylori transporter protein 233 8.50E-27 11132778.aa.
f598.aa W20861 H. pylori cell envelope transporter 233 9.60E-27
protein, 12ge10305orf16. f598.aa W34202 Streptomyces efflux pump
protein 196 2.80E-21 (frenolicin gene D product). f598.aa R71091 C.
jejuni PEB1A antigen from ORF3. 168 1.20E-17 f600.aa W25527
Staphylococcus aureus Gene #20 209 3.40E-26 polypeptide sequence 2.
f600.aa W34201 Streptomyces efflux pump protein 169 6.50E-19
(frenolicin gene C product). f600.aa W20639 H. pylori transporter
protein, 127 1.10E-14 02ce11022orf7. f603.aa W34200 Streptomyces
efflux pump protein 155 7.40E-32 (frenolicin gene B product).
f604.aa R48035 Hyaluronic acid synthase of 110 2.30E-20
Streptococcus equisimilis. f606.aa R48035 Hyaluronic acid synthase
of 116 1.20E-25 Streptococcus equisimilis. f607.aa R48035
Hyaluronic acid synthase of 141 1.50E-26 Streptococcus equisimilis.
f631.aa W15192 Staphylococcus aureus cell surface 160 7.30E-29
protein. f664.aa W20105 H. pylori flagella-associated protein, 202
3.20E-46 1171928.aa. f664.aa W20688 H. pylori flagella-associated
protein 202 2.60E-45 04ge11713orf5. f664.aa R97245 Virulence gene
cluster polypeptide 158 3.90E-13 product. f704.aa R60153
Nematode-inducible transmembrane 104 2.50E-18 pore protein. f704.aa
R33913 Sequence encoded by TobRB7-5A 104 2.50E-18 which encodes a
membrane channel f704.aa R77082 Tobacco root specific promoter RB7
104 2.50E-18 from clone lambda5A (TobRB7-5A). f742.aa W46499 Amino
acid sequence of the spindly 101 2.50E-14 (SPY) protein of
Arabidopsis. f752.aa W20733 H. pylori cell envelope protein, 141
3.00E-37 06cp11722orf15. f752.aa W20358 H. pylori cell envelope
protein 110 4.20E-18 26366312.aa. f814.aa W20753 H. pylori
transporter protein, 178 7.90E-35 06gp11202orf7. f814.aa W20420 H.
pylori cell envelope transporter 160 2.30E-21 protein 33399142.aa.
f843.aa R14319 Human T-cell immunosuppressive 167 1.20E-19 factor.
f860.aa W21894 Asparaginyl-tRNA synthetase from 245 2.30E-38
Staphylococcus aureus. f860.aa W33903 Streptococcus pneumoniae
asparaginyl 177 1.10E-22 tRNA synthetase. f867.aa W34261 An alpha
subunit of a thermostable 592 1.30E-161 ATPase. f867.aa R10098
Alpha subunit of ATP-synthase. 741 4.90E-144 f867.aa R31522 Carrot
reverse transcriptase. 311 4.60E-130 f867.aa R10099 Beta subunit of
ATP-synthase. 121 7.90E-14 f867.aa W34262 A beta subunit of a
thermostable 116 1.00E-12 ATPase. f868.aa W34262 A beta subunit of
a thermostable 151 6.10E-109 ATPase. f868.aa R10099 Beta subunit of
ATP-synthase. 172 1.90E-106 f868.aa W34261 An alpha subunit of a
thermostable 117 3.10E-19 ATPase. f868.aa R10098 Alpha subunit of
ATP-synthase. 113 2.00E-18 f868.aa R31522 Carrot reverse
transcriptase. 101 7.10E-15 f874.aa R10591 L-lactic acid
dehyrogenase. 538 7.20E-109 f874.aa R08355 Recombinant thermophilic
NAD- 455 9.80E-99 dependant dehydrogenase. f874.aa R09295
Recombinant thermophilic NAD- 455 9.80E-99 dependant dehydrogenase.
f874.aa R15736 L-lactic acid dehydrogenase. 426 1.60E-85 f874.aa
P91948 Pig H4 isoenzyme. 393 2.00E-82 f874.aa W33108 Chicken lactic
acid dehydrogenase type 390 2.20E-80 B subunit. f874.aa W33107
Chicken lactic acid dehydrogenase type 385 1.10E-79 B subunit.
f874.aa P80891 Testis-specific lactate dehydrogenase 339 5.50E-74
subunit LDH-C4. f874.aa R94013 Heat resistant maleate
dehydrogenase. 255 1.30E-55 f874.aa R11119 Recombinant
L-2-hydroxyisocaproic 224 7.90E-49 acid dehydrogenase. f874.aa
R62605 P. falciparum lactate dehydrogenase. 255 2.00E-44 f874.aa
W11476 Eimeria lactate dehydrogenase. 203 1.10E-25 f943.aa P91223
Coenzyme-independent L-sorbosone 175 4.30E-16 dehydrogenase from
Gluconobacter
[0297]
3TABLE 3 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0298]
4TABLE 4 Residues Comprising Epito-Bearing Fragments Query Residues
Comprising Epito-Bearing Fragments f101.aa from about Lys-62 to
about Gly-64, from about Ser-111 to about Asp-113, from about
Arg-136 to about Arg-139, from about Pro-189 to about Asn-193.
f11.aa from about Pro-38 to about Lys-40, from about Glu-92 to
about Lys-96. f12.aa from about Pro-288 to about Asp-290, from
about Asn-336 to about Gly-338, from about Tyr-410 to about
Gly-413, from about Asp-418 to about Arg-420, from about Pro-552 to
about Val-555, from about Gln-643 to about Asp-645, from about
Gln-1061 to about Arg-1063, from about Asn-1130 to about Lys-1132.
f129.aa from about Glu-76 to about Arg-81, from about Lys-144 to
about Asn-146. f147.aa from about Gln-94 to about Thr-96. f152.aa
from about Gly-35 to about Gly-37, from about Gln-321 to about
Gly-323. f154.aa from about Asn-39 to about Lys-41, from about
Ser-74 to about Lys-77, from about Ser-213 to about Gly-215, from
about Ser-303 to about Asp-306, from about Asp-422 to about
Asn-424. f157.aa from about Lys-21 to about Asp-24, from about
Ser-45 to about Tyr-47. f17.aa from about Arg-17 to about Asn-20,
from about Thr-94 to about Gly-96. f186.aa from about Lys-305 to
about Tyr-308. f196.aa from about Lys-121 to about Asn-123, from
about Pro-278 to about Lys-282, from about Glu-576 to about
Tyr-578. f899.aa from about Asn-174 to about Asp-177. f925.aa from
about Lys-201 to about Asp-204, from about Phe-291 to about
Lys-294. f929.aa from about Pro-139 to about Asn-141, from about
Arg-211 to about Glu-214, from about Thr-370 to about Asn-375.
f933.aa from about Ser-139 to about Lys-143. f940.aa from about
Gly-143 to about Asn-148. f943.aa from about Asp-58 to about
Asp-60, from about Lys-157 to about Asn-159, from about Asp-217 to
about Asp-221, from about Lys-250 to about Asn-254, from about
Pro-262 to about Asn-264, from about Gly-305 to about Trp-307.
f952.aa from about Ser-52 to about Ser-54. f4.aa from about Arg-64
to about Arg-67. f43.aa from about Ser-84 to about Gln-87, from
about Asp-231 to about Tyr-233, from about Arg-296 to about
Asp-300. f50.aa from about Glu-136 to about Gly-138, from about
Asp-153 to about Lys-155, from about Asp-289 to about Asp-291, from
about Glu-458 to about Asn-461. f65.aa from about Glu-120 to about
Asp-122, from about Pro-204 to about Tyr-206. f8.aa from about
Pro-263 to about Arg-265, from about Asp-274 to about Lys-278.
f82.aa from about Tyr-66 to about Gly-68, from about Ser-116 to
about Lys-119, from about Asp-121 to about Gly-123, from about
Pro-128 to about Gly-131. f86.aa from about Asn-179 to about
Asn-181, from about Lys-192 to about Asn-194, from about Lys-270 to
about Asn-272, from about Lys-279 to about Lys-282, from about
Asp-331 to about Asn-333. f477.aa from about Pro-250 to about
Lys-253. f488.aa from about Lys-76 to about Lys-79, from about
Asn-486 to about Asp-489, from about Lys-508 to about Gly-510, from
about Asn-559 to about Gly-562. f494.aa from about Lys-76 to about
Asn-78. f516.aa from about Lys-32 to about Asp-34. f523.aa from
about Pro-202 to about Asn-206, from about Lys-255 to about
Tyr-258. f526.aa from about Asn-85 to about Lys-88, from about
Asp-136 to about Gly-138. f577.aa from about Cys-18 to about
Lys-22, from about Asn-297 to about Gln-300. f584.aa from about
Pro-131 to about Lys-133, from about Pro-200 to about Ser-202.
f596.aa from about Arg-42 to about Asp-44, from about Asp-117 to
about Tyr-119, from about Pro-205 to about Asp-207. f600.aa from
about Pro-143 to about Asp-145. f603.aa from about Phe-35 to about
Ser-37. f607.aa from about Gln-67 to about Lys-70, from about
Asp-273 to about Tyr-275, from about Asp-333 to about Gly-338, from
about Pro-359 to about Lys-362, from about Arg-409 to about
Gly-411. f611.aa from about Arg-133 to about Gly-135. f631.aa from
about Pro-132 to about Asn-136, from about Asn-159 to about
Tyr-161, from about Pro-216 to about Asp-218, from about Pro-220 to
about Lys-223. f688.aa from about Lys-266 to about Asp-268, from
about Lys-271 to about Asn-273, from about Lys-315 to about
Lys-318. f704.aa from about Lys-250 to about Lys-253. f707.aa from
about Lys-131 to about Asp-134, from about Asp-246 to about
Asn-249. f709.aa from about Tyr-39 to about Gly-42, from about
Lys-148 to about Gly-150, from about Arg-269 to about Gly-272, from
about Ser-466 to about Tyr-468, from about Asn-489 to about
Asn-491, from about Lys-575 to about Asp-578, from about Pro-642 to
about Lys-644. f197.aa from about Pro-217 to about Asp-219, from
about Glu-675 to about Asp-678, from about Pro-687 to about
Asn-689, from about Glu-694 to about Gln-696. f200.aa from about
Arg-174 to about Phe-179. f208.aa from about Arg-326 to about
Ser-328. f210.aa from about Pro-191 to about Ile-194. f221.aa from
about Asn-133 to about Asn-135. f253.aa from about Arg-191 to about
Gly-194. f269.aa from about Ser-271 to about Thr-273, from about
Asp-284 to about Gly-286. f29.aa from about Pro-159 to about
Ser-161. f290.aa from about Pro-240 to about Gly-244. f291.aa from
about Gln-267 to about Lys-269. f296.aa from about Glu-98 to about
Lys-101. f3.aa from about Asn-241 to about Lys-245. f30.aa from
about Asn-156 to about Tyr-159, from about Asn-178 to about
Lys-180. f939.aa from about Ser-245 to about Asn-249. f739.aa from
about Asn-80 to about Tyr-82, from about Lys-208 to about Ser-210.
f742.aa from about Ser-141 to about Asp-145, from about Asn-222 to
about Gln-225, from about Asp-243 to about Tyr-247, from about
Asn-249 to about Asn-251. f743.aa from about Arg-111 to about
Gly-114, from about Pro-131 to about Asp-134. f790.aa from about
Thr-40 to about Asn-42, from about Ser-53 to about Ser-55, from
about Lys-215 to about Asp-218, from about Asn-274 to about
Gly-277. f792.aa from about Val-82 to about Ser-84, from about
Ser-102 to about Asn-104, from about Gln-127 to about Tyr-130, from
about Lys-309 to about Asn-314, from about Lys-375 to about
Thr-377, from about Pro-511 to about His-513, from about Thr-515 to
about Asp-517. f797.aa from about Pro-119 to about Gly-122, from
about Lys-166 to about Asn-169. f799.aa from about Asn-31 to about
Asn-34, from about Gln-44 to about Asn-47, from about Pro-123 to
about Gly-125. f814.aa from about Ser-120 to about Ser-122, from
about Arg-636 to about Asn-638, from about Cys-967 to about
Ser-969. f820.aa from about Thr-563 to about Tyr-565. f850.aa from
about Tyr-159 to about Tyr-164, from about Gln-375 to about
Asp-379. f853.aa from about Thr-180 to about Lys-184, from about
Arg-231 to about Asp-233, from about Asn-252 to about Gly-254.
f859.aa from about Lys-46 to about Ser-52, from about Pro-88 to
about Asn-91, from about Asn-117 to about Asp-120. f861.aa from
about Asp-38 to about Lys-40, from about Lys-219 to about Asn-225.
f368.aa from about Gln-228 to about Asn-231. f371.aa from about
Tyr-109 to about Asn-111, from about Asn-162 to about Gln-164.
f502.aa from about Asn-118 to about Lys-122, from about Ser-269 to
about Gly-271, from about Lys-370 to about Asp-373, from about
Asn-509 to about Lys-511, from about Lys-705 to about Arg-707, from
about Thr-912 to about Gly-914, from about Pro-1213 to about
Asp-1216, from about Asn-1491 to about Arg-1493. f527.aa from about
Cys-20 to about Gln-22, from about Asn-38 to about Asn-40, from
about Phe-112 to about Asp-114, from about Lys-160 to about
Asn-162, from about Ser-199 to about Asp-201, from about Gln-258 to
about Gly-261, from about Arg-282 to about Asn-284, from about
Ser-297 to about Asp-299. f541.aa from about Ser-68 to about
Asn-71. f604.aa from about Lys-77 to about Gly-79, from about
Lys-201 to about Asn-203, from about Asp-252 to about Asp-254, from
about Tyr-347 to about Gly-350, from about Asp-514 to about
Trp-516. f736.aa from about Lys-20 to about Asn-24, from about
Arg-147 to about Ser-153, from about Ser-231 to about Lys-233.
f752.aa from about Thr-119 to about Lys-122, from about Pro-420 to
about Gly-422. f798.aa from about Asp-33 to about Thr-36, from
about Lys-180 to about His-183. f635.aa from about Pro-100 to about
Asn-102, from about Asp-145 to about Phe-147. f32.aa from about
Lys-18 to about Asn-20. f320.aa from about Asn-193 to about
Leu-195, from about Gln-248 to about Lys-250. f352.aa from about
Ser-46 to about Asn-49. f301.aa from about Lys-178 to about
Lys-180, from about Ser-401 to about Tyr-404. f373.aa from about
Gly-88 to about Lys-90, from about Asn-539 to about Lys-542, from
about Glu-654 to about Ser-657. f384.aa from about Pro-250 to about
Asn-252, from about Asp-266 to about Lys-268. f446.aa from about
Asp-20 to about Ser-26, from about Asn-146 to about Lys-149.
f542.aa from about Arg-86 to about Gly-88, from about Arg-163 to
about Asn-165. f93.aa from about Asn-152 to about Asp-155. f105.aa
from about Asp-48 to about Phe-50. f150.aa from about Thr-214 to
about Asp-218, from about Asp-256 to about Asp-259. f219.aa from
about Asn-77 to about Asn-81, from about Asp-111 to about Asn-115.
f229.aa from about Gln-61 to about Asn-63. f32.aa from about Lys-18
to about Asn-20. f186.aa from about Lys-305 to about Tyr-308.
f216.aa from about Ser-105 to about Asn-107. f328.aa from about
Asn-105 to about Asp-107. f352.aa from about Ser-46 to about
Asn-49. f867.aa from about Thr-3 to about Gly-5, from about Lys-156
to about Ser-159. f868.aa from about Arg-94 to about Gly-96, from
about Pro-257 to about Gly-261, from about Pro-295 to about
Asp-297, from about Arg-340 to about Asp-342. f872.aa from about
Ser-19 to about Lys-23, from about Thr-139 to about Asp-142, from
about Ser-282 to about Tyr-286, from about Ser-311 to about
Ser-313. f886.aa from about Thr-83 to about Asp-85, from about
Asp-106 to about Lys-108, from about Lys-143 to about Gly-147, from
about Asp-186 to about Asn-191. f888.aa from about Asn-65 to about
Asp-67. f893.aa from about Asn-203 to about Asn-207, from about
Thr-446 to about Asn-450. f605.aa from about Arg-31 to about
Asp-33. f606.aa from about Asn-68 to about Gly-71, from about
Asn-136 to about Lys-139, from about Asn-223 to about Tyr-226, from
about Ser-276 to about Tyr-279, from about Pro-362 to about
Asn-365, from about Arg-503 to about Trp-507. f679.aa from about
Lys-154 to about Asp-156, from about Lys-224 to about Arg-226, from
about Asn-260 to about Asp-264, from about Glu-363 to about
Lys-366, from about Asp-387 to about Gly-389, from about Tyr-441 to
about Lys-443, from about Arg-501 to about Tyr-504. f11-12.aa from
about Pro-91 to about Asn-93, from about Pro-181 to about Asp-186,
from about Lys-244 to about Ser-248. f11-4.aa from about Asn-160 to
about Lys-163. f14-8.aa from about Pro-92 to about Gln-95, from
about Lys-123 to about Thr-125, from about Lys-215 to about
Asp-219. f17-6.aa from about Pro-36 to about Glu-38. f19-2.aa from
about Ser-104 to about Ser-106, from about Gln-230 to about
Asn-232. f19-4.aa from about Val-79 to about Thr-82, from about
Pro-195 to about Gly-201. f19-6.aa from about Asp-24 to about
Lys-30, from about Pro-36 to about Glu-38. f21-4.aa from about
Cys-24 to about Asn-26. f28-2.aa from about Ser-77 to about Lys-80,
from about Tyr-274 to about Asn-277. f28-3.aa from about Glu-53 to
about Arg-57, from about Gln-82 to about Asn-85, from about Gln-157
to about Asn-159. f31-2.aa from about Arg-95 to about Arg-97, from
about Asn-297 to about Asn-299. f4-15.aa from about Pro-182 to
about Asp-184, from about Lys-220 to about Asp-222. f4-50.aa from
about Thr-109 to about Asn-111. f42-1.aa from about Asn-55 to about
Asn-57, from about Arg-81 to about Ser-84, from about Asp-94 to
about Asn-97. f45-2.aa from about Asn-83 to about Gly-86. f47-2.aa
from about Ser-29 to about Asp-33, from about Asn-94 to about
Lys-99, from about Pro-152 to about Lys-157. f49-2.aa from about
Asn-452 to about Gly-454. f5-14.aa from about Glu-102 to about
Asp-106, from about Thr-272 to about Asn-275, from about Glu-313 to
about Asn-315, from about Ser-370 to about Ser-372. f5-15.aa from
about Lys-170 to about Gly-173, from about Asn-194 to about
Gly-196. f51-2.aa from about Asp-302 to about Lys-304. f6-21.aa
from about Glu-38 to about Asn-42, from about Arg-84 to about
Gly-87. f6-27.aa from about Asp-67 to about Asn-69, from about
Arg-85 to about Asn-89, from about Lys-168 to about Gly-171, from
about Lys-179 to about Asn-181, from about Ser-380 to about
His-382. f6-5.aa from about Ser-67 to about Asn-71. f7-30.aa from
about Pro-94 to about Asp-96, from about Lys-144 to about Arg-147.
f76-1.aa from about Asn-30 to about Lys-35, from about Lys-113 to
about Gly-116, from about Glu-119 to about Lys-121. f8-10.aa from
about Pro-25 to about Lys-32, from about Ser-168 to about Thr-172.
f01a.aa_bb001 from about Pro-123 to about Asp-125, from about
Ser-179 to about Asp-181, from about Lys-255 to about Gly-259.
_bb0011 from about Ala8 about Arg 17, from about Tyr31 to about
Gly40, from about Ser65 to about Lys78, from about Val93 to about
Asp102, from about Ser120 to about Ile129, from about Pro156 to
about Glu170, from about Lys187 to about Asn 196, from about His205
to about Lys214, from about Gly226 to about Glu235, fro about
Gln253 to about Asn266, from about Glu283 to about Glu293, from
about Leu311 to about Ile320, from about Arg326 to about Gly335,
from about Pro340 to about Ala349 f02a.aa_bb002 from about Tyr-169
to about Asn-171, from about Tyr-242 to about Asn-245, from about
Lys-264 to about Asp-267. _bb9 from about Met7 to about Lys16, from
about Lys47 to about Ser57, from about Asn80 to about Ser89, from
about Gly103 to about Glu113, from about Lys125 to about Pro133,
from about Lys138 to about Ala147 f03a.aa_bb006 from about Asp-54
to about Thr-57, from about Lys-201 to about His-204. _bb014 from
about Pro23 to about Gln31, from about Ser37 to about Asp45, from
about Leu76 to about Asn84, from about Leu76 to about Val84, from
about Ser89 to about Asn97, from about Ser105 to about Lys113, from
about Asn120 to about Met128, from about Asn159 to about Gly 167,
from about Lys173 to about Bal181 _bb023 from about Asp17 to about
Gly27, from about Arg40 to about Asp48, from about Val64 to about
Asp72, from about Glu105 to about Thr113, from about Ser141 to
about Gly150, from about Asp155 to about Ile163, from about Asn184
to about Lys198, from about Ile219 to about Pro227, from about
Ser230 to about Phe238, from about Ser241 to about Asn250, from
about Asp270 to about Val278, from about Ser285 to about Leu293,
from about Glyu307 to about Ser315, from about Lys327 to about
Asn335 f08a.aa_bb024 from about Asn-30 to about Asp-33, from about
Ser-116 to about Asn-118, from about Asn-154 to about Gly-156.
f09a.aa_bb025 from about Asn-30 to about Ser-35, from about Thr-145
to about Asn-148.
[0299]
Sequence CWU 0
0
* * * * *