U.S. patent application number 11/825796 was filed with the patent office on 2008-01-10 for immunization of dairy cattle with chimeric gapc protein against streptococcus infection.
This patent application is currently assigned to University of Saskatchewan. Invention is credited to Michael Fontaine, Jose Perez-Casal, Andrew A. Potter.
Application Number | 20080009022 11/825796 |
Document ID | / |
Family ID | 22786123 |
Filed Date | 2008-01-10 |
United States Patent
Application |
20080009022 |
Kind Code |
A1 |
Potter; Andrew A. ; et
al. |
January 10, 2008 |
Immunization of dairy cattle with chimeric gapC protein against
streptococcus infection
Abstract
The recombinant production of Gap4, a chimeric GapC plasmin
binding protein comprising the entire amino acid sequence of the
Streptococcus dysgalactiae GapC protein in addition to unique amino
acid sequences from the Streptococcus parauberis and Streptococcus
agalactiae GapC proteins, is described. Also described is the use
of Gap4 chimeric GapC protein in vaccine compositions to prevent or
treat streptococcal infections in general and mastitis in
particular.
Inventors: |
Potter; Andrew A.;
(Saskatoon, CA) ; Perez-Casal; Jose; (Saskatoon,
CA) ; Fontaine; Michael; (Saskatoon, CA) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD
SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
University of Saskatchewan
Saskatoon
CA
|
Family ID: |
22786123 |
Appl. No.: |
11/825796 |
Filed: |
July 9, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11066594 |
Feb 25, 2005 |
7258992 |
|
|
11825796 |
Jul 9, 2007 |
|
|
|
10650369 |
Aug 27, 2003 |
6875853 |
|
|
11066594 |
Feb 25, 2005 |
|
|
|
09878766 |
Jun 11, 2001 |
6660270 |
|
|
10650369 |
Aug 27, 2003 |
|
|
|
60211247 |
Jun 12, 2000 |
|
|
|
Current U.S.
Class: |
435/7.34 ;
530/387.1; 530/388.2; 530/389.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
G01N 33/56944 20130101; A61K 39/00 20130101; A61K 2039/53 20130101;
G01N 2469/20 20130101; A61K 48/00 20130101; A61P 31/04 20180101;
A61K 38/00 20130101; C07K 14/315 20130101 |
Class at
Publication: |
435/007.34 ;
530/387.1; 530/388.2; 530/389.5 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C07K 16/18 20060101 C07K016/18 |
Claims
1. A method of detecting Streptococcus antibodies in a biological
sample, comprising: (a) reacting said biological sample with a
multiple epitope fusion polypeptide comprising more than one
Streptococcus GapC epitope from more than one Streptococcus
species, under conditions which allow said Streptococcus
antibodies, when present in the biological sample, to bind to said
sequence to form an antibody/antigen complex; and (b) detecting the
presence or absence of said complex, and thereby detecting the
presence or absence of Streptococcus antibodies in said sample.
2. The method of claim 1, wherein said multiple epitope fusion
polypeptide comprises a GapC epitope from more than one
Streptococcus species selected from the group consisting of
Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus
uberis, Streptococcus parauberis, and Streptococcus iniae.
3. The method of claim 2, wherein said multiple epitope fusion
polypeptide comprises GapC epitopes from Streptococcus
dysgalactiae, Streptococcus agalactiae and Streptococcus
parauberis.
4. The method of claim 3, wherein the GapC epitopes in said
multiple epitope fusion polypeptide are separated by a spacer amino
acid sequence.
5. The method of claim 1, wherein said multiple epitope fusion
polypeptide comprises an epitope from a Streptococcus GapC protein
corresponding to (a) the amino acid sequences shown at amino acid
positions 62 to 81, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and
20; (b) the amino acid sequences shown at about amino acid
positions 102 to 112, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and
20; (c) the amino acid sequences shown at about amino acid
positions 165 to 172, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and
20; (d) the amino acid sequences shown at about amino acid
positions 248 to 271, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and
20; and (e) the amino acid sequences shown at about amino acid
positions 286 to 305, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and
20.
6. The method of claim 1, wherein said multiple epitope fusion
polypeptide comprises an amino acid sequence having at least 80%
sequence identity to the contiguous sequence of amino acids
depicted at positions 27-448 of the amino acid sequence depicted in
SEQ ID NO:22.
7. The method of claim 1, wherein said multiple epitope fusion
polypeptide comprises an amino acid sequence having at least 90%
sequence identity to the contiguous sequence of amino acids
depicted at positions 27-448 of the amino acid sequence depicted in
SEQ ID NO:22.
8. The method of claim 1, wherein said multiple epitope fusion
polypeptide comprises an amino acid sequence having at least 95%
sequence identity to the contiguous sequence of amino acids
depicted at positions 27-448 of the amino acid sequence depicted in
SEQ ID NO:22.
9. The method of claim 1, wherein said multiple epitope fusion
polypeptide comprises the amino acid sequence of SEQ ID NO:22.
10. The method of claim 6, wherein said multiple epitope fusion
polypeptide further comprises a signal sequence.
11. The method of claim 9, wherein said multiple epitope fusion
polypeptide further comprises a signal sequence.
12. The method of claim 10, wherein said signal sequence comprises
the amino acid sequence depicted at positions 1-26 of SEQ ID
NO:22.
13. The method of claim 11, wherein said signal sequence comprises
the amino acid sequence depicted at positions 1-26 of SEQ ID
NO:22.
14. An immunodiagnostic test kit for detecting Streptococcus
infection, said test kit comprising a multiple epitope fusion
polypeptide comprising more than one Streptococcus GapC epitope
from more than one Streptococcus species, and instructions for
conducting the immunodiagnostic test.
15. The immunodiagnostic test kit of claim 14, wherein said
multiple epitope fusion polypeptide comprises a GapC epitope from
more than one Streptococcus species selected from the group
consisting of Streptococcus dysgalactiae, Streptococcus agalactiae,
Streptococcus uberis, Streptococcus parauberis, and Streptococcus
iniae.
16. The immunodiagnostic test kit of claim 15, wherein said
multiple epitope fusion polypeptide comprises GapC epitopes from
Streptococcus dysgalactiae, Streptococcus agalactiae and
Streptococcus parauberis.
17. The immunodiagnostic test kit of claim 16, wherein said
multiple epitope fusion polypeptide comprises an epitope from a
Streptococcus GapC protein corresponding to (a) the amino acid
sequences shown at amino acid positions 62 to 81, inclusive, of SEQ
ID NOS: 12, 14, 16, 18 and 20; (b) the amino acid sequences shown
at about amino acid positions 102 to 112, inclusive, of SEQ ID NOS:
12, 14, 16, 18 and 20; (c) the amino acid sequences shown at about
amino acid positions 165 to 172, inclusive, of SEQ ID NOS: 12, 14,
16, 18 and 20; (d) the amino acid sequences shown at about amino
acid positions 248 to 271, inclusive, of SEQ ID NOS: 12, 14, 16, 18
and 20; and (e) the amino acid sequences shown at about amino acid
positions 286 to 305, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and
20.
18. The immunodiagnostic test kit of claim 14, wherein said
multiple epitope fusion polypeptide comprises an amino acid
sequence having at least 80% sequence identity to the contiguous
sequence of amino acids depicted at positions 27-448 of the amino
acid sequence depicted in SEQ ID NO:22.
19. The immunodiagnostic test kit of claim 14, wherein said
multiple epitope fusion polypeptide comprises an amino acid
sequence having at least 90% sequence identity to the contiguous
sequence of amino acids depicted at positions 27-448 of the amino
acid sequence depicted in SEQ ID NO:22.
20. The immunodiagnostic test kit of claim 14, wherein said
multiple epitope fusion polypeptide comprises an amino acid
sequence having at least 95% sequence identity to the contiguous
sequence of amino acids depicted at positions 27-448 of the amino
acid sequence depicted in SEQ ID NO:22.
21. The immunodiagnostic test kit of claim 14, wherein said
multiple epitope fusion polypeptide comprises the amino acid
sequence of SEQ ID NO:22.
22. The immunodiagnostic test kit of claim 18, wherein said
multiple epitope fusion polypeptide further comprises a signal
sequence.
23. The immunodiagnostic test kit of claim 21, wherein said
multiple epitope fusion polypeptide further comprises a signal
sequence.
24. The immunodiagnostic test kit of claim 22, wherein said signal
sequence comprises the amino acid sequence depicted at positions
1-26 of SEQ ID NO:22.
25. The immunodiagnostic test kit of claim 23, wherein said signal
sequence comprises the amino acid sequence depicted at positions
1-26 of SEQ ID NO:22.
26. Antibodies directed against a multiple epitope fusion
polypeptide comprising more than one Streptococcus GapC epitope
from more than one Streptococcus species.
27. The antibodies of claim 26, wherein said antibodies are
polyclonal.
28. The antibodies of claim 26, wherein said antibodies are
monoclonal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
11/066,594, filed Feb. 25, 2005 which is a continuation of U.S.
Ser. No. 10/650,369, filed Aug. 27, 2003, now U.S. Pat. No.
6,875,853, which is a continuation of U.S. Ser. No. 09/878,766,
filed Jun. 11, 2001, now U.S. Pat. No. 6,660,270, from which
applications priority is claimed pursuant to 35 U.S.C. .sctn.120.
U.S. Ser. No. 09/878,766 claims the benefit under 35 U.S.C.
.sctn.119(e)(1) of provisional Ser. No. 60/211,247, filed Jun. 12,
2000. All of the above-mentioned applications are hereby
incorporated by reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates generally to bacterial
antigens and genes encoding the same. More particularly, the
present invention pertains to the construction of a chimeric
plasmin binding protein gene comprising the entire S. dysgalactiae
gapC coding sequence as well as coding sequences for unique regions
from several Streptococcus bacteria species, and the use of the
same in vaccine compositions.
BACKGROUND
[0003] Mastitis, an infection of the mammary gland usually caused
by bacteria or fungus, results in major economic losses to the
dairy industry yearly. Among the bacterial species most commonly
associated with mastitis are various species of the genus
Streptococcus, including S. aureus, S. uberis, (untypeable), S.
agalactiae (Lancefield group B), S. dysgalactiae (Lancefield group
C), S. zooepidemicus, and the Lancefield groups D, G, L and N
streptococci. Some of those species are contagions (e.g. S.
agalactiae), while others are considered environmental pathogens
(e.g. S. dysgalactiae and S. uberis). The environmental pathogen S.
uberis is responsible for about 20% of all clinical cases of
mastitis (Bramley, A. J. and Dodd, F. H. (1984) J. Dairy Res.
51:481-512; Bramley, A. J. (1987) Animal Health Nutrition 42:12-16;
Watts, J. L. (1988) J. Dairy Sci. 71:1616-1624); it is the
predominant organism isolated from mammary glands during the
non-lactating period (Bramley, A. J. (1984) Br. Vet. J.
140:328-335; Bramley and Dodd (1984) J. Dairy Res. 51:481-512;
Oliver, S. P. (1988) Am. J. Vet. Res. 49:1789-1793).
[0004] Mastitis resulting from infection with S. uberis is commonly
subclinical, characterized by apparently normal milk with an
increase in somatic cell counts due to the influx of leukocytes.
The chemical composition of milk is changed due to suppression of
secretion with the transfer of sodium chloride and bicarbonate from
blood to milk, causing a shift of pH to a more alkaline level. S.
uberis mastitis may also take the form of an acute clinical
condition, with obvious signs of disease such as clots or
discoloration of the milk and swelling or hardness of the mammary
gland. Some cases of the clinical disease can be severe and pyrexia
may be present. For a review of the clinical manifestations of S.
uberis mastitis, see, Bramley (1991) Mastitis: physiology or
pathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and
A. W. Hill (ed.), New insights into the pathogenesis of mastitis.
Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The
mastitis complex--A brief summary. p. 1-3. In Bovine Mastitis. Lea
& Febiger, Philadelphia
[0005] Conventional antibacterial control methods such as teat
dipping and antibiotic therapy are effective in the control of many
types of contagious mastitis, but the environmental organisms
typically found in all dairy barns are often resistant to such
measures. Vaccination is therefore an attractive strategy to
prevent infections of the mammary glands, and has been shown to be
beneficial in the case of some contagious mastitis pathogens.
[0006] The literature is limited regarding vaccination studies with
S. dysgalactiae and S. uberis, and variable results have been
observed. In some cases, immunization has resulted in increased
sensitivity to the specific organism and in other cases
strain-specific protection has been obtained.
[0007] For example, previous studies have shown that primary
infection with S. uberis can considerably reduce the rate of
infection following a second challenge with the same strain (Hill,
A. W. (1988) Res. Vet. Sci. 44:386-387). Local vaccination with
killed S. uberis protects the bovine mammary gland against
intramammary challenge with the homologous strain (Finch et al.
(1994) Infect. Immun. 62:3599-3603). Similarly, subcutaneous
vaccination with live S. uberis has been shown to cause a dramatic
modification of the pathogenesis of mastitis with the same strain
(Hill et al. (1994) FEMS Immunol. Med. Microbiol. 8:109-118).
Animals vaccinated in this way shed fewer bacteria in their milk
and many quarters remain free of infection.
[0008] Nonetheless, vaccination with live or attenuated bacteria
can pose risks to the recipient. Further, it is clear that
conventional killed vaccines are in general largely ineffective
against S. uberis and S. agalactiae, either due to lack of
protective antigens on in vitro-grown cells or masking of these
antigens by molecular mimicry.
[0009] The current lack of existing mastitis vaccines against S.
agalactiae or the contagious streptococcus strains is due at least
in part to a lack of knowledge regarding the virulence determinants
and protective antigens produced by those organisms which are
involved in invasion and protection of the mammary gland (Collins
et al. (1988) J. Dairy Res. 55:25-32; Leigh et al. (1990) Res. Vet.
Sci. 49: 85-87; Marshall et al. (1986) J. Dairy Res. 53:
507-514).
[0010] S. dysgalactiae is known to bind several extracellular and
plasma-derived proteins such as fibronectin, fibrinogen, collagen,
alpha-II-macroglobulin, IgG, albumin and other compounds. The
organism also produces hyaluronidase and fibrinolysin and is
capable of adhering to and invading bovine mammary epithelial
cells. However, the exact roles of the bacterial components
responsible for these phenotypes in pathogenesis is not known.
[0011] Similarly, the pathogenesis of S. uberis infection is poorly
understood. Furthermore, the influence of S. uberis virulence
factors on host defense mechanisms and mammary gland physiology is
not well defined. Known virulence factors associated with S. uberis
include a hyaluronic acid capsule (Hill, A. W. (1988) Res. Vet.
Sci. 45:400-404), hyaluronidase (Schaufuss et al. (1989) Zentralbl.
Bakteriol. Ser. A 271:46-53), R-like protein (Groschup, M. H. and
Timoney, J. F. (1993) Res. Vet. Sci. 54:124-126), and a
cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka,
B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A 249:190-194),
R-like protein, plasminogen activator and CAMP factor. However,
very little is known of their roles in pathogenicity.
[0012] The use of virulence determinants from Streptococcus as
immunogenic agents has been proposed. For example, the CAMP factor
of S. uberis has been shown to protect vertebrate subjects from
infection by that organism (Jiang, U.S. Pat. No. 5,863,543).
[0013] The .gamma. antigen of the group B Streptococci strain A909
(ATCC No. 27591) is a component of the c protein marker complex,
which additionally comprises an .alpha. and .beta. subunit (Boyle,
U.S. Pat. No. 5,721,339). Subsets of serotype Ia, II, and virtually
all serotype Ib cells of group B streptococci, have been reported
to express components of the c protein. Use of the .gamma. subunit
as an immunogenic agent against infections by Lancefield Group B
Streptococcus infection has been proposed. However, its use to
prevent or treat bacterial infections in animals, including
mastitis in cattle, has not been studied.
[0014] A GapC plasmin binding protein from a strain of Group A
Streptococcus has previously been identified and characterized, and
its use in thrombolytic therapies has been described (Boyle, et
al., U.S. Pat. No. 5,237,050; Boyle, et al., U.S. Pat. No.
5,328,996). However, the use of GapC as an immunogenic agent to
treat or prevent mastitis was neither described nor suggested.
[0015] The group A streptococcal M protein is considered to be one
of the major virulence factors of this organism by virtue of its
ability to impede attack by human phagocytes (Lancefield, R. C.
(1962) J. Immunol. 89:307-313). The bacteria persist in the
infected tissue until antibodies are produced against the M
molecule. Type-specific antibodies to the M protein are able to
reverse the antiphagocytic effect of the molecule and allow
efficient clearance of the invading organism.
[0016] M proteins are one of the key virulence factors of
Streptococcus pyogenes, due to their involvement in mediating
resistance to phagocytosis (Kehoe, M. A. (1991) Vaccine 9:797-806)
and their ability to induce potentially harmful host immune
responses via their superantigenicity and their capacity to induce
host-cross-reactive antibody responses (Bisno, A. L. (1991) New
Engl. J. Med. 325:783-793; Froude et al. (1989) Curr. Top.
Microbiol. Immunol. 145:5-26; Stollerman, G. H. (1991) Clin.
Immunol. Immunopathol. 61:131-142).
[0017] However, obstacles exist to using intact M proteins as
vaccines. The protein's opsonic epitopes are extremely
type-specific, resulting in narrow, type-specific protection.
Further, some M proteins appear to contain epitopes that cross
react with tissues of the immunized subject, causing a harmful
autoimmune response (See e.g., Dale, J. L. and Beached, G. H.
(1982) J. Exp. Med. 156:1165-1176; Dale, J. L. and Beached, G. H.
(1985) J. Exp. Med. 161:113-122; Baird, R. W., Bronze, M. S.,
Drabs, W., Hill, H. R., Veasey, L. G. and Dale, J. L. (1991) J.
Immun. 146:3132-3137; Bronze, M. S. and Dale, J. L. (1993) J. Immun
151:2820-2828; Cunningham, M. W. and Russell, S. M. (1983) Infect.
Immun. 42:531-538).
[0018] An octavalent M protein vaccine has been constructed and was
tested for protective immunogenicity against multiple serotypes of
group A streptococci infection in rabbits. However, the immune
response obtained was serotype-specific, conferring protection only
against those bacterial strains exhibiting the M protein epitopes
present in the chimeric protein (Dale, J. B., Simmons, M., Chiang,
E. C., and Chiang, E. Y. (1996) Vaccine 14:944-948).
[0019] Chimeric proteins containing three different fibronectin
binding domains (FNBDs) derived from fibronectin binding proteins
of S. dysgalactiae and Staphylococcus aureus have been expressed on
the surface of Staph. carnosus cells. In the case of one of these
proteins, intranasal immunizations with live recombinant Staph.
carnosus cells expressing the chimeric protein on their surface
resulted in an improved antibody response to a model immunogen
present within the chimeric surface protein.
[0020] A chimeric Protein G molecule (a type III Fc binding protein
specific for the Fc region of all subclasses of IgG antibody
molecules) is known, but its use as an immunogenic agent has not
been described or suggested (Bjorck, et al. (1992) U.S. Pat. No.
5,108,894).
[0021] Until now, the protective capability of GapC multiple
epitope fusion proteins has not been studied.
SUMMARY OF THE INVENTION
[0022] Accordingly, the present invention provides GapC multiple
epitope fusion proteins and polynucleotides encoding the same. In
one embodiment, the invention is directed to a multiple epitope
fusion polypeptide comprising the general structural formula (I):
(A).sub.x-(B).sub.y(C).sub.z (I) wherein
[0023] (I) is a linear amino acid sequence;
[0024] B comprises an amino acid sequence containing at least five
amino acids which amino acids correspond to an antigenic
determinant of a GapC protein;
[0025] A and C each comprise an amino acid sequence that is [0026]
(i) different from B, [0027] (ii) different from the other, and
[0028] (iii) an amino acid sequence containing at least five amino
acids, which amino acid sequence corresponds to an antigenic
determinant of a GapC protein wherein said antigenic determinant is
not adjacent to B in nature;
[0029] y is an integer of 1 or more; and
[0030] x and z are each independently integers wherein x+z is 1 or
more.
[0031] In certain embodiments, the multiple epitope fusion
polypeptide further comprises a signal sequence and/or a
transmembrane sequence. Further, A, B, and/or C of the multiple
epitope fusion polypeptide may linked by one or more spacer
sequences, wherein the spacers
[0032] (i) are amino acid sequences of from 1 to 1,000 amino acids,
inclusive;
[0033] (ii) can be the same or different as A, B, or C; and
[0034] (iii) can be the same or different as each other.
[0035] In certain embodiments, A, B, and C each comprise epitopes
from one or more species of bacteria, such as from one or more
bacterial species of the genus Streptococcus, including but not
limited to one or more bacterial species selected from the group
consisting of Streptococcus dysgalactiae, Streptococcus agalactiae,
Streptococcus uberis, Streptococcus parauberis, and Streptococcus
iniae.
[0036] In yet another embodiment, A, B, and C each comprise amino
acid sequences selected from the group consisting of
[0037] (a) the amino acid sequence shown at about amino acid
positions 61 to 81, inclusive, of FIGS. 1 through 5, or any amino
acid sequence having at least about 80% identity thereto;
[0038] (b) the amino acid sequences shown at about amino acid
positions 102 to 112, inclusive, of FIGS. 1 through 5, or any amino
acid sequence having at least about 80% identity thereto;
[0039] (c) the amino acid sequences shown at about amino acid
positions 165 to 172, inclusive, of FIGS. 1 through 5, or any amino
acid sequence having at least about 80% identity thereto;
[0040] (d) the amino acid sequences shown at about amino acid
positions 248 to 271, inclusive, of Figures through 5, or any amino
acid sequence having at least about 80% identity thereto; and
[0041] (e) the amino acid sequences shown at about amino acid
positions 286 to 305, inclusive, of FIGS. 1 through 5, or any amino
acid sequence having at least about 80% identity thereto.
[0042] In another embodiment, the multiple epitope fusion
polypeptide comprises the amino acid sequence depicted in FIG. 6
(SEQ ID NO:22).
[0043] In yet further embodiments, the invention is directed to
polynucleotide sequences encoding the multiple epitope fusion
polypeptide sequence described above or compliments thereof, as
well as recombinant vectors comprising the polynucleotide, host
cells comprising the recombinant vectors and methods of
recombinantly producing the polypeptides.
[0044] In another embodiment, the invention is directed to a
vaccine composition comprising a pharmaceutically acceptable
vehicle and a multiple epitope fusion polypeptide as described
above. In certain embodiments, the vaccine compositions comprise an
adjuvant.
[0045] In still a further embodiment, the invention is directed to
a method of producing a vaccine composition comprising the steps
of
[0046] (1) providing the multiple epitope fusion polypeptide;
and
[0047] (2) combining the polypeptide with a pharmaceutically
acceptable vehicle.
[0048] In another embodiment, the invention is directed to a method
of treating or preventing a bacterial infection in a vertebrate
subject comprising administering to the subject a therapeutically
effective amount of a vaccine composition as described above.
[0049] In certain embodiments, the bacterial infection is a
streptococcal infection. Further, the bacterial infection may cause
mastitis.
[0050] In yet another embodiment, the invention is directed to a
method of treating or preventing a bacterial infection in a
vertebrate subject comprising administering to the subject a
therapeutically effective amount of a polynucleotide as described
herein.
[0051] In certain embodiments, the bacterial infection is a
streptococcal infection. Further, the bacterial infection may cause
mastitis.
[0052] In further embodiments, the invention is directed to
antibodies directed against the above multiple epitope fusion
polypeptides. The antibodies may be polyclonal or monoclonal.
[0053] In another embodiment, the invention is directed to a method
of detecting Streptococcus antibodies in a biological sample,
comprising:
[0054] (a) reacting said biological sample with a multiple epitope
fusion polypeptide under conditions which allow said Streptococcus
antibodies, when present in the biological sample, to bind to said
sequence to form an antibody/antigen complex; and
[0055] (b) detecting the presence or absence of said complex, and
thereby detecting the presence or absence of Streptococcus
antibodies in said sample.
[0056] In still a further embodiment, the invention is directed to
an immunodiagnostic test kit for detecting Streptococcus infection.
The test kit comprises a multiple epitope fusion polypeptide as
described herein and instructions for conducting the
immunodiagnostic test.
[0057] These and other embodiments of the subject invention will
readily occur to those of skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0058] FIGS. 1A-1B depict the isolated nucleotide sequence and
deduced amino acid sequence of the gapC gene for S. dysgalactiae
(SEQ ID NO:11 and SEQ ID NO:12). In the figure, the asterisk
represents a stop codon, and the underlined regions represent
nucleotide sequences complementary to the primers used to isolate
the genes from the bacterial chromosomes.
[0059] FIGS. 2A-2B depict the isolated nucleotide sequence and
deduced amino acid sequence of the gapC gene for S. agalactiae (SEQ
ID NO:13 and SEQ ID NO:14). In the figure, the asterisk represents
a stop codon, and the underlined regions represent nucleotide
sequences complementary to the primers used to isolate the genes
from the bacterial chromosomes.
[0060] FIGS. 3A-3B depict the isolated nucleotide sequence and
deduced amino acid sequence of the gapC gene for S. uberis (SEQ ID
NO:15) and SEQ ID NO:16). In the figure, the asterisk represents a
stop codon, and the underlined regions represent nucleotide
sequences complementary to the primers used to isolate the genes
from the bacterial chromosomes.
[0061] FIGS. 4A-4B depict the isolated nucleotide sequence and
deduced amino acid sequence of the gapC gene for S. parauberis (SEQ
ID NO:17 and SEQ ID NO:18). In the figure, the asterisk represents
a stop codon, and the underlined regions represent nucleotide
sequences complementary to the primers used to isolate the genes
from the bacterial chromosomes.
[0062] FIGS. 5A-5B depict the isolated nucleotide sequence and
deduced amino acid sequence of the gapC gene for S. iniae (SEQ ID
NO:19 and SEQ ID NO:20). In the figure, the asterisk represents a
stop codon, and the underlined regions represent nucleotide
sequences complementary to the primers used to isolate the genes
from the bacterial chromosomes.
[0063] FIG. 6 depicts the nucleotide sequence (SEQ ID NO:21) and
deduced amino acid sequence (SEQ ID NO:22) of the GapC multiple
epitope fusion protein of the present invention.
[0064] FIGS. 7A-7E show a DNA alignment chart created by PileUp and
displayed by Pretty software (a component of the GCG Wisconsin
Package, version 10, provided by the SeqWeb sequence analysis
package, version 1.1, of the Canadian Bioinformatics Resource). The
figure depicts the isolated nucleotide sequences of the gapC genes
from S. dysgalactiae (DysGapC, Check 9344) (SEQ ID NO:11); S.
agalactiae (AgalGapC. Check 2895) (SEQ ID NO:13); S. uberis
(UberGapC, Check 5966) (SEQ ID NO:15); S. parauberis (PUberGapC,
Check 9672) (SEQ ID NO:17); and S. iniae (IniaeGapC, Check 990)
(SEQ ID NO:19). The previously known sequences of S. equisimilis
(SeqGapC, Check 5841) (SEQ ID NO:25), S. pyogenes (SpyGapC, Check
4037) (SEQ ID NO:23), and a bovine GAPDH protein (BovGapC, check
5059) (SEQ ID NO:27) are also included. The length and weight
parameters were the same for all sequences (1018 and 1.00,
respectively). The parameters used in the DNA sequence comparison
were as follows: Plurality--2.00; Threshold--1; AveWeight--1.00;
AveMatch--1.00; AvMisMatch--0.00; Symbol comparison
table--pileupdna.cmp; CompCheck--6876; GapWeight--5;
GapLengthWeight--1; PileUp MSF--1018; Type--N; Check--3804. In the
figure, dashes represent identical nucleotides; dots represent gaps
introduced by the software used to generate the alignment chart,
and tildes represent regions not included in the overall alignment
due to differences in the length of the gene sequences.
[0065] FIGS. 8A-8C show an amino acid sequence alignment chart
created by PileUp and displayed by Pretty (as above) that depicts
the alignment of PolyGap4 (SEQ ID NO:22), the multiple epitope
fusion polypeptide of the present invention, with the deduced amino
acid sequences of the native GapC proteins isolated from S.
dysgalactiae (DysGapC, Check 6731) (SEQ ID NO:12), S. agalactiae
(AgalGapC, Check 1229) (SEQ ID NO:14), S. uberis (UberGapC, Check
8229) (SEQ ID NO:16), S. parauberis (PUberGapC, Check 8889) (SEQ ID
NO:18), and S. iniae (IniaeGapC, check 8785) (SEQ ID NO:20). The
previously known sequences of S. equisimilis (SeqGapC, Check 8252)
(SEQ ID NO:26), S. pyogenes (SpyGapC, Check 6626) (SEQ ID NO:24)
and a bovine GAPDH protein (BovGapC, Check 8479) (SEQ ID NO:28) are
also included. In the figure, dashes represent identical amino acid
residues; dots represent gaps introduced by the PileUp software,
and tildes represent regions not included in the overall alignment
due to differences in the length of the gene sequences.
[0066] FIG. 9 shows a Kyte-Doolittle hydropathy plot, averaged over
a window of 7, an Emini surface probability plot, a Karplus-Schulz
chain flexibility plot, a Jameson-Wolf antigenic index plot, and
both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure
plots for the GapC protein isolated from S. dysgal.
[0067] FIG. 10 shows a Kyte-Doolittle hydropathy plot, averaged
over a window of 7, an Emini surface probability plot, a
Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic
index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson
secondary structure plots for the GapC protein isolated from S.
agal.
[0068] FIG. 11 shows a Kyte-Doolittle hydropathy plot, averaged
over a window of 7, an Emini surface probability plot, a
Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic
index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson
secondary structure plots for the GapC protein isolated from S.
uberis.
[0069] FIG. 12 shows a Kyte-Doolittle hydropathy plot, averaged
over a window of 7, an Emini surface probability plot, a
Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic
index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson
secondary structure plots for the GapC protein isolated from S.
parauberis.
[0070] FIG. 13 shows a Kyte-Doolittle hydropathy plot, averaged
over a window of 7, an Emini surface probability plot, a
Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic
index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson
secondary structure plots for the GapC protein isolated from S.
iniae.
[0071] FIG. 14 shows a Kyte-Doolittle hydropathy plot, averaged
over a window of 7, an Emini surface probability plot, a
Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic
index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson
secondary structure plots for LipoFGAP4 (SEQ ID NO:22), the
chimeric GapC protein.
[0072] FIG. 15 is a diagrammatic representation of the Chou-Fasman
secondary structure plot for the GapC protein isolated from S.
dysgal.
[0073] FIG. 16 is a diagrammatic representation of the Chou-Fasman
secondary structure plot for the GapC protein isolated from S.
agal.
[0074] FIG. 17 is a diagrammatic representation of the Chou-Fasman
secondary structure plot for the GapC protein isolated from S.
uberis.
[0075] FIG. 18 is a diagrammatic representation of the Chou-Fasman
secondary structure plot for the GapC protein isolated from S.
parauberis.
[0076] FIG. 19 is a diagrammatic representation of the Chou-Fasman
secondary structure plot for the GapC protein isolated from and S.
iniae.
[0077] FIG. 20 is a diagrammatic representation of the Chou-Fasman
secondary structure plot for LipoFGAP4 (SEQ ID NO:22), the chimeric
GapC protein.
[0078] FIG. 21 is a diagram of plasmid pPolyGap.1.
[0079] FIG. 22 is a diagram of plasmid pPolyGap.2.
[0080] FIG. 23 is a diagram of plasmid pPolyGap.3.
[0081] FIG. 24 is a diagram of plasmid pPolyGap.4
[0082] FIG. 25 is a diagram of plasmid polygap4.
DETAILED DESCRIPTION
[0083] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA technology, and immunology, which are
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Sambrook, Fritsch & Maniatis,
Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second
Edition (1989); Perbal, B., A Practical Guide to Molecular Cloning
(1984); the series, Methods In Enzymology (S. Colowick and N.
Kaplan eds., Academic Press, Inc.); and Handbook of Experimental
Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986,
Blackwell Scientific Publications).
[0084] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0085] The following amino acid abbreviations are used throughout
the text: [0086] Alanine: Ala (A) Arginine: Arg (R) [0087]
Asparagine: Asn (N) Aspartic acid: Asp (D) [0088] Cysteine: Cys (C)
Glutamine: Gln (Q) [0089] Glutamic acid: Glu (E) Glycine: Gly (G)
[0090] Histidine: H is (H) Isoleucine: Ile (I) [0091] Leucine: Leu
(L) Lysine: Lys (K) [0092] Methionine: Met (M) Phenylalanine: Phe
(F) [0093] Proline: Pro (P) Serine: Ser (S) [0094] Threonine: Thr
(T) Tryptophan: Trp (W) [0095] Tyrosine: Tyr (Y) Valine: Val
(V)
A. DEFINITIONS
[0096] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0097] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a Streptococcus GapC protein"
includes a mixture of two or more such proteins, and the like.
[0098] The terms "GapC protein" and "GapC plasmin binding protein"
(used interchangeably herein) or a nucleotide sequence encoding the
same, intends a protein or a nucleotide sequence, respectively,
which is derived from a GapC gene found in a variety of
Streptococcus species, including, without limitation certain
strains of group A streptococci (Lottenbery, R., et al., (1987)
Infect. Immun. 55:1914-1918). The nucleotide sequence of
representative Streptococcus gapC genes, and the corresponding
amino acid sequence of the GapC proteins encoded by these genes,
are depicted in the Figures. In particular, FIGS. 1 through 5
depict the isolated nucleotide sequences and isolated amino acid
sequences of S. dysgalactiae (SEQ ID NO:11 and SEQ ID NO:12,
respectively), S. agalactiae(SEQ ID NO:13 and SEQ ID NO:14,
respectively), S. uberis (SEQ ID NO: 15 and SEQ ID NO:16,
respectively), S. parauberis (SEQ ID NO:17 and SEQ ID NO:18,
respectively,), and S. iniae (SEQ ID NO:19 and SEQ ID NO:20,
respectively). However, a GapC protein as defined herein is not
limited to the depicted sequences as subtypes of each of these
Streptococcus species are known and variations in GapC proteins
will occur between them.
[0099] Representative gapC genes, derived from S. dysgalactiae, S.
agalactiae, S. uberis, and S. parauberis, are found in the plasmids
pET15bgapC, pMF521c, pMF521a, pMF521d, and pMF521e,
respectively.
[0100] Furthermore, the derived protein or nucleotide sequences
need not be physically derived from the gene described above, but
may be generated in any manner, including for example, chemical
synthesis, isolation (e.g., from S. dysgalactiae) or by recombinant
production, based on the information provided herein. Additionally,
the term intends proteins having amino acid sequences substantially
homologous (as defined below) to contiguous amino acid sequences
encoded by the genes, which display immunological and/or
plasmin-binding activity.
[0101] Thus, the terms intend full-length, as well as immunogenic,
truncated and partial sequences, and active analogs and precursor
forms of the proteins. Also included in the term are nucleotide
fragments of the gene that include at least about 8 contiguous base
pairs, more preferably at least about 10-20 contiguous base pairs,
and most preferably at least about 25 to 50, or more, contiguous
base pairs of the gene, or any integers between these values. Such
fragments are useful as probes and in diagnostic methods, discussed
more fully below.
[0102] The terms also include those forms possessing, as well as
lacking, a signal sequence, if such is present, as well as the
nucleic acid sequences coding therefore. Additionally, the term
intends forms of the GapC proteins which lack a membrane anchor
region, and nucleic acid sequences encoding proteins with such
deletions. Such deletions may be desirable in systems that do not
provide for secretion of the protein. Furthermore, the
plasmin-binding domains of the proteins, may or may not be present.
Thus, for example, if the GapC plasmin-binding protein will be used
to purify plasmin, the plasmin-binding domain will generally be
retained. If the protein is to be used in vaccine compositions,
immunogenic epitopes which may or may not include the
plasmin-binding domain, will be present.
[0103] The terms also include proteins in neutral form or in the
form of basic or acid addition salts depending on the mode of
preparation. Such acid addition salts may involve free amino groups
and basic salts may be formed with free carboxyls. Pharmaceutically
acceptable basic and acid addition salts are discussed further
below. In addition, the proteins may be modified by combination
with other biological materials such as lipids (both those
occurring naturally with the molecule or other lipids that do not
destroy immunological activity) and saccharides, or by side chain
modification, such as acetylation of amino groups, phosphorylation
of hydroxyl side chains, oxidation of sulfhydryl groups,
glycosylation of amino acid residues, as well as other
modifications of the encoded primary sequence.
[0104] The term therefore intends deletions, additions and
substitutions to the sequence, so long as the polypeptide functions
to produce an immunological response as defined herein. In this
regard, particularly preferred substitutions will generally be
conservative in nature, i.e., those substitutions that take place
within a family of amino acids. For example, amino acids are
generally divided into four families: (1) acidic--aspartate and
glutamate; (2) basic--lysine, arginine, histidine; (3)
non-polar--alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar--glycine, asparagine, glutamine, cystine, serine threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified as aromatic amino acids. For example, it is reasonably
predictable that an isolated replacement of leucine with isoleucine
or valine, or vice versa; an aspartate with a glutamate or vice
versa; a threonine with a serine or vice versa; or a similar
conservative replacement of an amino acid with a structurally
related amino acid, will not have a major effect on the biological
activity. Proteins having substantially the same amino acid
sequence as the reference molecule, but possessing minor amino acid
substitutions that do not substantially affect the immunogenicity
and/or plasmin-binding affinity of the protein, are therefore
within the definition of the reference polypeptide.
[0105] For example, the polypeptide of interest may include up to
about 5-10 conservative or non-conservative amino acid
substitutions, or even up to about 15-25 or 20-50 conservative or
non-conservative amino acid substitutions, or any integer between
these values, so long as the desired function of the molecule
remains intact.
[0106] In this regard, GapC proteins isolated from streptococci
exhibit several variable regions in their amino acid sequences,
located at amino acid positions 62 to 81; 102 to 112; 165 to 172;
248 to 271; and 286 to 305. These regions, which in S.
dysgalactiae, S. agalactiae, S. uberis, S. parauberis and S. iniae
exhibit from 1 to 9 amino acid substitutions, are likely to be
amenable to variation without substantially affecting immunogenic
or enzymatic function.
[0107] Similarly, substitutions occurring in the transmembrane
binding domain, if present, and the signal sequence, if present,
normally will not affect immunogenicity. One of skill in the art
may readily determine other regions of the molecule of interest
that can tolerate change by reference to the protein structure
plots shown in FIGS. 9 to 20 herein.
[0108] The term "streptococcal GapC protein" intends a GapC
plasmin-binding protein, as defined above, derived from a
streptococcal species that produces the same, including, but not
limited to S. dysgalactiae, S. agalactiae, S. uberis, S.
parauberis, and S. iniae. For example, a "S. dysgalactiae GapC
protein" is a GapC plasmin-binding protein as defined above,
derived from S. dysgalactiae. Similarly, an "S. agalactiae GapC
protein" intends a gapC binding protein derived from S.
agalactiae.
[0109] "Wild type" or "native" proteins or polypeptides refer to
proteins or polypeptides isolated from the source in which the
proteins naturally occur. "Recombinant" polypeptides refer to
polypeptides produced by recombinant DNA techniques; i.e., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide. "Synthetic" polypeptides are those prepared by
chemical synthesis.
[0110] An "isolated" protein or polypeptide is a protein or
polypeptide molecule separate and discrete from the whole organism
with which the molecule is found in nature; or a protein or
polypeptide devoid, in whole or part, of sequences normally
associated with it in nature; or a sequence, as it exists in
nature, but having heterologous sequences (as defined below) in
association therewith.
[0111] The term "functionally equivalent" intends that the amino
acid sequence of a GapC plasmin-binding protein is one that will
elicit a substantially equivalent or enhanced immunological
response, as defined above, as compared to the response elicited by
a GapC plasmin-binding protein having identity with the reference
GapC plasmin-binding protein, or an immunogenic portion
thereof.
[0112] The term "epitope" refers to the site on an antigen or
hapten to which specific B cells and/or T cells respond. The term
is also used interchangeably with "antigenic determinant" or
"antigenic determinant site." Antibodies that recognize the same
epitope can be identified in a simple immunoassay showing the
ability of one antibody to block the binding of another antibody to
a target antigen. Epitopes may include 3 to 5 amino acids, more
preferably 5 to 10 amino acids, up to the full length of the
reference molecule.
[0113] The term "multiple epitope" protein or polypeptide specifies
a sequence of amino acids comprising an epitope as defined herein,
which contains at least one epitope repeated two or more times
within a linear molecule. The repeating sequence need not be
directly connected to itself, is not repeated in nature in the same
manner and, further, may be present within a larger sequence which
includes other amino acids that are not repeated. For the purposes
of this invention, the epitope sequence may either be an exact copy
of a wild-type epitope sequence, or a sequence which is
"functionally equivalent" as defined herein. refers to a multiple
epitope protein or polypeptide as defined herein that is produced
by recombinant or synthetic methods.
[0114] A "fusion" or "chimeric" protein or polypeptide is one in
which amino acid sequences from more than one source are joined.
Such molecules may be produced synthetically or recombinantly, as
described further herein (see the section entitled "Production of
GapC Plasmin-Binding Proteins" infra). Hence, the term "multiple
epitope fusion protein or polypeptide" refers to a multiple epitope
protein or polypeptide as defined herein which is made by either
synthetic or recombinant means.
[0115] In this regard, a multiple epitope fusion protein comprising
the variable regions in the amino acid sequences of the GapC
proteins referred to above may be produced. The amino acid sequence
for a representative GapC multiple epitope fusion protein, and a
corresponding polynucleotide coding sequence, is depicted in FIGS.
6A-6C herein (SEQ ID NO:22). Methods for recombinantly producing
the protein, including a method for constructing the polyGap4
plasmid containing the chimeric coding sequence (diagramed in FIG.
25) and a method for expressing the protein from the polyGap4
plasmid, are described in Examples 4 and 5 infra.
[0116] The terms "immunogenic" protein or polypeptide refer to an
amino acid sequence which elicits an immunological response as
described herein. An "immunogenic" protein or polypeptide, as used
herein, includes the full-length sequence of the GapC
plasmin-binding protein in question, with or without the signal
sequence, membrane anchor domain and/or plasmin-binding domain,
analogs thereof, or immunogenic fragments thereof. By "immunogenic
fragment" is meant a fragment of a GapC plasmin-binding protein
which includes one or more epitopes and thus elicits the
immunological response described above. Such fragments can be
identified using any number of epitope mapping techniques, well
known in the art. See, e.g., Epitope Mapping Protocols in Methods
in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana
Press, Totowa, N.J. For example, linear epitopes may be determined
by concurrently synthesizing large numbers of peptides on solid
supports, the peptides corresponding to portions of the protein
molecule, and reacting the peptides with antibodies while the
peptides are still attached to the supports. Such techniques are
known in the art and described in, e.g., U.S. Pat. No. 4,708,871;
Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;
Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated
herein by reference in their entireties. Similarly, conformational
epitopes are readily identified by determining spatial conformation
of amino acids such as by, e.g., x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols, supra. Antigenic regions of proteins can also be
identified using standard antigenicity and hydropathy plots, such
as those calculated using, e.g., the Omiga version 1.0 software
program available from the Oxford Molecular Group. This computer
program employs the Hopp/Woods method, Hopp et al., Proc. Natl.
Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity
profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol.
Biol. (1982) 157:105-132 for hydropathy plots. FIGS. 9 to 20 herein
depict Kyte-Doolittle profiles for representative proteins
encompassed by the invention.
[0117] Immunogenic fragments, for purposes of the present
invention, will usually include at least about 3 amino acids,
preferably at least about 5 amino acids, more preferably at least
about 10-15 amino acids, and most preferably 25 or more amino
acids, of the parent GapC plasmin-binding-binding protein molecule.
There is no critical upper limit to the length of the fragment,
which may comprise nearly the full-length of the protein sequence,
or even a fusion protein comprising two or more epitopes of
GapC.
[0118] An "immunogenic composition" is a composition that comprises
an antigenic molecule where administration of the composition to a
subject results in the development in the subject of a humoral
and/or a cellular immune response to the antigenic molecule of
interest.
[0119] By "subunit vaccine composition" is meant a composition
containing at least one immunogenic polypeptide, but not all
antigens, derived from or homologous to an antigen from a pathogen
of interest. Such a composition is substantially free of intact
pathogen cells or particles, or the lysate of such cells or
particles. Thus, a "subunit vaccine composition" is prepared from
at least partially purified (preferably substantially purified)
immunogenic polypeptides from the pathogen, or recombinant analogs
thereof. A subunit vaccine composition can comprise the subunit
antigen or antigens of interest substantially free of other
antigens or polypeptides from the pathogen.
[0120] By "pharmaceutically acceptable" or "pharmacologically
acceptable" is meant a material which is not biologically or
otherwise undesirable, i.e., the material may be administered to an
individual in a formulation or composition without causing any
undesirable biological effects or interacting in a deleterious
manner with any of the components of the composition in which it is
contained.
[0121] An "immunological response" to a composition or vaccine is
the development in the host of a cellular and/or antibody-mediated
immune response to the composition or vaccine of interest. Usually,
an "immunological response" includes but is not limited to one or
more of the following effects: the production of antibodies, B
cells, helper T cells, suppressor T cells, and/or cytotoxic T cells
and/or .gamma..delta. T cells, directed specifically to an antigen
or antigens included in the composition or vaccine of interest.
Preferably, the host will display either a therapeutic or
protective immunological response such that resistance of the
mammary gland to new infection will be enhanced and/or the clinical
severity of the disease reduced. Such protection will be
demonstrated by either a reduction or lack of symptoms normally
displayed by an infected host and/or a quicker recovery time.
[0122] By "nucleic acid immunization" is meant the introduction of
a nucleic acid molecule encoding one or more selected antigens into
a host cell, for the in vivo expression of an antigen, antigens, an
epitope, or epitopes. The nucleic acid molecule can be introduced
directly into a recipient subject, such as by injection,
inhalation, oral, intranasal and mucosal administration, or the
like, or can be introduced ex vivo, into cells which have been
removed from the host. In the latter case, the transformed cells
are reintroduced into the subject where an immune response can be
mounted against the antigen encoded by the nucleic acid
molecule.
[0123] The term "treatment" as used herein refers to either (1) the
prevention of infection or reinfection (prophylaxis), or (2) the
reduction or elimination of symptoms of the disease of interest
(therapy).
[0124] By "mastitis" is meant an inflammation of the mammary gland
in mammals, including in cows, ewes, goats, sows, mares, and the
like, caused by the presence of pathogenic microorganisms, such as
S. uberis. The infection manifests itself by the infiltration of
phagocytic cells in the gland. Generally, 4 clinical types of
mastitis are recognized: (1) peracute, associated with swelling,
heat, pain, and abnormal secretion in the gland and accompanied by
fever and other signs of systemic disturbance, such as marked
depression, rapid weak pulse, sunken eyes, weakness and complete
anorexia; (2) acute, with changes in the gland similar to those
above but where fever, anorexia and depression are slight to
moderate; (3) subacute, where no systemic changes are displayed and
the changes in the gland and its secretion are less marked: and (4)
subclinical, where the inflammatory reaction is detectable only by
standard tests for mastitis.
[0125] Standard tests for the detection of mastitis include but are
not limited to, the California Mastitis Test, the Wisconsin
Mastitis Test, the Nagase test, the electronic cell count and
somatic cell counts used to detect a persistently high white blood
cell content in milk. In general, a somatic cell count of about
300,000 to about 500,000 cells per ml or higher, in milk will
indicate the presence of infection. Thus, a vaccine is considered
effective in the treatment and/or prevention of mastitis when, for
example, the somatic cell count in milk is retained below about
500,000 cells per ml. For a discussion of mastitis and the
diagnosis thereof, see, e.g., The Merck Veterinary Manual: A
Handbook of Diagnosis, Therapy, and Disease Prevention and Control
for the Veterinarian, Merck and Co., Rahway, N.J., 1991.
[0126] By the terms "vertebrate," "subject," and "vertebrate
subject" are meant any member of the subphylum Chordata, including,
without limitation, mammals such as cattle, sheep, pigs, goats,
horses, and humans; domestic animals such as dogs and cats; and
birds, including domestic, wild and game birds such as cocks and
hens including chickens, turkeys and other gallinaceous birds; and
fish. The term does not denote a particular age. Thus, both adult
and newborn animals, as well as fetuses, are intended to be
covered.
[0127] A "nucleic acid" molecule can include, but is not limited
to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic
mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA,
and even synthetic DNA sequences. The term also captures sequences
that include any of the known base analogs of DNA and RNA.
[0128] An "isolated" nucleic acid molecule is a nucleic acid
molecule separate and discrete from the whole organism with which
the molecule is found in nature; or a nucleic acid molecule devoid,
in whole or part, of sequences normally associated with it in
nature; or a sequence, as it exists in nature, but having
heterologous sequences (as defined below) in association therewith.
The term "isolated" in the context of a polynucleotide intends that
the polynucleotide is isolated from the chromosome with which it is
normally associated, and is isolated from the complete genomic
sequence in which it normally occurs.
[0129] "Purified polynucleotide" refers to a polynucleotide of
interest or fragment thereof which is essentially free, e.g.,
contains less than about 50%, preferably less than about 70%, and
more preferably less than about 90%, of the protein with which the
polynucleotide is naturally associated. Techniques for purifying
polynucleotides of interest are well-known in the art and include,
for example, disruption of the cell containing the polynucleotide
with a chaotropic agent and separation of the polynucleotide(s) and
proteins by ion-exchange chromatography, affinity chromatography
and sedimentation according to density.
[0130] A "coding sequence" or a "nucleotide sequence encoding" a
particular protein, is a nucleotide sequence which is transcribed
and translated into a polypeptide in vitro or in vivo when placed
under the control of appropriate regulatory elements. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxy) terminus. A coding sequence can include, but is not
limited to, procaryotic sequences, cDNA from eucaryotic mRNA,
genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A transcription termination sequence
will usually be located 3' to the coding sequence. A
"complementary" sequence is one in which the nitrogenous base at a
given nucleotide position is the complement of the nitrogenous base
appearing at the same position in the reference sequence. To
illustrate, the complement of adenosine is tyrosine, and vice
versa; similarly, cytosine is complementary to guanine, and vice
versa; hence, the complement of the reference sequence
5'-ATGCTGA-3' would be 5'-TACGACT-3'.
[0131] A "wild-type" or "native" sequence, as used herein, refers
to polypeptide encoding sequences that are essentially as they are
found in nature, e.g., the S. dysgalactiae GapC protein encoding
sequences depicted in FIGS. 1A-1B (SEQ ID NO:12).
[0132] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation:
(1) is not associated with all or a portion of the polynucleotide
with which it is associated in nature; and/or (2) is linked to a
polynucleotide other than that to which it is linked in nature. The
term "recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide. "Recombinant host cells," "host cells," "cells,"
"cell lines," "cell cultures," and other such terms denoting
procaryotic microorganisms or eucaryotic cell lines cultured as
unicellular entities, are used interchangeably, and refer to cells
which can be, or have been, used as recipients for recombinant
vectors or other transfer DNA, and include the progeny of the
original cell which has been transfected. It is understood that the
progeny of a single parental cell may not necessarily be completely
identical in morphology or in genomic or total DNA complement to
the original parent, due to accidental or deliberate mutation.
Progeny of the parental cell which are sufficiently similar to the
parent to be characterized by the relevant property, such as the
presence of a nucleotide sequence encoding a desired peptide, are
included in the progeny intended by this definition, and are
covered by the above terms.
[0133] "Homology" refers to the percent identity between two
polynucleotide or two polypeptide moieties. Two DNA, or two
polypeptide sequences are "substantially homologous" to each other
when the sequences exhibit at least about 80%-85%, preferably at
least about 90%, and most preferably at least about 95%-98%
sequence identity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified DNA or polypeptide sequence.
[0134] In general, "identity" refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively.
Percent identity can be determined by a direct comparison of the
sequence information between two molecules by aligning the
sequences, counting the exact number of matches between the two
aligned sequences, dividing by the length of the shorter sequence,
and multiplying the result by 100. Readily available computer
programs can be used to aid in the analysis, such as ALIGN,
Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O.
Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research
Foundation, Washington, D.C., which adapts the local homology
algorithm of Smith and Waterman (1981) Advances in Appl. Math.
2:482-489 for peptide analysis. Programs for determining nucleotide
sequence identity are available in the Wisconsin Sequence Analysis
Package, Version 8 (available from Genetics Computer Group,
Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,
which also rely on the Smith and Waterman algorithm. These programs
are readily utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis
Package referred to above. For example, percent identity of a
particular nucleotide sequence to a reference sequence can be
determined using the homology algorithm of Smith and Waterman with
a default scoring table and a gap penalty of six nucleotide
positions.
[0135] Another method of establishing percent identity in the
context of the present invention is to use the MPSRCH package of
programs copyrighted by the University of Edinburgh, developed by
John F. Collins and Shane S. Sturrok, and distributed by
IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of
packages the Smith-Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open
penalty of 12, gap extension penalty of one, and a gap of six).
From the data generated the "Match" value reflects "sequence
identity." Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs
can be found at the following internet address:
http://www.ncbi.nlm.gov/cgi-bin/BLAST.
[0136] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0137] By the term "degenerate variant" is intended a
polynucleotide containing changes in the nucleic acid sequence
thereof, that encodes a polypeptide having the same amino acid
sequence as the polypeptide encoded by the polynucleotide from
which the degenerate variant is derived.
[0138] Techniques for determining amino acid sequence "similarity"
are well known in the art. In general, "similarity" means the exact
amino acid to amino acid comparison of two or more polypeptides at
the appropriate place, where amino acids are identical or possess
similar chemical and/or physical properties such as charge or
hydrophobicity. A so-termed "percent similarity" then can be
determined between the compared polypeptide sequences. Techniques
for determining nucleic acid and amino acid sequence identity also
are well known in the art and include determining the nucleotide
sequence of the mRNA for that gene (usually via a cDNA
intermediate) and determining the amino acid sequence encoded
thereby, and comparing this to a second amino acid sequence. In
general, "identity" refers to an exact nucleotide to nucleotide or
amino acid to amino acid correspondence of two polynucleotides or
polypeptide sequences, respectively.
[0139] A "heterologous" region of a DNA construct is an
identifiable segment of DNA within or attached to another DNA
molecule that is not found in association with the other molecule
in nature. Thus, when the heterologous region encodes a bacterial
gene, the gene will usually be flanked by DNA that does not flank
the bacterial gene in the genome of the source bacteria. Another
example of the heterologous coding sequence is a construct where
the coding sequence itself is not found in nature (e.g., synthetic
sequences having codons different from the native gene). Allelic
variation or naturally occurring mutational events do not give rise
to a heterologous region of DNA, as used herein.
[0140] A "vector" is a replicon, such as a plasmid, phage, or
cosmid, to which another DNA segment may be attached so as to bring
about the replication of the attached segment. A vector is capable
of transferring gene sequences to target cells (e.g., bacterial
plasmid vectors, viral vectors, non-viral vectors, particulate
carriers, and liposomes).
[0141] Typically, the terms "vector construct," "expression
vector," "gene expression vector," "gene delivery vector," "gene
transfer vector," and "expression cassette" all refer to an
assembly which is capable of directing the expression of a sequence
or gene of interest. Thus, the terms include cloning and expression
vehicles, as well as viral vectors.
[0142] These assemblies include a promoter which is operably linked
to the sequences or gene(s) of interest. Other control elements may
be present as well. The expression cassettes described herein may
be contained within a plasmid construct. In addition to the
components of the expression cassette, the plasmid construct may
also include a bacterial origin of replication, one or more
selectable markers, a signal which allows the plasmid construct to
exist as single-stranded DNA (e.g., a M13 origin of replication), a
multiple cloning site, and a "mammalian" origin of replication
(e.g., a SV40 or adenovirus origin of replication).
[0143] DNA "control elements" refers collectively to transcription
promoters, transcription enhancer elements, transcription
termination sequences, polyadenylation sequences (located 3' to the
translation stop codon), sequences for optimization of initiation
of translation (located 5' to the coding sequence), translation
termination sequences, upstream regulatory domains, ribosome
binding sites and the like, which collectively provide for the
transcription and translation of a coding sequence in a host cell.
See e.g., McCaughan et al. (1995) PNAS USA 92:5431-5435; Kochetov
et al (1998) FEBS Letts. 440:351-355. Not all of these control
sequences need always be present in a recombinant vector so long as
the desired gene is capable of being transcribed and
translated.
[0144] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control elements operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control elements need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter and the
coding sequence and the promoter can still be considered "operably
linked" to the coding sequence. Similarly, "control elements
compatible with expression in a subject" are those which are
capable of effecting the expression of the coding sequence in that
subject.
[0145] A control element, such as a promoter, "directs the
transcription" of a coding sequence in a cell when RNA polymerase
will bind the promoter and transcribe the coding sequence into
mRNA, which is then translated into the polypeptide encoded by the
coding sequence.
[0146] A "host cell" is a cell which has been transformed, or is
capable of transformation, by an exogenous nucleic acid
molecule.
[0147] A cell has been "transformed" by exogenous DNA when such
exogenous DNA has been introduced inside the cell membrane.
Exogenous DNA may or may not be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell. In procaryotes
and yeasts, for example, the exogenous DNA may be maintained on an
episomal element, such as a plasmid. With respect to eucaryotic
cells, a stably transformed cell is one in which the exogenous DNA
has become integrated into the chromosome so that it is inherited
by daughter cells through chromosome replication. This stability is
demonstrated by the ability of the eucaryotic cell to establish
cell lines or clones comprised of a population of daughter cells
containing the exogenous DNA.
[0148] As used herein, a "biological sample" refers to a sample of
tissue or fluid isolated from a subject, including but not limited
to, for example, blood, plasma, serum, fecal matter, urine, bone
marrow, bile, spinal fluid, lymph fluid, samples of the skin,
external secretions of the skin, respiratory, intestinal, and
genitourinary tracts, tears, saliva, milk, blood cells, organs,
biopsies and also samples of in vitro cell culture constituents
including but not limited to conditioned media resulting from the
growth of cells and tissues in culture medium, e.g., recombinant
cells, and cell components.
[0149] As used herein, the terms "label" and "detectable label"
refer to a molecule capable of detection, including, but not
limited to, radioactive isotopes, fluorescers, chemiluminescers,
enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,
chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin
or haptens) and the like. The term "fluorescer" refers to a
substance or a portion thereof which is capable of exhibiting
fluorescence in the detectable range. Particular examples of labels
which may be used under the invention include fluorescein,
rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH and
.alpha.-.beta.-galactosidase.
2. MODES OF CARRYING OUT THE INVENTION
[0150] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0151] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
General Overview of the Invention
[0152] Central to the present invention is the discovery that the
GapC protein is capable of eliciting an immune response in a
vertebrate subject. Experiments performed in support of the present
invention have demonstrated that immunization of dairy cattle with
the GapC protein of S. dysgalactiae conferred protection against
experimental infection with this organism, and furthermore,
conferred cross-protection against infection by S. uberis.
[0153] GapC is produced by a number of different streptococcus
species. With the exception of several localized variable regions,
the amino acid sequences of the GapC proteins produced by those
strains are highly conserved. Therefore, it is desirable to
construct multiple epitope GapC fusion proteins comprising
antigenic determinants taken from both the highly conserved regions
of GapC, and the unique regions of GapC proteins from several
streptococcal species. Experiments performed in support of the
present invention have demonstrated that such a protein is capable
of eliciting broad immunity against a variety of streptococcal
infections while providing the additional economic advantage of
minimizing the number of antigens present in the final formulation,
and concomitantly reducing the cost of producing that
formulation.
[0154] The GapC multiple epitope fusion proteins of the present
invention are described by the general structural formula
(A).sub.x-(B).sub.y-(C).sub.z representing a linear amino acid
sequence. B is an amino acid sequence of at least five and not more
than 1,000 amino acids of an antigenic determinant from a GapC
protein, and y is an integer of 2 or more. A and C are each
different from B, as well as being different from each other, and
are independently an amino acid sequence of an antigenic
determinant containing at least five and not more than 1,000 amino
acids not immediately adjacent to B in nature. x and z are each
independently an integer of 0 or more, wherein at least one of x
and z is 1 or more.
[0155] Typically, A, B, and C are antigenic determinants from the
GapC proteins of one or more bacterial species. In a preferred
embodiment, A, B, and C are amino acid sequences comprising one or
more antigenic determinants from the GapC protein of one or more of
the following species of streptococcus: S. dysgalactiae; S.
agalactiae; S. uberis; S. parauberis, and S. iniae.
[0156] In this regard, FIGS. 9 through 13 show plots of the
following for the streptococcal GapC proteins employed by the
present invention: Kyte-Doolittle hydropathy, averaged over a
window of 7; surface probability according to Emini; chain
flexibility according to Karplus-Schulz; antigenicity index
according to Jameson-Wolf; secondary structure according to
Garnier-Osguthorpe-Robson; secondary structure according to
Chou-Fasman; and predicted glycosylation sites. FIGS. 15 through 19
show plots of secondary structure according to Chou-Fasman for the
aforementioned proteins. One of skill in the art can readily use
the information presented in FIGS. 9 through 13 and 15 to 19 in
view of the teachings of the present specification to identify
antigenic regions which may be employed in constructing the
chimeric protein of the present invention.
[0157] Most preferably, A, B, and/or C include one or more variable
regions of the GapC proteins from more than one streptococcus
species. In this regard, FIGS. 8A-8C show an amino acid sequence
alignment which illustrates regions of homology and variability
that exist among GapC proteins from S. dysgalactiae, S. agalactiae,
S. uberis, S. parauberis, and S. iniae. Amino acid sequences for
the GapC proteins of S. pyogenes and S. equisimilis, S. pyogenes
are also included. In particular, several variable regions are
located at amino acid positions 62 to 81; 102 to 112; 165 to 172;
248 to 271; and 286 to 305.
[0158] The multiple epitope fusion protein of the present invention
may also include spacer sequences interposed between A, B, and/or
C. The spacer sequences are typically amino acid sequences of from
1 to 1,000 amino acids, may be the same or different as A, B, or C,
and may be the same or different as each other.
[0159] The present invention may also include a signal sequence
and/or a transmembrane sequence. Examples of suitable signal
sequences include the E. coli LipoF signal sequence, and the OmpF
signal sequence. Examples of suitable transmembrane sequences
include those associated with LipoF and OmpF.
[0160] An especially preferred embodiment of the present invention
is the multiple epitope fusion protein Gap4. The amino acid
sequence of Gap4 (SEQ ID NO:22), a representative multiple epitope
GapC fusion protein, is shown in FIGS. 6A-6C, as is the
polynucleotide sequence which encodes it (SEQ ID NO:21). Gap4 is a
47.905 kDa chimeric protein of 448 amino acids. Residues 1 to 27
are identical to amino acid residues 1 to 27 of the E. coli LipoF
signal sequence. Residues 28 to 123 are identical to residues 1 to
96 of the S. dysgalactiae GapC protein. Residues 124 (leucine) and
125 (glutamic acid) are spacer amino acids. They are followed by
residues 126 to 165, which are identical to residues 56 to 95 of S.
parauberis as well as to the same residues of S. uberis. Residue
166 (isoleucine) is a spacer amino acid. Residues 167 to 208 are
identical to residues 55 to 96 of the S. agalactiae GapC protein.
Residues 209 (threonine) and 210 (serine) are spacer amino acids.
Residues 211 to 448 are identical to residues 99 to 336 of the S.
dysgalactiae GapC protein.
[0161] As expressed, Gap4 has a cysteine residue present at the
amino terminal end of the mature protein. The LipoF signal sequence
and cysteine residue are present to ensure that the chimeric
molecule is efficiently secreted from the bacterial host cell and
becomes bound to the host cell membrane via the lipid-moiety. The
protein may then be extracted from the cell surface via
differential solubilization with a detergent such as Sarkosyl or
TritonX-100.RTM. (see Example 5 infra).
[0162] The GapC chimeric proteins of the present invention or
antigenic fragments thereof can be provided in subunit vaccine
compositions. In addition to use in vaccine compositions, the
proteins or antibodies thereto can be used as diagnostic reagents
to detect the presence of infection in a vertebrate subject.
Similarly, the genes encoding the proteins can be cloned and used
to design probes to detect and isolate homologous genes in other
bacterial strains. For example, fragments comprising at least about
15-20 nucleotides, more preferably at least about 20-50
nucleotides, and most preferably about 60-100 nucleotides, or any
integer between these values, will find use in these
embodiments.
[0163] The vaccine compositions of the present invention can be
used to treat or prevent a wide variety of bacterial infections in
vertebrate subjects. For example, vaccine compositions including
GapC multiple epitope fusion proteins comprising antigenic
determinants from S. dysgalactiae, S. uberis, S. parauberis, S.
iniae, and/or group B streptococci (GBS) such as S. agalactiae, can
be used to treat streptococcal infections in vertebrate subjects
that are caused by these or other species. In particular, S. uberis
and S. agalactiae are common bacterial pathogens associated with
mastitis in bovine, equine, ovine and goat species. Additionally,
group B streptococci, such as S. agalactiae, are known to cause
numerous other infections in vertebrates, including septicemia,
meningitis, bacteremia, impetigo, arthritis, urinary tract
infections, abscesses, spontaneous abortion etc. Hence, vaccine
compositions containing chimeric GapC proteins will find use in
treating and/or preventing a wide variety of streptococcal
infections.
[0164] Similarly, GapC multiple epitope fusion proteins comprising
antigenic determinants derived from other bacterial genera such as
Staphylococcus, Mycobacterium, Escherichia, Pseudomonas, Nocardia,
Pasteurella, Clostridium and Mycoplasma will find use for treating
bacterial infections caused by species belonging to those genera.
Thus, it is readily apparent that chimeric GapC proteins can be
used to treat and/or prevent a wide variety of bacterial infections
in numerous species.
[0165] The GapC multiple epitope fusion proteins of the present
invention can be used in vaccine compositions either alone or in
combination with other bacterial, fungal, viral or protozoal
antigens. These other antigens can be provided separately or even
as fusion proteins comprising the GapC chimeric protein fused to
one or more of these antigens. For example, other immunogenic
proteins from S. uberis, such as the CAMP factor, hyaluronic acid
capsule, hyaluronidase, R-like protein and plasminogen activator,
can be administered with the chimeric GapC protein. Additionally,
immunogenic proteins from other organisms involved in mastitis,
such as from the genera Staphylococcus, Corynebacterium,
Pseudomonas, Nocardia, Clostridium, Mycobacterium, Mycoplasma,
Pasteurella, Prototheca, other streptococci, coliform bacteria, as
well as yeast, can be administered along with the GapC fusion
proteins described herein to provide a broad spectrum of
protection. Thus, for example, immunogenic proteins from
Staphylococcus aureus, Str. agalactiae, Str. dysgalactiae, Str.
zooepidemicus, Corynebacterium pyogenes, Pseudomonas aeruginosa,
Nocardia asteroides, Clostridium perfringens, Escherichia coli,
Enterobacter aerogenes and Klebsiella spp. can be provided along
with the GapC plasmin-binding proteins of the present
invention.
Production of GapC Multiple Epitope Fusion Proteins
[0166] The above-described chimeric proteins and active fragments
and analogs derived from the same, can be produced by recombinant
methods as described herein. These recombinant products can take
the form of partial protein sequences, full-length sequences,
precursor forms that include signal sequences, or mature forms
without signals.
[0167] The GapC plasmin-binding protein DNA sequences used to
construct the chimeric proteins of the present invention can be
isolated by a variety of methods known to those of skill in the
art. See, e.g., Sambrook et al., supra. Methods for isolating,
cloning and sequencing the gene sequences encoding GapC proteins
from S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis, and
S. iniae are detailed in Examples 1, 2 and 3, infra.
[0168] After isolating and cloning the desired GapC protein
sequences, polynucleotide sequences encoding the chimeric proteins
are constructed using standard recombinant techniques including PCR
amplification, restriction endonuclease digestion and ligation.
See, e.g., Sambrook et al., supra. Methods for constructing Gap4,
an especially preferred embodiment of the present invention, are
detailed in Example 4, infra.
[0169] Alternatively, the DNA sequences encoding the proteins of
interest can be prepared synthetically rather than cloned. The DNA
sequences can be designed with the appropriate codons for the
particular amino acid sequence. In general, one will select
preferred codons for the intended host if the sequence will be used
for expression. The complete sequence is assembled from overlapping
oligonucleotides prepared by standard methods and assembled into a
complete coding sequence. See, e.g., Edge (1981) Nature 292:756;
Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol.
Chem. 259:6311.
[0170] Once coding sequences for the desired proteins have been
prepared, they can be cloned into any suitable vector or replicon.
Numerous cloning vectors are known to those of skill in the art,
and the selection of an appropriate cloning vector is a matter of
choice. Examples of recombinant DNA vectors for cloning and host
cells which they can transform include the bacteriophage .lamda.
(E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230
(gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1
(gram-negative bacteria), pME290 (non-E. coli gram-negative
bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus),
pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces),
YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells).
See, Sambrook et al., supra; DNA Cloning, supra; B. Perbal,
supra.
[0171] The gene can be placed under the control of a promoter,
ribosome binding site (for bacterial expression) and, optionally,
an operator (collectively referred to herein as "control"
elements), so that the DNA sequence encoding the desired protein is
transcribed into RNA in the host cell transformed by a vector
containing this expression construction. The coding sequence may or
may not contain a signal peptide or leader sequence. If a signal
sequence is included, it can either be the native, homologous
sequence, or a heterologous sequence. For example, the LipoF signal
sequence is added to the amino-terminal region of the chimeric
protein Gap4 to permit secretion of the protein after expression.
See Examples 4E and 5, infra. Leader sequences can be removed by
the host in post-translational processing. See, e.g., U.S. Pat.
Nos. 4,431,739; 4,425,437; 4,338,397.
[0172] Other regulatory sequences which allow for regulation of
expression of the protein sequences relative to the growth of the
host cell may also be desirable. Regulatory sequences are known to
those of skill in the art, and examples include those which cause
the expression of a gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Other types of regulatory elements may also be
present in the vector, for example, enhancer sequences.
[0173] The control sequences and other regulatory sequences may be
ligated to the coding sequence prior to insertion into a vector,
such as the cloning vectors described above. Alternatively, the
coding sequence can be cloned directly into an expression vector
which already contains the control sequences and an appropriate
restriction site.
[0174] In some cases it may be necessary to modify the coding
sequence so that it may be attached to the control sequences with
the appropriate orientation; i.e., to maintain the proper reading
frame. It may also be desirable to produce mutants or analogs of
the GapC plasmin-binding protein. Mutants or analogs may be
prepared by the deletion of a portion of the sequence encoding the
protein, by insertion of a sequence, and/or by substitution of one
or more nucleotides within the sequence. Techniques for modifying
nucleotide sequences, such as site-directed mutagenesis, are
described in, e.g., Sambrook et al., supra; DNA Cloning, supra;
Nucleic Acid Hybridization, supra.
[0175] The expression vector is then used to transform an
appropriate host cell. A number of mammalian cell lines are known
in the art and include immortalized cell lines available from the
American Type Culture Collection (ATCC), such as, but not limited
to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK")
cells, as well as others. Similarly, bacterial hosts such as E.
coli, Bacillus subtilis, and Streptococcus spp., will find use with
the present expression constructs. Yeast hosts useful in the
present invention include inter alia, Saccharomyces cerevisiae,
Candida albicans, Candida maltosa, Hansenula polymorpha,
Kluyveromyces fragilis, Kluyveromyces lactis, Pichia
guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and
Yarrowia lipolytica. Insect cells for use with baculovirus
expression vectors include, inter alia, Aedes aegypti, Autographa
californica, Bombyx mori, Drosophila melanogaster, Spodoptera
frugiperda, and Trichoplusia ni.
[0176] Depending on the expression system and host selected, the
proteins of the present invention are produced by culturing host
cells transformed by an expression vector described above under
conditions whereby the protein of interest is expressed. The
protein is then isolated from the host cells and purified. If the
expression system secretes the protein into the growth media, the
protein can be purified directly from the media. If the protein is
not secreted, it is isolated from cell lysates. The selection of
the appropriate growth conditions and recovery methods are within
the skill of the art.
[0177] The proteins of the present invention may also be produced
by chemical synthesis such as solid phase peptide synthesis, using
known amino acid sequences or amino acid sequences derived from the
DNA sequence of the genes of interest. Such methods are known to
those skilled in the art. See, e.g., J. M. Stewart and J. D. Young,
Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,
Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The
Peptides: Analysis, Synthesis, Biology, editors E. Gross and J.
Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254,
for solid phase peptide synthesis techniques; and M. Bodansky,
Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and
E. Gross and J. Meienhofer, Eds., The Peptides: Analysis,
Synthesis, Biology, supra, Vol. 1, for classical solution
synthesis. Chemical synthesis of peptides may be preferable if a
small fragment of the antigen in question is capable of raising an
immunological response in the subject of interest.
[0178] The chimeric GapC plasmin-binding proteins of the present
invention, or their fragments, can be used to produce antibodies,
both polyclonal and monoclonal. If polyclonal antibodies are
desired, a selected mammal, (e.g., mouse, rabbit, goat, horse,
etc.) is immunized with an antigen of the present invention, or its
fragment, or a mutated antigen. Serum from the immunized animal is
collected and treated according to known procedures. See, e.g.,
Jurgens et al. (1985) J. Chrom. 348:363-370. If serum containing
polyclonal antibodies is used, the polyclonal antibodies can be
purified by immunoaffinity chromatography, using known
procedures.
[0179] Monoclonal antibodies to the chimeric GapC plasmin-binding
proteins and to the fragments thereof, can also be readily produced
by one skilled in the art. The general methodology for making
monoclonal antibodies by using hybridoma technology is well known.
Immortal antibody-producing cell lines can be created by cell
fusion, and also by other techniques such as direct transformation
of B lymphocytes with oncogenic DNA, or transfection with
Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma
Techniques (1980); Hammerling et al., Monoclonal Antibodies and
T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies
(1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;
4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and
4,493,890. Panels of monoclonal antibodies produced against the
chimeric GapC plasmin-binding proteins, or fragments thereof, can
be screened for various properties; i.e., for isotype, epitope,
affinity, etc. Monoclonal antibodies are useful in purification,
using immunoaffinity techniques, of the individual antigens which
they are directed against. Both polyclonal and monoclonal
antibodies can also be used for passive immunization or can be
combined with subunit vaccine preparations to enhance the immune
response. Polyclonal and monoclonal antibodies are also useful for
diagnostic purposes.
Vaccine Formulations and Administration
[0180] The GapC multiple epitope fusion proteins of the present
invention can be formulated into vaccine compositions, either alone
or in combination with other antigens, for use in immunizing
subjects as described below. Methods of preparing such formulations
are described in, e.g., Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., 18 Edition, 1990. Typically, the
vaccines of the present invention are prepared as injectables,
either as liquid solutions or suspensions. Solid forms suitable for
solution in or suspension in liquid vehicles prior to injection may
also be prepared. The preparation may also be emulsified or the
active ingredient encapsulated in liposome vehicles. The active
immunogenic ingredient is generally mixed with a compatible
pharmaceutical vehicle, such as, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vehicle may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents and pH
buffering agents.
[0181] Adjuvants which enhance the effectiveness of the vaccine may
also be added to the formulation. Adjuvants may include for
example, muramyl dipeptides, pyridine, aluminum hydroxide,
dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water
emulsions, saponins, cytokines, and other substances known in the
art.
[0182] The chimeric GapC plasmin-binding protein may be linked to a
carrier in order to increase the immunogenicity thereof. Suitable
carriers include large, slowly metabolized macromolecules such as
proteins, including serum albumins, keyhole limpet hemocyanin,
immunoglobulin molecules, thyroglobulin, ovalbumin, and other
proteins well known to those skilled in the art; polysaccharides,
such as sepharose, agarose, cellulose, cellulose beads and the
like; polymeric amino acids such as polyglutamic acid, polylysine,
and the like; amino acid copolymers; and inactive virus
particles.
[0183] The chimeric GapC plasmin-binding proteins may be used in
their native form or their functional group content may be modified
by, for example, succinylation of lysine residues or reaction with
Cys-thiolactone. A sulfhydryl group may also be incorporated into
the carrier (or antigen) by, for example, reaction of amino
functions with 2-iminothiolane or the N-hydroxysuccinimide ester of
3-(4-dithiopyridyl propionate. Suitable carriers may also be
modified to incorporate spacer arms (such as hexamethylene diamine
or other bifunctional molecules of similar size) for attachment of
peptides.
[0184] Other suitable carriers for the chimeric GapC
plasmin-binding proteins of the present invention include VP6
polypeptides of rotaviruses, or functional fragments thereof, as
disclosed in U.S. Pat. No. 5,071,651, incorporated herein by
reference. Also useful is a fusion product of a viral protein and
the subject chimeric proteins made by methods disclosed in U.S.
Pat. No. 4,722,840. Still other suitable carriers include cells,
such as lymphocytes, since presentation in this form mimics the
natural mode of presentation in the subject, which gives rise to
the immunized state. Alternatively, the proteins of the present
invention may be coupled to erythrocytes, preferably the subject's
own erythrocytes. Methods of coupling peptides to proteins or cells
are known to those of skill in the art.
[0185] Furthermore, the chimeric GapC plasmin-binding proteins (or
complexes thereof) may be formulated into vaccine compositions in
either neutral or salt forms. Pharmaceutically acceptable salts
include the acid addition salts (formed with the free amino groups
of the active polypeptides) and which are formed with inorganic
acids such as, for example, hydrochloric or phosphoric acids, or
such organic acids as acetic, oxalic, tartaric, mandelic, and the
like. Salts formed from free carboxyl groups may also be derived
from inorganic bases such as, for example, sodium, potassium,
ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
procaine, and the like.
[0186] Vaccine formulations will contain a "therapeutically
effective amount" of the active ingredient, that is, an amount
capable of eliciting an immune response in a subject to which the
composition is administered. In the treatment and prevention of
mastitis, for example, a "therapeutically effective amount" would
preferably be an amount that enhances resistance of the mammary
gland to new infection and/or reduces the clinical severity of the
disease. Such protection will be demonstrated by either a reduction
or lack of symptoms normally displayed by an infected host, a
quicker recovery time and/or a lowered somatic cell count in milk
from the infected quarter. For example, the ability of the
composition to retain or bring the somatic cell count (SCC) in milk
below about 500,000 cells per ml, the threshold value set by the
International Dairy Federation, above which, animals are considered
to have clinical mastitis, will be indicative of a therapeutic
effect.
[0187] The exact amount is readily determined by one skilled in the
art using standard tests. The chimeric GapC plasmin-binding protein
concentration will typically range from about 1% to about 95% (w/w)
of the composition, or even higher or lower if appropriate. With
the present vaccine formulations, 5 to 500 .mu.g of active
ingredient per ml of injected solution should be adequate to raise
an immunological response when a dose of 1 to 3 ml per animal is
administered.
[0188] To immunize a subject, the vaccine is generally administered
parenterally, usually by intramuscular injection. Other modes of
administration, however, such as subcutaneous, intraperitoneal and
intravenous injection, are also acceptable. The quantity to be
administered depends on the animal to be treated, the capacity of
the animal's immune system to synthesize antibodies, and the degree
of protection desired. Effective dosages can be readily established
by one of ordinary skill in the art through routine trials
establishing dose response curves. The subject is immunized by
administration of the vaccine in at least one dose, and preferably
two doses. Moreover, the animal may be administered as many doses
as is required to maintain a state of immunity to infection.
[0189] Additional vaccine formulations which are suitable for other
modes of administration include suppositories and, in some cases,
aerosol, intranasal, oral formulations, and sustained release
formulations. For suppositories, the vehicle composition will
include traditional binders and carriers, such as, polyalkaline
glycols, or triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of about
0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral
vehicles include such normally employed excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium,
stearate, sodium saccharin cellulose, magnesium carbonate, and the
like. These oral vaccine compositions may be taken in the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations, or powders, and contain from about 10% to about 95%
of the active ingredient, preferably about 25% to about 70%.
[0190] Intranasal formulations will usually include vehicles that
neither cause irritation to the nasal mucosa nor significantly
disturb ciliary function. Diluents such as water, aqueous saline or
other known substances can be employed with the subject invention.
The nasal formulations may also contain preservatives such as, but
not limited to, chlorobutanol and benzalkonium chloride. A
surfactant may be present to enhance absorption of the subject
proteins by the nasal mucosa.
[0191] Controlled or sustained release formulations are made by
incorporating the protein into carriers or vehicles such as
liposomes, nonresorbable impermeable polymers such as ethylenevinyl
acetate copolymers and Hytrel.RTM. copolymers, swellable polymers
such as hydrogels, or resorbable polymers such as collagen and
certain polyacids or polyesters such as those used to make
resorbable sutures. The chimeric GapC plasmin-binding proteins can
also be delivered using implanted mini-pumps, well known in the
art.
[0192] The chimeric GapC plasmin-binding proteins of the instant
invention can also be administered via a carrier virus which
expresses the same. Carrier viruses which will find use with the
instant invention include but are not limited to the vaccinia and
other pox viruses, adenovirus, and herpes virus. By way of example,
vaccinia virus recombinants expressing the novel proteins can be
constructed as follows. The DNA encoding the particular protein is
first inserted into an appropriate vector so that it is adjacent to
a vaccinia promoter and flanking vaceinia DNA sequences, such as
the sequence encoding thymidine kinase (TK). This vector is then
used to transfect cells which are simultaneously infected with
vaccinia. Homologous recombination serves to insert the vaccinia
promoter plus the gene encoding the instant protein into the viral
genome. The resulting TK recombinant can be selected by culturing
the cells in the presence of 5-bromodeoxyuridine and picking viral
plaques resistant thereto.
[0193] An alternative route of administration involves gene therapy
or nucleic acid immunization. Thus, nucleotide sequences (and
accompanying regulatory elements) encoding the subject chimeric
GapC plasmin-binding proteins can be administered directly to a
subject for in vivo translation thereof. Alternatively, gene
transfer can be accomplished by transfecting the subject's cells or
tissues ex vivo and reintroducing the transformed material into the
host. DNA can be directly introduced into the host organism, i.e.,
by injection (see International Publication No. WO/90/11092; and
Wolff et al. (1990) Science 247:1465-1468). Liposome-mediated gene
transfer can also be accomplished using known methods. See, e.g.,
Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol. 4:206-209;
Brigham et al. (1989) Am. J. Med. Sci. 298:278-281; Canonico et al.
(1991) Clin. Res. 39:219A; and Nabel et al. (1990) Science
249:1285-1288. Targeting agents, such as antibodies directed
against surface antigens expressed on specific cell types, can be
covalently conjugated to the liposomal surface so that the nucleic
acid can be delivered to specific tissues and cells susceptible to
infection.
Diagnostic Assays
[0194] As explained above, the chimeric GapC plasmin-binding
proteins of the present invention may also be used as diagnostics
to detect the presence of reactive antibodies of streptococcus, for
example S. dysgalactiae, in a biological sample in order to
determine the presence of streptococcus infection. For example, the
presence of antibodies reactive with chimeric GapC plasmin-binding
proteins can be detected using standard electrophoretic and
immunodiagnostic techniques, including immunoassays such as
competition, direct reaction, or sandwich type assays. Such assays
include, but are not limited to, Western blots; agglutination
tests; enzyme-labeled and mediated immunoassays, such as ELISAs;
biotin/avidin type assays; radioimmunoassays;
immunoelectrophoresis; immunoprecipitation, etc. The reactions
generally include revealing labels such as fluorescent,
chemiluminescent, radioactive, enzymatic labels or dye molecules,
or other methods for detecting the formation of a complex between
the antigen and the antibody or antibodies reacted therewith.
[0195] The aforementioned assays generally involve separation of
unbound antibody in a liquid phase from a solid phase support to
which antigen-antibody complexes are bound. Solid supports which
can be used in the practice of the invention include substrates
such as nitrocellulose (e.g., in membrane or microtiter well form);
polyvinylchloride (e.g., sheets or microtiter wells); polystyrene
latex (e.g., beads or microtiter plates); polyvinylidine fluoride;
diazotized paper; nylon membranes; activated beads, magnetically
responsive beads, and the like.
[0196] Typically, a solid support is first reacted with a solid
phase component (e.g., one or more chimeric GapC plasmin-binding
proteins) under suitable binding conditions such that the component
is sufficiently immobilized to the support. Sometimes,
immobilization of the antigen to the support can be enhanced by
first coupling the antigen to a protein with better binding
properties. Suitable coupling proteins include, but are not limited
to, macromolecules such as serum albumins including bovine serum
albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules,
thyroglobulin, ovalbumin, and other proteins well known to those
skilled in the art. Other molecules that can be used to bind the
antigens to the support include polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers,
and the like. Such molecules and methods of coupling these
molecules to the antigens, are well known to those of ordinary
skill in the art. See, e.g., Brinkley, M. A. Bioconjugate Chem.
(1992) 3:2-13; Hashida et al., J. Appl. Biochem. (1984) 6:56-63;
and Anjaneyulu and Staros, International J. of Peptide and Protein
Res. (1987) 30:117-124.
[0197] After reacting the solid support with the solid phase
component, any non-immobilized solid-phase components are removed
from the support by washing, and the support-bound component is
then contacted with a biological sample suspected of containing
ligand moieties (e.g., antibodies toward the immobilized antigens)
under suitable binding conditions. After washing to remove any
non-bound ligand, a secondary binder moiety is added under suitable
binding conditions, wherein the secondary binder is capable of
associating selectively with the bound ligand. The presence of the
secondary binder can then be detected using techniques well known
in the art.
[0198] More particularly, an ELISA method can be used, wherein the
wells of a microtiter plate are coated with a chimeric GapC
plasmin-binding protein. A biological sample containing or
suspected of containing anti-chimeric GapC plasmin-binding protein
immunoglobulin molecules is then added to the coated wells. After a
period of incubation sufficient to allow antibody binding to the
immobilized antigen, the plate(s) can be washed to remove unbound
moieties and a detectably labeled secondary binding molecule added.
The secondary binding molecule is allowed to react with any
captured sample antibodies, the plate washed and the presence of
the secondary binding molecule detected using methods well known in
the art.
[0199] Thus, in one particular embodiment, the presence of bound
anti-chimeric GapC plasmin-binding antigen ligands from a
biological sample can be readily detected using a secondary binder
comprising an antibody directed against the antibody ligands. A
number of anti-bovine immunoglobulin (Ig) molecules are known in
the art which can be readily conjugated to a detectable enzyme
label, such as horseradish peroxidase, alkaline phosphatase or
urease, using methods known to those of skill in the art. An
appropriate enzyme substrate is then used to generate a detectable
signal. In other related embodiments, competitive-type ELISA
techniques can be practiced using methods known to those skilled in
the art.
[0200] Assays can also be conducted in solution, such that the
chimeric GapC plasmin-binding proteins and antibodies specific for
those proteins form complexes under precipitating conditions. In
one particular embodiment, chimeric GapC plasmin-binding proteins
can be attached to a solid phase particle (e.g., an agarose bead or
the like) using coupling techniques known in the art, such as by
direct chemical or indirect coupling. The antigen-coated particle
is then contacted under suitable binding conditions with a
biological sample suspected of containing antibodies for the
chimeric GapC plasmin-binding proteins. Cross-linking between bound
antibodies causes the formation of particle-antigen-antibody
complex aggregates which can be precipitated and separated from the
sample using washing and/or centrifugation. The reaction mixture
can be analyzed to determine the presence or absence of
antibody-antigen complexes using any of a number of standard
methods, such as those immunodiagnostic methods described
above.
[0201] In yet a further embodiment, an immunoaffinity matrix can be
provided, wherein a polyclonal population of antibodies from a
biological sample suspected of containing anti-chimeric GapC
plasmin-binding molecules is immobilized to a substrate. In this
regard, an initial affinity purification of the sample can be
carried out using immobilized antigens. The resultant sample
preparation will thus only contain anti-streptococcus moieties,
avoiding potential nonspecific binding properties in the affinity
support. A number of methods of immobilizing immunoglobulins
(either intact or in specific fragments) at high yield and good
retention of antigen binding activity are known in the art. Not
being limited by any particular method, immobilized protein A or
protein G can be used to immobilize immunoglobulins.
[0202] Accordingly, once the immunoglobulin molecules have been
immobilized to provide an immunoaffinity matrix, labeled chimeric
GapC plasmin-binding proteins are contacted with the bound
antibodies under suitable binding conditions. After any
non-specifically bound antigen has been washed from the
immunoaffinity support, the presence of bound antigen can be
determined by assaying for label using methods known in the
art.
[0203] Additionally, antibodies raised to the chimeric GapC
plasmin-binding proteins, rather than the chimeric GapC
plasmin-binding proteins themselves, can be used in the
above-described assays in order to detect the presence of
antibodies to the proteins in a given sample. These assays are
performed essentially as described above and are well known to
those of skill in the art.
[0204] The above-described assay reagents, including the chimeric
GapC plasmin-binding proteins, or antibodies thereto, can be
provided in kits, with suitable instructions and other necessary
reagents, in order to conduct immunoassays as described above. The
kit can also contain, depending on the particular immunoassay used,
suitable labels and other packaged reagents and materials (i.e.
wash buffers and the like). Standard immunoassays, such as those
described above, can be conducted using these kits.
Deposits of Strains Useful in Practicing the Invention
[0205] A deposit of biologically pure cultures of the following
strains was made with the American Type Culture Collection, 10801
University Boulevard, Manassas, Va., under the provisions of the
Budapest Treaty. The accession number indicated was assigned after
successful viability testing, and the requisite fees were paid. The
designated deposits will be maintained for a period of thirty (30)
years from the date of deposit, or for five (5) years after the
last request for the deposit, whichever is longer. Should a culture
become nonviable or be inadvertently destroyed, or, in the case of
plasmid-containing strains, lose its plasmid, it will be replaced
with a viable culture(s) of the same taxonomic description.
[0206] Should there be a discrepancy between the sequence presented
in the present application and the sequence of the gene of interest
in the deposited plasmid due to routine sequencing errors, the
sequence in the deposited plasmid controls. TABLE-US-00001
Bacterial Strain Plasmid Deposit Date ATCC No. XLI Blue MRF
pPolyGap.1 May 31, 2000 PTA-1981 XLI Blue MRF pPolyGap.2 May 31,
2000 PTA-1974 XLI Blue MRF pPolyGap.3 May 31, 2000 PTA-1979 XLI
Blue MRF pPolyGap.4 May 31, 2000 PTA-1980 XLI Blue MRF polygap4 May
31, 2000 PTA-1978
[0207] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
C. EXPERIMENTAL
Example 1
Preparation of Chromosomal DNA
[0208] A clinical S. dysgalactiae isolate from a case of bovine
mastitis (ATCC Accession No. ATCC43078) was obtained from the
American Type Culture Collection (10801 University Boulevard,
Manassas, Va. 20110-2209), and was used as a source of DNA. The
organism was routinely grown on TSA sheep blood agar plates (PML
Microbiologicals, Mississauga, Ontario) at 37.degree. C. for 18
hours, or in Todd-Hewitt broth (Oxoid Ltd., Hampshire, England)
supplemented with 0.3% yeast extract (THB-YE) at 37.degree. C., 5%
CO.sub.2.
[0209] Chromosomal DNA was prepared from S. dysgalactiae grown in
100 ml of THB-YE supplemented with 20 mM glycine for approximately
6 hours, until an A.sub.600 of 0.8 to 1.0 was reached. Cells were
harvested and re-suspended in 50 mM EDTA, 50 mM Tris-HCl, 0.5%
Tween-20.RTM. (Sigma, St. Louis, Mo.) and supplemented with RNase A
(200 mg/ml), proteinase K (20 mg/ml), lysozyme (100 mg/ml) and
mutanolysin (100 mg/ml). (all enzymes purchased from SIGMA, St.
Louis, Mo.). Following bacterial lysis for 30 minutes at 37.degree.
C. with vigorous shaking, guanidine hydrochloride and Tween-2.RTM.,
pH 5.5, were mixed with the lysate to give a final concentration of
0.8 M and 5%, respectively. This mixture was incubated at
50.degree. C. for 30 minutes. The chromosomal DNA was then purified
using a Qiagen genomic-tip 100 g (Qiagen, Santa Clarita, Calif.)
and precipitated using 0.7 volumes of isopropanol. The resulting
pellet was washed in 70% ethanol and re-suspended in 0.5 ml 10 mM
Tris-HCl, pH 8.8.
[0210] Chromosomal DNA from S. agalactiae, S. uberis and, S.
parauberis was isolated essentially as described above, from
strains designated ATCC 27541, 9927, and 13386, respectively.
Chromosomal DNA from S. iniae was also isolated as above from a
strain designated 9117 obtained from Mount Sinai Hospital,
University of Toronto, Canada.
Example 2
Amplification and Cloning of gapC Genes from S. dysgalactiae, S.
uberis, S. parauberis, S. agalactiae and S. iniae
[0211] The polynucleotide sequences encoding GapC from S.
dysgalactiae, S. uberis, S. parauberis, S. agalactiae and S. iniae
were initially isolated from chromosomal DNA by PCR amplification.
The primers used to PCR-amplify the gapC genes from all species
were gapC1 (SEQ ID NO:1) and gapC1r (SEQ ID NO:2), shown in Table
1. In the table, underlining denotes nucleotides added to the
original sequences (i.e., nucleotides added to the 5' end of the
original sense strand sequence and to the 3' end of the original
anti-sense strand sequence, respectively, of the gapC coding region
being amplified), and bolding indicates the location of restriction
endonuclease recognition sites.
[0212] PCR was carried out using Vent DNA polymerase (New England
Biolabs, Mississauga, ON, Canada). A reaction mixture containing
0.2 .mu.g of genomic DNA, 1 pM of each of the preceding primers,
100 pM each of dATP, dTTP, dCTP and dGTP, 10 mM Tris HCL, pH9; 1.5
mM MgCl.sub.2, 50 mM HCL, 1.5 units Taq DNA polymerase (Pharmacia,
Quebec, Canada) was incubated for 40 amplification cycles of 40
seconds at 94.degree. C., 40 seconds at 55.degree. C., and 1
minute, 20 seconds at 72.degree. C., and then for a single cycle of
10 minutes at 72.degree. C.
[0213] The resulting PCR reaction products were then digested with
NdeI and BamHI. In the case of the S. dysgalactiae gapC product,
the fragment was cloned directly into the same sites of pET15b
(Novagen, Madison, Wis.) after the plasmid was digested with the
same enzymes. The resulting construct was denominated pET15bgapC.
In the case of the S. agalactiae, S. uberis, S. parauberis and S.
iniae sequences, each was first cloned into pPCR-Script using the
cloning protocol described in the PCR-Script Amp Cloning Kit
(Stratagene, La Jolla, Calif.), subsequently excised using NdeI and
BamHI, and finally re-cloned into the corresponding sites of pET15b
using conventional cloning protocols (see e.g., Sambrook et al.,
supra).
[0214] The plasmids containing the S. agalactiae, S. uberis, S.
parauberis and S. iniae sequences were designated pMF521c-inv,
pMF521a-inv, pMF521d-inv, and pMF521e-inv, respectively.
TABLE-US-00002 TABLE 1 Sequence Identification Numbers and
Correspond- ing Nucleotide and Amino Acid Sequences SEQ ID NO. Name
Nucleotide Sequence (5' to 3') gapC1 GG CGG CGG CAT ATG GTA GTT AAA
GTT GGT ATT AAC GG gapC1r GC GGA TCC TTA TTT AGC GAT TTT TGC AAA
GTA CTC Gap-1 AAA AAA GGA TCC GGT ATG GTA GTT AAA GTT GG Gap-2 AAA
AAA CCA TGG TTA CTC GAG TGC TTC CAG AAC GAT TTC Gap-3 AAA AAA CTC
GAG GGT ACT GTA GAA GTT AAA G Gap-4 AAA AAA CCA TGG TTA ATC GAT TTC
AAG AAC GAT TTC AAC ACC GTC Gap-5 AAA AAA ATC GAT GGT ACT GTT GAA
GTT AAA GAA G Gap-6 AAA AAA CCA TGG TTA ACT AGT TGC TTC AAG AAC GAT
TTC TAC GCC Gap-7 AAA AAA ACT AGT TTC TTT GCT AAA AAA GAA GCT GC
Gap-8 AAA AAA CCA TGG CTA TTA TTT AGC GAT TTT TGC AAA ATA CTC
Streptococcus (see FIG. 1) dysgalactiae gapC gene Streptococcus
dysgalactiae GapC protein Streptococcus (see FIG. 2) agalactiae
gapC gene Streptococcus agalactiae GapC protein Streptococcus (see
FIG. 3) uberis gapC gene Streptococcus uberis GapC protein
Streptococcus (see FIG. 4) parauberis gapC gene Streptococcus
parauberis GapC protein Streptococcus (see FIG. 5) iniae gapC gene
Streptococcus iniae GapC protein Gap4 chimeric (see FIG. 6) gapC
gene Gap4 chimeric GapC protein
Example 3
Sequencing of gapC Genes
[0215] The genes isolated and cloned in the preceding examples were
sequenced using fluorescence tag terminators on an ABI 373 DNA
automatic sequencer (Applied Biosystems, Emeryville, Calif.) at the
Plant Biotechnology Institute (PBI, Saskatoon, Saskatchewan,
Canada).
[0216] The nucleotide sequences so determined, and the
corresponding amino acid sequences deduced therefrom, are shown in
FIGS. 1 through 5.
Example 4
Construction of a Chimeric gapC Gene
[0217] A chimeric gapC gene composed of sequences from S.
dysgalactiae, S. parauberis, and S. agalactiae was constructed in a
three-step process using pAA556, a standard tac-inducible
expression plasmid derived from the plasmid pGH432 that contains
the signal sequence from the E. Coli ompF gene.
[0218] The partial gapC gene sequences used to construct the
chimeric gene were prepared by PCR amplification of selected
polynucleotide sequences from the genomic gapC genes isolated
above, using the primers Gap-1 through Gap-8. The primer sequences
are depicted in Table 1.
[0219] After assembly, the chimeric gene, sans the ompF signal
sequence, was then excised from pAA556 and inserted into the
plasmid pAA555, a pGH432 derivative that is a standard
tac-inducible expression plasmid containing the signal sequence
from the E. coli ompF gene.
A. Construction of pPolyGap.1
[0220] In the first step, the first 288 bases of the S.
dysgalactiae gapC gene were PCR amplified using the primers Gap-1
and Gap-2.
[0221] PCR amplification was carried out as follows: 1.6 .mu.g of
template DNA was combined in a reaction mixture containing 20 pM
each of primer Gap-1 (SEQ ID NO:1) and Gap-2 (SEQ ID NO:2), 200
.mu.m each of dATP, dCTP, dGTP and dTTP, 2.5 mM MgSO.sub.4, PCR
Buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), and 1 unit Taq DNA
polymerase (Pharmacia, Quebec, Canada). The mix was amplified for 1
cycle of 1 minute at 95.degree. C., then for 29 cycles of 1 minute
at 95.degree. C., 1 minute at 55.degree. C., and 30 seconds at
72.degree. C., and finally for 1 cycle of 10 minutes at 4.degree.
C.
[0222] The amplification product was then digested with BamHI and
NcoI and inserted into the same sites of an pAA556 vector. The
resulting plasmid construct, designated pPolyGap.1, is illustrated
in FIG. 21.
B. Construction of pPolyGap.2
[0223] A PCR product representing bases 170-285 of the S.
parauberis gapC gene was then obtained using the primers Gap-3 (SEQ
ID NO:5) and Gap-4 (SEQ ID NO:6). This product codes for an amino
acid sequence identical to the corresponding amino acid sequence
found in the S. uberis gapC gene. PCR amplification was carried out
essentially as above, except using 2 .mu.g of template DNA.
[0224] The S. parauberis PCR product and the pPolyGap1plasmid were
both digested with Xho1 and Nco1, and the PCR product was ligated
into the corresponding sites in the vector. This construct, called
pPolyGap.2, is illustrated in FIG. 22.
C. Construction of pPolyGap.3
[0225] Nucleotides 166-288 of the S. agalactiae gapC gene were
amplified using PCR primers Gap-5 (SEQ ID NO:7) and Gap-6 (SEQ ID
NO:8) as in Example 4B above.
[0226] The PCR product obtained was digested with ClaI and NcoI,
then inserted into the same sites of pPolyGap2 immediately
downstream of the S. parauberis sequence. pPolyGap3 is diagramed in
FIG. 23.
D. Construction of pPolyGap.4
[0227] The final step in constructing the chimeric gene involved
the insertion of the remaining S. dysgalactiae gapC sequence
(nucleotides 295-1011) in-frame and immediately downstream of the
S. agalactiae sequence.
[0228] The S. dysgalactiae sequence was first PCR amplified using
the primers Gap-7 (SEQ ID NO:9) and Gap-8 (SEQ ID NO:10) as in
Example 4A above. The amplification product was then digested with
the enzyme GamHi/NcoI, as was the pPolyGap.3 vector, and the
fragment was then ligated into the corresponding vector sites.
[0229] This final step resulted in the plasmid pPolyGap.4
containing the complete gapC chimeric gene construct comprising an
S. dysgalactiae gapC backbone with unique sequences from S.
parauberis as well as S. agalactiae. See FIG. 24.
E. Cloning of the Chimeric gapC Gene into pAA55: Construction of
PolyGap.4
[0230] The chimeric gapC gene constructed in the preceding steps
was excised from pAA556 by digestion with BamH1 and NcoI and
inserted into the plasmid pAA555 digested with the same enzymes.
pAA555 is identical to pAA556 except that the former plasmid
contains the LipoF signal sequence, and provides for the addition
of a cysteine at the amino terminal end of the mature GapC protein.
The N-terminal cysteine was added to insure the chimeric protein's
efficient secretion of from the cell and binding to the membrane
via the lipid-moiety. The coding sequence of the PolyGap4 plasmid
construct is shown in FIG. 25.
Example 5
Expression and Isolation of the Chimeric GapC Protein
[0231] PolyGap4 is used to transform E. coli J5 in the presence of
polyethlene glycol (Kurien and Scofield (1995) BioTechniques
18:1023-1026).
[0232] The transformed cells carrying pPolyGap4 are grown to
logarithmic phase in LB media at 37.degree. C. with shaking.
Expression of the chimeric GapC protein is then induced by adding
IPTG to a final concentration of 1 mM and incubating the cells at
37.degree. C. for an additional 4 hours.
[0233] The chimeric GapC protein is then extracted from the cell
surface by differential solubilization. The cells are collected by
centrifugation and re-suspended in a volume of resuspension buffer
(0.85% NaCl solution containing 0.6% sarkosyl) equal to 1/10th the
original culture volume. The suspension is incubated at room
temperature for 30 minutes with gentle shaking. The cells are
collected by centrifugation and the supernatant containing the
chimeric GapC protein is passed through a 0.2 .mu.m membrane
filter. Aliquots of the sterile supernatant are analyzed by
SDS-PAGE and Western blots using a rabbit anti-GapC polyclonal
antibody.
Example 6
Immunization of Animals with the Chimeric GapC Protein
[0234] Vaccines were formulated in such a fashion that they
contained 100 .mu.g/ml of purified chimeric GapC protein in the
oil-based adjuvant VSA3 (VIDO, Saskatoon, Saskatchewan, Canada).
VSA3 is a combination of Emulsigen Plus.TM. (MVP Laboratories,
Ralston, Nebr.) and dimethyldioctadecyl ammonium bromide (Kodak,
Rochester, N.Y.).
[0235] Non-lactating Holstein cows with no history of S.
dysgalactiae infection are obtained. Two weeks prior to
vaccination, all animals are treated with 300 mg of Cephapirin per
quarter (Cepha-dry.TM., Ayerst Laboratories, Montreal, Canada), in
order to resolve any pre-existing udder infection prior to the
vaccination step.
[0236] Groups of experimental animals are immunized subcutaneously
with two doses of vaccines containing the chimeric GapC protein or
a placebo with a three-week interval between immunizations. Ten
days to two weeks following the second immunization, animals are
exposed to 500-1,000 colony forming units of S. dysgalactiae
delivered into three quarters with an udder infusion cannula. The
fourth quarter on each animal serves as an un-infective
control.
[0237] All animals are examined daily for clinical signs of disease
and samples from all udder quarters are collected on each day.
Samples are observed for consistency and antibody titre, somatic
cell counts, and bacterial numbers are determined.
Example 7
Determination of Antibodies Specific for the Chimeric GapC
Protein
[0238] GapC-specific antibodies in bovine serum are measured using
an enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre
plates (NUNC, Naperville, Ill.) are coated by adding 0.1 ml
microgram per well purified chimeric protein in 50 mM sodium
carbonate buffer, pH 9.6, incubated overnight at 4.degree. C. The
liquid is removed and the wells are blocked with 3% bovine serum
albumin for 1 hr at 37.degree. C. Serial dilutions of bovine serum
(from 1:4 to 1:64,000) are added to the wells and incubated for 2
hours at room temperature. The wells are aspirated, washed and
incubated with 100 .mu.l of alkaline phosphatase-conjugated goat
anti-bovine IgG (Kirkgaard & Perry Laboratories Inc.,
Gaithersburg, Md.) for 1 hr at room temperature. The wells are
washed again, and 100 .mu.l of p-nitrophenol phosphate (Sigma, St.
Louis, Mo.) is added as a substrate to detect alkaline phosphatase
activity. The absorbance at 405 nanometers is recorded following 1
hr incubation with the substrate at room temperature.
Example 8
Bacterial Colonization
[0239] Bacteria are enumerated by spreading serial dilutions
(10.sup.0 to 10.sup.-6) directly onto TSA sheep blood agar plates
followed by overnight incubation at 37.degree. C., 5% CO.sub.2.
Colonization is defined as >500 cfu/ml of the challenge organism
recovered.
[0240] To confirm that the bacteria recovered from milk secretions
are S. dysgalactiae, selected colonies recovered from each animal
are tested using an API strep-20 test (bioMerieux SA, Hazelwood,
Mo.) according to the manufacturer's instructions. This test
identifies Streptococcus species according to an analytical profile
compiled on the basis of enzymatic activity and sugar fermentation,
using either an analytical profile index or identification
software.
[0241] The relationship between anti-GapC titer and bacterial
colonization is also determined.
Example 9
Determination of Inflammatory Response
[0242] Inflammatory response is measured as a function of mammary
gland somatic cell count i.e., lymphocytes, neutrophils, and
monocytes). Somatic cell counts are measured using standard
techniques recommended by Agriculture and AgriFood Canada (IDF50B
(1985): Milk and Milk Products--Methods of Sampling in a Coulter
counter). Samples are read within 48 hours of collection and
fixation, at days 1 through 7 post challenge.
[0243] The numbers of somatic cells present in the gland are
determined on each day post challenge.
[0244] Although preferred embodiments of the subject invention have
been described in some detail, it is understood that obvious
variations can be made without departing from the spirit and the
scope of the invention as defined by the appended claims.
Sequence CWU 1
1
28 1 37 DNA Artificial primer gapC1 1 ggcggcggca tatggtagtt
aaagttggta ttaacgg 37 2 35 DNA Artificial primer gapC1r 2
gcggatcctt atttagcgat ttttgcaaag tactc 35 3 32 DNA Artificial
primer gap-1 3 aaaaaaggat ccggtatggt agttaaagtt gg 32 4 39 DNA
Artificial primer Gap-2 4 aaaaaaccat ggttactcga gtgcttccag
aacgatttc 39 5 31 DNA Artificial primer Gap-3 5 aaaaaactcg
agggtactgt agaagttaaa g 31 6 45 DNA Artificial primer Gap-4 6
aaaaaaccat ggttaatcga tttcaagaac gatttcaaca ccgtc 45 7 34 DNA
Artificial primer Gap-5 7 aaaaaaatcg atggtactgt tgaagttaaa gaag 34
8 45 DNA Artificial primer Gap-6 8 aaaaaaccat ggttaactag ttgcttcaag
aacgatttct acgcc 45 9 35 DNA Artificial primer Gap-7 9 aaaaaaacta
gtttctttgc taaaaaagaa gctgc 35 10 42 DNA Artificial primer Gap-8 10
aaaaaaccat ggctattatt tagcgatttt tgcaaaatac tc 42 11 1011 DNA
Streptococcus dysgalactiae CDS (1)..(1011) 11 atg gta gtt aaa gtt
ggt att aac ggt ttc ggt cgt atc gga cgt ctt 48 Met Val Val Lys Val
Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15 gca ttc cgt
cgt att caa aat gtt gaa ggt gtt gaa gta act cgt atc 96 Ala Phe Arg
Arg Ile Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20 25 30 aac
gac ctt aca gat cca aac atg ctt gca cac ttg ttg aaa tac gat 144 Asn
Asp Leu Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35 40
45 aca act caa gga cgt ttt gac gga act gtt gaa gtt aaa gaa ggt gga
192 Thr Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys Glu Gly Gly
50 55 60 ttt gaa gta aac gga aac ttc atc aaa gtt tct gct gaa cgt
gat cca 240 Phe Glu Val Asn Gly Asn Phe Ile Lys Val Ser Ala Glu Arg
Asp Pro 65 70 75 80 gaa aac atc gac tgg gca act gac ggt gtt gaa atc
gtt ctg gaa gca 288 Glu Asn Ile Asp Trp Ala Thr Asp Gly Val Glu Ile
Val Leu Glu Ala 85 90 95 act ggt ttc ttt gct aaa aaa gaa gct gct
gaa aaa cac tta cat gct 336 Thr Gly Phe Phe Ala Lys Lys Glu Ala Ala
Glu Lys His Leu His Ala 100 105 110 aac ggt gct aaa aaa gtt gtt atc
aca gct cct ggt gga aac gac gtt 384 Asn Gly Ala Lys Lys Val Val Ile
Thr Ala Pro Gly Gly Asn Asp Val 115 120 125 aaa aca gtt gtt ttc aac
act aac cac gac att ctt gac ggt act gaa 432 Lys Thr Val Val Phe Asn
Thr Asn His Asp Ile Leu Asp Gly Thr Glu 130 135 140 aca gtt atc tca
ggt gct tca tgt act aca aac tgt tta gct cct atg 480 Thr Val Ile Ser
Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150 155 160 gct
aaa gct ctt cac gat gca ttt ggt atc caa aaa ggt ctt atg act 528 Ala
Lys Ala Leu His Asp Ala Phe Gly Ile Gln Lys Gly Leu Met Thr 165 170
175 aca atc cac gct tat act ggt gac caa atg atc ctt gac gga cca cac
576 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Leu Asp Gly Pro His
180 185 190 cgt ggt ggt gac ctt cgt cgt gct cgt gct ggt gct gca aac
att gtt 624 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Ala Asn
Ile Val 195 200 205 cct aac tca act ggt gct gct aaa gct atc ggt ctt
gtt atc cca gaa 672 Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu
Val Ile Pro Glu 210 215 220 ttg aat ggt aaa ctt gat ggt gct gca caa
cgt gtt cct gtt cca act 720 Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln
Arg Val Pro Val Pro Thr 225 230 235 240 gga tca gta act gag ttg gtt
gta act ctt gat aaa aac gtt tct gtt 768 Gly Ser Val Thr Glu Leu Val
Val Thr Leu Asp Lys Asn Val Ser Val 245 250 255 gac gaa atc aac gct
gct atg aaa gct gct tca aac gac agt ttc ggt 816 Asp Glu Ile Asn Ala
Ala Met Lys Ala Ala Ser Asn Asp Ser Phe Gly 260 265 270 tac act gaa
gat cca att gtt tct tca gat atc gta ggc gtg tca tac 864 Tyr Thr Glu
Asp Pro Ile Val Ser Ser Asp Ile Val Gly Val Ser Tyr 275 280 285 ggt
tca ttg ttt gac gca act caa act aaa gtt atg gaa gtt gac gga 912 Gly
Ser Leu Phe Asp Ala Thr Gln Thr Lys Val Met Glu Val Asp Gly 290 295
300 tca caa ttg gtt aaa gtt gta tca tgg tat gac aat gaa atg tct tac
960 Ser Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr
305 310 315 320 act gct caa ctt gtt cgt aca ctt gag tac ttt gca aaa
atc gct aaa 1008 Thr Ala Gln Leu Val Arg Thr Leu Glu Tyr Phe Ala
Lys Ile Ala Lys 325 330 335 taa 1011 12 336 PRT Streptococcus
dysgalactiae 12 Met Val Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile
Gly Arg Leu 1 5 10 15 Ala Phe Arg Arg Ile Gln Asn Val Glu Gly Val
Glu Val Thr Arg Ile 20 25 30 Asn Asp Leu Thr Asp Pro Asn Met Leu
Ala His Leu Leu Lys Tyr Asp 35 40 45 Thr Thr Gln Gly Arg Phe Asp
Gly Thr Val Glu Val Lys Glu Gly Gly 50 55 60 Phe Glu Val Asn Gly
Asn Phe Ile Lys Val Ser Ala Glu Arg Asp Pro 65 70 75 80 Glu Asn Ile
Asp Trp Ala Thr Asp Gly Val Glu Ile Val Leu Glu Ala 85 90 95 Thr
Gly Phe Phe Ala Lys Lys Glu Ala Ala Glu Lys His Leu His Ala 100 105
110 Asn Gly Ala Lys Lys Val Val Ile Thr Ala Pro Gly Gly Asn Asp Val
115 120 125 Lys Thr Val Val Phe Asn Thr Asn His Asp Ile Leu Asp Gly
Thr Glu 130 135 140 Thr Val Ile Ser Gly Ala Ser Cys Thr Thr Asn Cys
Leu Ala Pro Met 145 150 155 160 Ala Lys Ala Leu His Asp Ala Phe Gly
Ile Gln Lys Gly Leu Met Thr 165 170 175 Thr Ile His Ala Tyr Thr Gly
Asp Gln Met Ile Leu Asp Gly Pro His 180 185 190 Arg Gly Gly Asp Leu
Arg Arg Ala Arg Ala Gly Ala Ala Asn Ile Val 195 200 205 Pro Asn Ser
Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Ile Pro Glu 210 215 220 Leu
Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg Val Pro Val Pro Thr 225 230
235 240 Gly Ser Val Thr Glu Leu Val Val Thr Leu Asp Lys Asn Val Ser
Val 245 250 255 Asp Glu Ile Asn Ala Ala Met Lys Ala Ala Ser Asn Asp
Ser Phe Gly 260 265 270 Tyr Thr Glu Asp Pro Ile Val Ser Ser Asp Ile
Val Gly Val Ser Tyr 275 280 285 Gly Ser Leu Phe Asp Ala Thr Gln Thr
Lys Val Met Glu Val Asp Gly 290 295 300 Ser Gln Leu Val Lys Val Val
Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310 315 320 Thr Ala Gln Leu
Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 325 330 335 13 1011
DNA Streptococcus agalactiae CDS (1)..(1011) 13 atg gta gtt aaa gtt
ggt att aac ggt ttc ggt cgt atc ggt cgt ctt 48 Met Val Val Lys Val
Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15 gca ttc cgt
cgc atc caa aac gta gaa ggt gtt gaa gtt act cgt atc 96 Ala Phe Arg
Arg Ile Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20 25 30 aac
gac ctt aca gat cca aac atg ctt gca cac ttg ttg aaa tat gac 144 Asn
Asp Leu Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35 40
45 aca act caa ggt cgt ttc gac ggt act gtt gaa gtt aaa gaa ggt gga
192 Thr Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys Glu Gly Gly
50 55 60 ttc gaa gtt aac ggt caa ttt gtt aaa gtt tct gct gaa cgc
gaa cca 240 Phe Glu Val Asn Gly Gln Phe Val Lys Val Ser Ala Glu Arg
Glu Pro 65 70 75 80 gca aac att gac tgg gct act gat ggc gta gaa atc
gtt ctt gaa gca 288 Ala Asn Ile Asp Trp Ala Thr Asp Gly Val Glu Ile
Val Leu Glu Ala 85 90 95 act ggt ttc ttt gca tca aaa gaa aaa gct
gga caa cac atc cat gaa 336 Thr Gly Phe Phe Ala Ser Lys Glu Lys Ala
Gly Gln His Ile His Glu 100 105 110 aat ggt gct aaa aaa gtt gtt atc
aca gct cct ggt gga aac gac gtt 384 Asn Gly Ala Lys Lys Val Val Ile
Thr Ala Pro Gly Gly Asn Asp Val 115 120 125 aaa aca gtt gtt ttc aac
act aac cac gat atc ctt gat gga act gaa 432 Lys Thr Val Val Phe Asn
Thr Asn His Asp Ile Leu Asp Gly Thr Glu 130 135 140 aca gtt atc tca
ggt gct tca tgt act aca aac tgt ctt gct cca atg 480 Thr Val Ile Ser
Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150 155 160 gct
aaa gct tta caa gac aac ttt ggt gtt aaa caa ggt ttg atg act 528 Ala
Lys Ala Leu Gln Asp Asn Phe Gly Val Lys Gln Gly Leu Met Thr 165 170
175 act atc cac gca tac act ggt gac caa atg atc ctt gac gga cca cac
576 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Leu Asp Gly Pro His
180 185 190 cgt ggt ggt gac ctt cgt cgt gct cgt gca ggt gct gca aac
atc gtt 624 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Ala Asn
Ile Val 195 200 205 cct aac tca act ggt gct gca aaa gct atc gga ctt
gtt atc cca gaa 672 Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu
Val Ile Pro Glu 210 215 220 ttg aac ggt aaa ctt gat ggt gct gca caa
cgt gtt cct gtt cca act 720 Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln
Arg Val Pro Val Pro Thr 225 230 235 240 gga tca gta act gaa ttg gtt
gca act ctt gaa aaa gac gta act gtc 768 Gly Ser Val Thr Glu Leu Val
Ala Thr Leu Glu Lys Asp Val Thr Val 245 250 255 gaa gaa gta aat gca
gct atg aaa gca gca gct aac gat tca tac ggt 816 Glu Glu Val Asn Ala
Ala Met Lys Ala Ala Ala Asn Asp Ser Tyr Gly 260 265 270 tat act gaa
gat cca atc gta tca tct gat atc gtt ggt att tca tac 864 Tyr Thr Glu
Asp Pro Ile Val Ser Ser Asp Ile Val Gly Ile Ser Tyr 275 280 285 ggt
tca ttg ttt gat gct act caa act aaa gtt caa act gtt gac ggt 912 Gly
Ser Leu Phe Asp Ala Thr Gln Thr Lys Val Gln Thr Val Asp Gly 290 295
300 aac caa ttg gtt aaa gtt gtt tca tgg tac gat aac gaa atg tca tac
960 Asn Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr
305 310 315 320 act tca caa ctt gtt cgt aca ctt gag tac ttt gca aaa
atc gct aaa 1008 Thr Ser Gln Leu Val Arg Thr Leu Glu Tyr Phe Ala
Lys Ile Ala Lys 325 330 335 taa 1011 14 336 PRT Streptococcus
agalactiae 14 Met Val Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile
Gly Arg Leu 1 5 10 15 Ala Phe Arg Arg Ile Gln Asn Val Glu Gly Val
Glu Val Thr Arg Ile 20 25 30 Asn Asp Leu Thr Asp Pro Asn Met Leu
Ala His Leu Leu Lys Tyr Asp 35 40 45 Thr Thr Gln Gly Arg Phe Asp
Gly Thr Val Glu Val Lys Glu Gly Gly 50 55 60 Phe Glu Val Asn Gly
Gln Phe Val Lys Val Ser Ala Glu Arg Glu Pro 65 70 75 80 Ala Asn Ile
Asp Trp Ala Thr Asp Gly Val Glu Ile Val Leu Glu Ala 85 90 95 Thr
Gly Phe Phe Ala Ser Lys Glu Lys Ala Gly Gln His Ile His Glu 100 105
110 Asn Gly Ala Lys Lys Val Val Ile Thr Ala Pro Gly Gly Asn Asp Val
115 120 125 Lys Thr Val Val Phe Asn Thr Asn His Asp Ile Leu Asp Gly
Thr Glu 130 135 140 Thr Val Ile Ser Gly Ala Ser Cys Thr Thr Asn Cys
Leu Ala Pro Met 145 150 155 160 Ala Lys Ala Leu Gln Asp Asn Phe Gly
Val Lys Gln Gly Leu Met Thr 165 170 175 Thr Ile His Ala Tyr Thr Gly
Asp Gln Met Ile Leu Asp Gly Pro His 180 185 190 Arg Gly Gly Asp Leu
Arg Arg Ala Arg Ala Gly Ala Ala Asn Ile Val 195 200 205 Pro Asn Ser
Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Ile Pro Glu 210 215 220 Leu
Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg Val Pro Val Pro Thr 225 230
235 240 Gly Ser Val Thr Glu Leu Val Ala Thr Leu Glu Lys Asp Val Thr
Val 245 250 255 Glu Glu Val Asn Ala Ala Met Lys Ala Ala Ala Asn Asp
Ser Tyr Gly 260 265 270 Tyr Thr Glu Asp Pro Ile Val Ser Ser Asp Ile
Val Gly Ile Ser Tyr 275 280 285 Gly Ser Leu Phe Asp Ala Thr Gln Thr
Lys Val Gln Thr Val Asp Gly 290 295 300 Asn Gln Leu Val Lys Val Val
Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310 315 320 Thr Ser Gln Leu
Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 325 330 335 15 1011
DNA Streptococcus uberis CDS (1)..(1011) 15 atg gta gtt aaa gtt ggt
att aac ggt ttc ggt cgt atc gga cgt ctt 48 Met Val Val Lys Val Gly
Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15 gca ttc cgt cgt
att caa aac gtt gaa ggt gtt gaa gta act cgt att 96 Ala Phe Arg Arg
Ile Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20 25 30 aac gat
ctt act gac cca aat atg ctt gca cac ttg ttg aaa tat gat 144 Asn Asp
Leu Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35 40 45
aca act caa ggt cgt ttc gac ggt aca gtt gaa gtt aaa gat ggt gga 192
Thr Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys Asp Gly Gly 50
55 60 ttc gaa gtt aac gga aac ttc atc aaa gtt tct gct gaa aaa gat
cca 240 Phe Glu Val Asn Gly Asn Phe Ile Lys Val Ser Ala Glu Lys Asp
Pro 65 70 75 80 gaa aac att gac tgg gca act gac ggt gta gaa atc gtt
ctt gaa gca 288 Glu Asn Ile Asp Trp Ala Thr Asp Gly Val Glu Ile Val
Leu Glu Ala 85 90 95 act ggt ttc ttt gct aaa aaa gca gct gct gaa
aaa cat tta cat gct 336 Thr Gly Phe Phe Ala Lys Lys Ala Ala Ala Glu
Lys His Leu His Ala 100 105 110 aac ggt gct aaa aaa gtt gtt atc aca
gct cct ggt gga gat gat gtt 384 Asn Gly Ala Lys Lys Val Val Ile Thr
Ala Pro Gly Gly Asp Asp Val 115 120 125 aaa act gtt gta ttt aac aca
aac cat gac att ctt gac ggt aca gaa 432 Lys Thr Val Val Phe Asn Thr
Asn His Asp Ile Leu Asp Gly Thr Glu 130 135 140 act gta att tca ggt
gct tca tgt act act aac tgt tta gct cca atg 480 Thr Val Ile Ser Gly
Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150 155 160 gct aaa
gct ttg caa gat aac ttt ggt gtt aaa caa ggt ttg atg aca 528 Ala Lys
Ala Leu Gln Asp Asn Phe Gly Val Lys Gln Gly Leu Met Thr 165 170 175
act atc cac gct tac act ggt gac caa atg atc ctt gac gga cca cac 576
Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Leu Asp Gly Pro His 180
185 190 cgt ggt ggt gac ctt cgt cgt gct cgt gct ggt gca agc aac att
gtt 624 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Ser Asn Ile
Val 195 200 205 cct aac tca act ggt gct gct aaa gca atc ggt ctt gta
atc cca gaa 672 Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu Val
Ile Pro Glu 210 215 220 tta aat ggt aaa ctt gac ggt gct gca caa cgt
gtt cct gtt cca act 720 Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg
Val Pro Val Pro Thr 225 230 235 240 gga tca gta act gaa tta gta gca
gtt ctt gaa aaa gaa act tca gtt 768 Gly Ser Val Thr Glu Leu Val Ala
Val Leu Glu Lys Glu Thr Ser Val 245 250 255 gaa gaa atc aac gca gca
atg aaa gca gct gca aac gat tca tac gga 816 Glu Glu Ile Asn Ala Ala
Met Lys Ala Ala Ala Asn Asp Ser Tyr Gly 260 265 270 tac act gaa gac
cca atc gta tct tct gat atc atc ggt atg gct tac 864 Tyr Thr Glu Asp
Pro Ile Val Ser Ser Asp Ile Ile Gly Met Ala Tyr 275 280 285 ggt tca
ttg ttt gat gct act caa act aaa gta caa act gtt gat gga 912 Gly Ser
Leu Phe Asp Ala Thr Gln Thr Lys Val Gln Thr Val Asp Gly 290 295 300
aat caa tta gtt aaa gtt gtt tca tgg tat gac aac gaa atg tct tac 960
Asn Gln Leu Val
Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310 315 320 act
gca caa ctt gtt cgt act ctt gag tac ttt gca aaa atc gct aaa 1008
Thr Ala Gln Leu Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 325
330 335 taa 1011 16 336 PRT Streptococcus uberis 16 Met Val Val Lys
Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15 Ala Phe
Arg Arg Ile Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20 25 30
Asn Asp Leu Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35
40 45 Thr Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys Asp Gly
Gly 50 55 60 Phe Glu Val Asn Gly Asn Phe Ile Lys Val Ser Ala Glu
Lys Asp Pro 65 70 75 80 Glu Asn Ile Asp Trp Ala Thr Asp Gly Val Glu
Ile Val Leu Glu Ala 85 90 95 Thr Gly Phe Phe Ala Lys Lys Ala Ala
Ala Glu Lys His Leu His Ala 100 105 110 Asn Gly Ala Lys Lys Val Val
Ile Thr Ala Pro Gly Gly Asp Asp Val 115 120 125 Lys Thr Val Val Phe
Asn Thr Asn His Asp Ile Leu Asp Gly Thr Glu 130 135 140 Thr Val Ile
Ser Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150 155 160
Ala Lys Ala Leu Gln Asp Asn Phe Gly Val Lys Gln Gly Leu Met Thr 165
170 175 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Leu Asp Gly Pro
His 180 185 190 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Ser
Asn Ile Val 195 200 205 Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly
Leu Val Ile Pro Glu 210 215 220 Leu Asn Gly Lys Leu Asp Gly Ala Ala
Gln Arg Val Pro Val Pro Thr 225 230 235 240 Gly Ser Val Thr Glu Leu
Val Ala Val Leu Glu Lys Glu Thr Ser Val 245 250 255 Glu Glu Ile Asn
Ala Ala Met Lys Ala Ala Ala Asn Asp Ser Tyr Gly 260 265 270 Tyr Thr
Glu Asp Pro Ile Val Ser Ser Asp Ile Ile Gly Met Ala Tyr 275 280 285
Gly Ser Leu Phe Asp Ala Thr Gln Thr Lys Val Gln Thr Val Asp Gly 290
295 300 Asn Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser
Tyr 305 310 315 320 Thr Ala Gln Leu Val Arg Thr Leu Glu Tyr Phe Ala
Lys Ile Ala Lys 325 330 335 17 1011 DNA Streptococcus parauberis
CDS (1)..(1011) 17 atg gta gtt aaa gtt ggt att aac ggt ttt ggc cgt
atc gga cgt ctt 48 Met Val Val Lys Val Gly Ile Asn Gly Phe Gly Arg
Ile Gly Arg Leu 1 5 10 15 gct ttc cgt cgt att caa aat gta gaa ggt
gtt gaa gtt act cgc atc 96 Ala Phe Arg Arg Ile Gln Asn Val Glu Gly
Val Glu Val Thr Arg Ile 20 25 30 aac gac ctt aca gat cca aat atg
ctt gca cac ttg tta aaa tac gat 144 Asn Asp Leu Thr Asp Pro Asn Met
Leu Ala His Leu Leu Lys Tyr Asp 35 40 45 aca act caa ggt cgt ttt
gac ggt act gta gaa gtt aaa gat ggt gga 192 Thr Thr Gln Gly Arg Phe
Asp Gly Thr Val Glu Val Lys Asp Gly Gly 50 55 60 ttt gac gtt aac
gga aaa ttc att aaa gtt tct gct gaa aaa gat cca 240 Phe Asp Val Asn
Gly Lys Phe Ile Lys Val Ser Ala Glu Lys Asp Pro 65 70 75 80 gaa caa
att gac tgg gca act gac ggt gtt gaa atc gtt ctt gaa gca 288 Glu Gln
Ile Asp Trp Ala Thr Asp Gly Val Glu Ile Val Leu Glu Ala 85 90 95
act ggt ttc ttt gct aaa aaa gca gct gct gaa aaa cat tta cat gaa 336
Thr Gly Phe Phe Ala Lys Lys Ala Ala Ala Glu Lys His Leu His Glu 100
105 110 aat ggt gct aaa aaa gtt gtt atc act gct cct ggt gga gat gac
gtg 384 Asn Gly Ala Lys Lys Val Val Ile Thr Ala Pro Gly Gly Asp Asp
Val 115 120 125 aaa aca gtt gta ttt aac act aac cat gat atc ctt gat
gga act gaa 432 Lys Thr Val Val Phe Asn Thr Asn His Asp Ile Leu Asp
Gly Thr Glu 130 135 140 aca gtt att tca ggt gct tca tgt act aca aac
tgt tta gct cca atg 480 Thr Val Ile Ser Gly Ala Ser Cys Thr Thr Asn
Cys Leu Ala Pro Met 145 150 155 160 gct aaa gct tta caa gat aac ttt
ggc gta aaa caa ggt tta atg act 528 Ala Lys Ala Leu Gln Asp Asn Phe
Gly Val Lys Gln Gly Leu Met Thr 165 170 175 aca atc cac gct tac act
ggt gat caa atg ctt ctt gat gga cct cac 576 Thr Ile His Ala Tyr Thr
Gly Asp Gln Met Leu Leu Asp Gly Pro His 180 185 190 cgt ggt ggt gac
tta cgt cgt gcc cgt gct ggt gct aac aat att gtt 624 Arg Gly Gly Asp
Leu Arg Arg Ala Arg Ala Gly Ala Asn Asn Ile Val 195 200 205 cct aac
tca act ggt gct gct aaa gca atc ggt ctt gtt atc cct gaa 672 Pro Asn
Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Ile Pro Glu 210 215 220
tta aat ggt aaa ctt gac ggt gct gca caa cgt gta cca gtt cca aca 720
Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg Val Pro Val Pro Thr 225
230 235 240 ggt tca gta aca gaa tta gta gca gtt ctt aat aaa gaa act
tca gta 768 Gly Ser Val Thr Glu Leu Val Ala Val Leu Asn Lys Glu Thr
Ser Val 245 250 255 gaa gaa att aac tca gta atg aaa gct gca gct aat
gat tca tat ggt 816 Glu Glu Ile Asn Ser Val Met Lys Ala Ala Ala Asn
Asp Ser Tyr Gly 260 265 270 tac act gaa gat cca atc gta tca tct gat
atc gtt ggt atg tct ttc 864 Tyr Thr Glu Asp Pro Ile Val Ser Ser Asp
Ile Val Gly Met Ser Phe 275 280 285 ggt tca tta ttc gat gct act caa
act aaa gta caa act gtt gat gga 912 Gly Ser Leu Phe Asp Ala Thr Gln
Thr Lys Val Gln Thr Val Asp Gly 290 295 300 aat caa tta gtt aaa gtt
gtt tca tgg tat gac aat gaa atg tct tac 960 Asn Gln Leu Val Lys Val
Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310 315 320 act gct caa
ctt gat cgt aca ctt gag tac ttt gca aaa atc gct aaa 1008 Thr Ala
Gln Leu Asp Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 325 330 335
taa 1011 18 336 PRT Streptococcus parauberis 18 Met Val Val Lys Val
Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15 Ala Phe Arg
Arg Ile Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20 25 30 Asn
Asp Leu Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35 40
45 Thr Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys Asp Gly Gly
50 55 60 Phe Asp Val Asn Gly Lys Phe Ile Lys Val Ser Ala Glu Lys
Asp Pro 65 70 75 80 Glu Gln Ile Asp Trp Ala Thr Asp Gly Val Glu Ile
Val Leu Glu Ala 85 90 95 Thr Gly Phe Phe Ala Lys Lys Ala Ala Ala
Glu Lys His Leu His Glu 100 105 110 Asn Gly Ala Lys Lys Val Val Ile
Thr Ala Pro Gly Gly Asp Asp Val 115 120 125 Lys Thr Val Val Phe Asn
Thr Asn His Asp Ile Leu Asp Gly Thr Glu 130 135 140 Thr Val Ile Ser
Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150 155 160 Ala
Lys Ala Leu Gln Asp Asn Phe Gly Val Lys Gln Gly Leu Met Thr 165 170
175 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Leu Leu Asp Gly Pro His
180 185 190 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Asn Asn
Ile Val 195 200 205 Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu
Val Ile Pro Glu 210 215 220 Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln
Arg Val Pro Val Pro Thr 225 230 235 240 Gly Ser Val Thr Glu Leu Val
Ala Val Leu Asn Lys Glu Thr Ser Val 245 250 255 Glu Glu Ile Asn Ser
Val Met Lys Ala Ala Ala Asn Asp Ser Tyr Gly 260 265 270 Tyr Thr Glu
Asp Pro Ile Val Ser Ser Asp Ile Val Gly Met Ser Phe 275 280 285 Gly
Ser Leu Phe Asp Ala Thr Gln Thr Lys Val Gln Thr Val Asp Gly 290 295
300 Asn Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr
305 310 315 320 Thr Ala Gln Leu Asp Arg Thr Leu Glu Tyr Phe Ala Lys
Ile Ala Lys 325 330 335 19 1011 DNA Streptococcus iniae CDS
(1)..(1011) 19 atg gta gtt aaa gtt ggt att aac ggt ttc gga cgt atc
ggt cgt ctt 48 Met Val Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile
Gly Arg Leu 1 5 10 15 gca ttc cgt cgt att caa aat gtt gaa ggt gtt
gaa gta act cgt atc 96 Ala Phe Arg Arg Ile Gln Asn Val Glu Gly Val
Glu Val Thr Arg Ile 20 25 30 aat gac ctt aca gat cct aac atg ctt
gca cac ttg ttg aaa tat gat 144 Asn Asp Leu Thr Asp Pro Asn Met Leu
Ala His Leu Leu Lys Tyr Asp 35 40 45 aca act caa ggt cgt ttt gac
ggt aca gtt gaa gtt aaa gat ggt gga 192 Thr Thr Gln Gly Arg Phe Asp
Gly Thr Val Glu Val Lys Asp Gly Gly 50 55 60 ttc gaa gtt aac gga
agc ttt gtt aaa gtt tct gca gaa cgc gaa cca 240 Phe Glu Val Asn Gly
Ser Phe Val Lys Val Ser Ala Glu Arg Glu Pro 65 70 75 80 gca aac att
gac tgg gct act gat ggt gta gac atc gtt ctt gaa gca 288 Ala Asn Ile
Asp Trp Ala Thr Asp Gly Val Asp Ile Val Leu Glu Ala 85 90 95 aca
ggt ttc ttc gct tct aaa gca gct gct gaa caa cac att cac gct 336 Thr
Gly Phe Phe Ala Ser Lys Ala Ala Ala Glu Gln His Ile His Ala 100 105
110 aac ggt gcg aaa aaa gtt gtt atc aca gct cct ggt gga aat gac gtt
384 Asn Gly Ala Lys Lys Val Val Ile Thr Ala Pro Gly Gly Asn Asp Val
115 120 125 aaa aca gtt gtt tac aac act aac cat gat att ctt gat gga
act gaa 432 Lys Thr Val Val Tyr Asn Thr Asn His Asp Ile Leu Asp Gly
Thr Glu 130 135 140 aca gtt atc tca ggt gct tca tgt act aca aac tgt
tta gct cca atg 480 Thr Val Ile Ser Gly Ala Ser Cys Thr Thr Asn Cys
Leu Ala Pro Met 145 150 155 160 gct aaa gca tta caa gat aac ttt ggt
gta aaa caa ggt tta atg act 528 Ala Lys Ala Leu Gln Asp Asn Phe Gly
Val Lys Gln Gly Leu Met Thr 165 170 175 act atc cat ggt tac act ggt
gac caa atg gtt ctt gac gga cca cac 576 Thr Ile His Gly Tyr Thr Gly
Asp Gln Met Val Leu Asp Gly Pro His 180 185 190 cgt ggt ggt gat ctt
cgt cgt gct cgt gca gct gca gca aac atc gtt 624 Arg Gly Gly Asp Leu
Arg Arg Ala Arg Ala Ala Ala Ala Asn Ile Val 195 200 205 cct aac tca
act ggt gct gct aaa gca atc ggt ctt gtt atc cca gaa 672 Pro Asn Ser
Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Ile Pro Glu 210 215 220 tta
aat ggt aaa ctt gac ggt gct gca caa cgt gtt cct gtt cca act 720 Leu
Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg Val Pro Val Pro Thr 225 230
235 240 gga tca gta act gaa tta gta gca gtt ctt gaa aaa gat act tca
gta 768 Gly Ser Val Thr Glu Leu Val Ala Val Leu Glu Lys Asp Thr Ser
Val 245 250 255 gaa gaa atc aat gca gct atg aaa gca gca gct aac gat
tca tac ggt 816 Glu Glu Ile Asn Ala Ala Met Lys Ala Ala Ala Asn Asp
Ser Tyr Gly 260 265 270 tac act gaa gat gct atc gta tca tca gat atc
gta ggt att tct tac 864 Tyr Thr Glu Asp Ala Ile Val Ser Ser Asp Ile
Val Gly Ile Ser Tyr 275 280 285 ggt tca tta ttt gat gct act caa act
aaa gta caa act gtt gat gga 912 Gly Ser Leu Phe Asp Ala Thr Gln Thr
Lys Val Gln Thr Val Asp Gly 290 295 300 aat caa ttg gtt aaa gtt gtt
tca tgg tat gac aat gaa atg tct tac 960 Asn Gln Leu Val Lys Val Val
Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310 315 320 act gct caa ctt
gtt cgt act ctt gag tac ttt gca aaa atc gct aaa 1008 Thr Ala Gln
Leu Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 325 330 335 taa
1011 20 336 PRT Streptococcus iniae 20 Met Val Val Lys Val Gly Ile
Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15 Ala Phe Arg Arg Ile
Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20 25 30 Asn Asp Leu
Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35 40 45 Thr
Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys Asp Gly Gly 50 55
60 Phe Glu Val Asn Gly Ser Phe Val Lys Val Ser Ala Glu Arg Glu Pro
65 70 75 80 Ala Asn Ile Asp Trp Ala Thr Asp Gly Val Asp Ile Val Leu
Glu Ala 85 90 95 Thr Gly Phe Phe Ala Ser Lys Ala Ala Ala Glu Gln
His Ile His Ala 100 105 110 Asn Gly Ala Lys Lys Val Val Ile Thr Ala
Pro Gly Gly Asn Asp Val 115 120 125 Lys Thr Val Val Tyr Asn Thr Asn
His Asp Ile Leu Asp Gly Thr Glu 130 135 140 Thr Val Ile Ser Gly Ala
Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150 155 160 Ala Lys Ala
Leu Gln Asp Asn Phe Gly Val Lys Gln Gly Leu Met Thr 165 170 175 Thr
Ile His Gly Tyr Thr Gly Asp Gln Met Val Leu Asp Gly Pro His 180 185
190 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Ala Ala Ala Asn Ile Val
195 200 205 Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Ile
Pro Glu 210 215 220 Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg Val
Pro Val Pro Thr 225 230 235 240 Gly Ser Val Thr Glu Leu Val Ala Val
Leu Glu Lys Asp Thr Ser Val 245 250 255 Glu Glu Ile Asn Ala Ala Met
Lys Ala Ala Ala Asn Asp Ser Tyr Gly 260 265 270 Tyr Thr Glu Asp Ala
Ile Val Ser Ser Asp Ile Val Gly Ile Ser Tyr 275 280 285 Gly Ser Leu
Phe Asp Ala Thr Gln Thr Lys Val Gln Thr Val Asp Gly 290 295 300 Asn
Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310
315 320 Thr Ala Gln Leu Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala
Lys 325 330 335 21 1347 DNA Artificial chimeric GapC protein CDS
(1)..(1347) 21 atg aaa aaa ata aca ggg att att tta ttg ctt ctt gca
gtc att att 48 Met Lys Lys Ile Thr Gly Ile Ile Leu Leu Leu Leu Ala
Val Ile Ile 1 5 10 15 ctg tct gca tgc cag gca aac tac gga tcc ggt
atg gta gtt aaa gtt 96 Leu Ser Ala Cys Gln Ala Asn Tyr Gly Ser Gly
Met Val Val Lys Val 20 25 30 ggt att aac ggt ttc ggt cgt atc gga
cgt ctt gca ttc cgt cgt att 144 Gly Ile Asn Gly Phe Gly Arg Ile Gly
Arg Leu Ala Phe Arg Arg Ile 35 40 45 caa aat gtt gaa ggt gtt gaa
gta act cgt atc aac gac ctt aca gat 192 Gln Asn Val Glu Gly Val Glu
Val Thr Arg Ile Asn Asp Leu Thr Asp 50 55 60 cca aac atg ctt gca
cac ttg ttg aaa tac gat aca act caa gga cgt 240 Pro Asn Met Leu Ala
His Leu Leu Lys Tyr Asp Thr Thr Gln Gly Arg 65 70 75 80 ttt gac gga
act gtt gaa gtt aaa gaa ggt gga ttt gaa gta aac gga 288 Phe Asp Gly
Thr Val Glu Val Lys Glu Gly Gly Phe Glu Val Asn Gly 85 90 95 aac
ttc atc aaa gtt tct gct gaa cgt gat cca gaa aac atc gac tgg 336 Asn
Phe Ile Lys Val Ser Ala Glu Arg Asp Pro Glu Asn Ile Asp Trp 100 105
110 gca act gac ggt gtt gaa atc gtt ctg gaa gca ctc gag ggt act gta
384 Ala Thr Asp Gly Val Glu Ile Val Leu Glu Ala Leu Glu Gly Thr Val
115 120 125 gaa gtt aaa gat ggt gga ttt gac gtt aac gga aaa ttc att
aaa gtt 432 Glu Val Lys Asp Gly Gly Phe Asp Val Asn Gly Lys Phe Ile
Lys Val 130 135 140 tct gct gaa aaa gat cca gaa caa att gac tgg gca
act gac ggt gtt 480 Ser Ala Glu Lys Asp Pro Glu Gln Ile Asp Trp Ala
Thr Asp Gly Val 145 150
155 160 gaa atc gtt ctt gaa atc gat ggt act gtt gaa gtt aaa gaa ggt
gga 528 Glu Ile Val Leu Glu Ile Asp Gly Thr Val Glu Val Lys Glu Gly
Gly 165 170 175 ttc gaa gtt aac ggt caa ttt gtt aaa gtt tct gct gaa
cgc gaa cca 576 Phe Glu Val Asn Gly Gln Phe Val Lys Val Ser Ala Glu
Arg Glu Pro 180 185 190 gca aac att gac tgg gct act gat ggc gta gaa
atc gtt ctt gaa gca 624 Ala Asn Ile Asp Trp Ala Thr Asp Gly Val Glu
Ile Val Leu Glu Ala 195 200 205 act agt ttc ttt gct aaa aaa gaa gct
gct gaa aaa cac tta cat gct 672 Thr Ser Phe Phe Ala Lys Lys Glu Ala
Ala Glu Lys His Leu His Ala 210 215 220 aac ggt gct aaa aaa gtt gtt
atc aca gct cct ggt gga aac gac gtt 720 Asn Gly Ala Lys Lys Val Val
Ile Thr Ala Pro Gly Gly Asn Asp Val 225 230 235 240 aaa aca gtt gtt
ttc aac act aac cac gac att ctt gac ggt act gaa 768 Lys Thr Val Val
Phe Asn Thr Asn His Asp Ile Leu Asp Gly Thr Glu 245 250 255 aca gtt
atc tca ggt gct tca tgt act aca aac tgt tta gct cct atg 816 Thr Val
Ile Ser Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 260 265 270
gct aaa gct ctt cac gat gca ttt ggt atc caa aaa ggt ctt atg act 864
Ala Lys Ala Leu His Asp Ala Phe Gly Ile Gln Lys Gly Leu Met Thr 275
280 285 aca atc cac gct tat act ggt gac caa atg atc ctt gac gga cca
cac 912 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Leu Asp Gly Pro
His 290 295 300 cgt ggt ggt gac ctt cgt cgt gct cgt gct ggt gct gca
aac att gtt 960 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Ala
Asn Ile Val 305 310 315 320 cct aac tca act ggt gct gct aaa gct atc
ggt ctt gtt atc cca gaa 1008 Pro Asn Ser Thr Gly Ala Ala Lys Ala
Ile Gly Leu Val Ile Pro Glu 325 330 335 ttg aat ggt aaa ctt gat ggt
gct gca caa cgt gtt cct gtt cca act 1056 Leu Asn Gly Lys Leu Asp
Gly Ala Ala Gln Arg Val Pro Val Pro Thr 340 345 350 gga tca gta act
gag ttg gtt gta act ctt gat aaa aac gtt tct gtt 1104 Gly Ser Val
Thr Glu Leu Val Val Thr Leu Asp Lys Asn Val Ser Val 355 360 365 gac
gaa atc aac gct gct atg aaa gct gct tca aac gac agt ttc ggt 1152
Asp Glu Ile Asn Ala Ala Met Lys Ala Ala Ser Asn Asp Ser Phe Gly 370
375 380 tac act gaa gat cca att gtt tct tca gat atc gta ggc gtg tca
tac 1200 Tyr Thr Glu Asp Pro Ile Val Ser Ser Asp Ile Val Gly Val
Ser Tyr 385 390 395 400 ggt tca ttg ttt gac gca act caa act aaa gtt
atg gaa gtt gac gga 1248 Gly Ser Leu Phe Asp Ala Thr Gln Thr Lys
Val Met Glu Val Asp Gly 405 410 415 tca caa ttg gtt aaa gtt gta tca
tgg tat gac aat gaa atg tct tac 1296 Ser Gln Leu Val Lys Val Val
Ser Trp Tyr Asp Asn Glu Met Ser Tyr 420 425 430 act gct caa ctt gtt
cgt aca ctt gag tat ttt gca aaa atc gct aaa 1344 Thr Ala Gln Leu
Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 435 440 445 taa
1347 22 448 PRT Artificial GapC multiple epitope fusion protein 22
Met Lys Lys Ile Thr Gly Ile Ile Leu Leu Leu Leu Ala Val Ile Ile 1 5
10 15 Leu Ser Ala Cys Gln Ala Asn Tyr Gly Ser Gly Met Val Val Lys
Val 20 25 30 Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu Ala Phe
Arg Arg Ile 35 40 45 Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile
Asn Asp Leu Thr Asp 50 55 60 Pro Asn Met Leu Ala His Leu Leu Lys
Tyr Asp Thr Thr Gln Gly Arg 65 70 75 80 Phe Asp Gly Thr Val Glu Val
Lys Glu Gly Gly Phe Glu Val Asn Gly 85 90 95 Asn Phe Ile Lys Val
Ser Ala Glu Arg Asp Pro Glu Asn Ile Asp Trp 100 105 110 Ala Thr Asp
Gly Val Glu Ile Val Leu Glu Ala Leu Glu Gly Thr Val 115 120 125 Glu
Val Lys Asp Gly Gly Phe Asp Val Asn Gly Lys Phe Ile Lys Val 130 135
140 Ser Ala Glu Lys Asp Pro Glu Gln Ile Asp Trp Ala Thr Asp Gly Val
145 150 155 160 Glu Ile Val Leu Glu Ile Asp Gly Thr Val Glu Val Lys
Glu Gly Gly 165 170 175 Phe Glu Val Asn Gly Gln Phe Val Lys Val Ser
Ala Glu Arg Glu Pro 180 185 190 Ala Asn Ile Asp Trp Ala Thr Asp Gly
Val Glu Ile Val Leu Glu Ala 195 200 205 Thr Ser Phe Phe Ala Lys Lys
Glu Ala Ala Glu Lys His Leu His Ala 210 215 220 Asn Gly Ala Lys Lys
Val Val Ile Thr Ala Pro Gly Gly Asn Asp Val 225 230 235 240 Lys Thr
Val Val Phe Asn Thr Asn His Asp Ile Leu Asp Gly Thr Glu 245 250 255
Thr Val Ile Ser Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 260
265 270 Ala Lys Ala Leu His Asp Ala Phe Gly Ile Gln Lys Gly Leu Met
Thr 275 280 285 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Leu Asp
Gly Pro His 290 295 300 Arg Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly
Ala Ala Asn Ile Val 305 310 315 320 Pro Asn Ser Thr Gly Ala Ala Lys
Ala Ile Gly Leu Val Ile Pro Glu 325 330 335 Leu Asn Gly Lys Leu Asp
Gly Ala Ala Gln Arg Val Pro Val Pro Thr 340 345 350 Gly Ser Val Thr
Glu Leu Val Val Thr Leu Asp Lys Asn Val Ser Val 355 360 365 Asp Glu
Ile Asn Ala Ala Met Lys Ala Ala Ser Asn Asp Ser Phe Gly 370 375 380
Tyr Thr Glu Asp Pro Ile Val Ser Ser Asp Ile Val Gly Val Ser Tyr 385
390 395 400 Gly Ser Leu Phe Asp Ala Thr Gln Thr Lys Val Met Glu Val
Asp Gly 405 410 415 Ser Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn
Glu Met Ser Tyr 420 425 430 Thr Ala Gln Leu Val Arg Thr Leu Glu Tyr
Phe Ala Lys Ile Ala Lys 435 440 445 23 1011 DNA Streptococcus
pyogenes 23 atggtagtta aagttggtat taacggtttc ggtcgtatcg gacgtcttgc
attccgccgt 60 attcaaaaca tcgaaggtgt tgaagtaact cgtatcaatg
accttacaga tccaaatatg 120 cttgcacact tgttgaaata cgatacaact
caaggacgtt ttgatggaac agttgaagtt 180 aaagaaggtg gatttgaagt
aaacggaaac ttcatcaaag tttctgctga acgtgatcca 240 gaaaacatcg
actgggcaac tgatggggtt gaaatcgttc tggaagcaac tggtttcttt 300
gctaaaaaag aagcagctga aaaacactta catgctaacg gtgctaaaaa agttgttatc
360 acagctcctg gtggaaacga tgttaaaaca gttgttttca acactaacca
cgacattctt 420 gacggtactg aaacagttat ctcaggtgct tcatgtacta
caaactgttt agctcctatg 480 gctaaagctc ttcacgatgc attcggtatc
caaaaaggtc ttatgactac aatccacgct 540 tatactggtg accaaatgat
ccttgacgga ccacaccgtg gtggtgacct tcgtcgtgca 600 cgcgctggtg
ctgcaaacat tgttcctaac tcaactggtg ctgctaaagc tatcggtctt 660
gttatcccag aacttaacgg taaacttgat ggtgctgcac aacgtgttcc tgttccaact
720 ggatcagtaa ctgagttggt tgtaactctt gacaaaaacg tttctgttga
cgaaatcaac 780 tctgctatga aagctgcttc aaacgacagc ttcggttaca
ctgaagatcc aattgtttct 840 tcagatatcg taggcgtatc atacggttca
ttgtttgacg caactcaaac taaagtaatg 900 gaagttgacg gatcacaatt
ggttaaagtt gtatcatggt atgacaacga aatgtcttac 960 actgctcaac
ttgtacgtac tcttgagtat ttcgcaaaaa ttgctaaata a 1011 24 336 PRT
Streptococcus pyogenes 24 Met Val Val Lys Val Gly Ile Asn Gly Phe
Gly Arg Ile Gly Arg Leu 1 5 10 15 Ala Phe Arg Arg Ile Gln Asn Ile
Glu Gly Val Glu Val Thr Arg Ile 20 25 30 Asn Asp Leu Thr Asp Pro
Asn Met Leu Ala His Leu Leu Lys Tyr Asp 35 40 45 Thr Thr Gln Gly
Arg Phe Asp Gly Thr Val Glu Val Lys Glu Gly Gly 50 55 60 Phe Glu
Val Asn Gly Asn Phe Ile Lys Val Ser Ala Glu Arg Asp Pro 65 70 75 80
Glu Asn Ile Asp Trp Ala Thr Asp Gly Val Glu Ile Val Leu Glu Ala 85
90 95 Thr Gly Phe Phe Ala Lys Lys Glu Ala Ala Glu Lys His Leu His
Ala 100 105 110 Asn Gly Ala Lys Lys Val Val Ile Thr Ala Pro Gly Gly
Asn Asp Val 115 120 125 Lys Thr Val Val Phe Asn Thr Asn His Asp Ile
Leu Asp Gly Thr Glu 130 135 140 Thr Val Ile Ser Gly Ala Ser Cys Thr
Thr Asn Cys Leu Ala Pro Met 145 150 155 160 Ala Lys Ala Leu His Asp
Ala Phe Gly Ile Gln Lys Gly Leu Met Thr 165 170 175 Thr Ile His Ala
Tyr Thr Gly Asp Gln Met Ile Leu Asp Gly Pro His 180 185 190 Arg Gly
Gly Asp Leu Arg Arg Ala Arg Ala Gly Ala Ala Asn Ile Val 195 200 205
Pro Asn Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Ile Pro Glu 210
215 220 Leu Asn Gly Lys Leu Asp Gly Ala Ala Gln Arg Val Pro Val Pro
Thr 225 230 235 240 Gly Ser Val Thr Glu Leu Val Val Thr Leu Asp Lys
Asn Val Ser Val 245 250 255 Asp Glu Ile Asn Ala Ala Met Lys Ala Ala
Ser Asn Asp Ser Phe Gly 260 265 270 Tyr Thr Glu Asp Pro Ile Val Ser
Ser Asp Ile Val Gly Val Ser Tyr 275 280 285 Gly Ser Leu Phe Asp Ala
Thr Gln Thr Lys Val Met Glu Val Asp Gly 290 295 300 Ser Gln Leu Val
Lys Val Val Ser Trp Tyr Asp Asn Glu Met Ser Tyr 305 310 315 320 Thr
Ala Gln Leu Val Arg Thr Leu Glu Tyr Phe Ala Lys Ile Ala Lys 325 330
335 25 1011 DNA Streptococcus equisimilis 25 atggtagtta aagttggtat
taacggtttc ggtcgtatcg gacgtcttgc attccgtcgt 60 attcaaaatg
ttgaaggtgt tgaagtaact cgtatcaacg accttacaga tccaaacatg 120
cttgcacact tgttgaaata cgatacaact caaggacgtt ttgacggaac tgttgaagtt
180 aaagaaggtg gatttgaagt aaacggaaac ttcatcaaag tttctgctga
acgtgatcca 240 gaaaacatcg actgggcaac tgacggtgtt gaaatcgttc
tggaagcaac tggtttcttt 300 gctaaaaaag aagctgctga aaaaccctta
catgctaacg gtgctaaaaa agttgttatc 360 acagctcctg gtggaaacga
cgttaaacag ttgttttcaa cactaaccac gagcattctt 420 gacggtactg
aaacagttat ctcaggtgct tcatgtacta caaactgttt agctcctatg 480
gctaaagctc ttcacgatgc atttggtatc caaaaaggtc ttatgactac aatccacgct
540 tatactggtg accaaatgat cgttgatgga caccgtggtg gtggtgatct
tcgtcgtgct 600 cgtgctggtg ctgcaaacat tgttcctaac tcaactggtg
ctcgtaaagc tatcggtctt 660 gttatcccag aattgaacgg taaacttgat
ggtgctgcac aacgtgttcc tgttccaact 720 ggatcagtaa ctgagttggt
tgtaactctt gacaaaaacg tttctgttga cgaaatcaac 780 gctgctatga
aagctgcttc aaacgacagc ttcggttaca ctgaagatcc aattgtttct 840
tcagatatcg taggcgtatc atacggttca ttgtttgacg caactcaaac taaagttatg
900 gaagttgatg gatcacaatt ggttaaagtt gtatcatggt atgacaacga
aatgtcttac 960 actgctcaac ttgttcgtac acttgagtat tttgcaaaaa
tcgctaaata a 1011 26 336 PRT Streptococcus equisimilis 26 Met Val
Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu 1 5 10 15
Ala Phe Arg Arg Ile Gln Asn Val Glu Gly Val Glu Val Thr Arg Ile 20
25 30 Asn Asp Leu Thr Asp Pro Asn Met Leu Ala His Leu Leu Lys Tyr
Asp 35 40 45 Thr Thr Gln Gly Arg Phe Asp Gly Thr Val Glu Val Lys
Glu Gly Gly 50 55 60 Phe Glu Val Asn Gly Asn Phe Ile Lys Val Ser
Ala Glu Arg Asp Pro 65 70 75 80 Glu Asn Ile Asp Trp Ala Thr Asp Gly
Val Glu Ile Val Leu Glu Ala 85 90 95 Thr Gly Phe Phe Ala Lys Lys
Glu Ala Ala Glu Lys Pro Leu His Ala 100 105 110 Asn Gly Ala Lys Lys
Val Val Ile Thr Ala Pro Gly Gly Asn Asp Val 115 120 125 Lys Gln Leu
Phe Ser Thr Leu Thr Thr Ser Ile Leu Asp Gly Thr Glu 130 135 140 Thr
Val Ile Ser Gly Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Met 145 150
155 160 Ala Lys Ala Leu His Asp Ala Phe Gly Ile Gln Lys Gly Leu Met
Thr 165 170 175 Thr Ile His Ala Tyr Thr Gly Asp Gln Met Ile Val Asp
Gly His Arg 180 185 190 Gly Gly Gly Asp Leu Arg Arg Ala Arg Ala Gly
Ala Ala Asn Ile Val 195 200 205 Pro Asn Ser Thr Gly Ala Arg Lys Ala
Ile Gly Leu Val Ile Pro Glu 210 215 220 Leu Asn Gly Lys Leu Asp Gly
Ala Ala Gln Arg Val Pro Val Pro Thr 225 230 235 240 Gly Ser Val Thr
Glu Leu Val Val Thr Leu Asp Lys Asn Val Ser Val 245 250 255 Asp Glu
Ile Asn Ala Ala Met Lys Ala Ala Ser Asn Asp Ser Phe Gly 260 265 270
Tyr Thr Glu Asp Pro Ile Val Ser Ser Asp Ile Val Gly Val Ser Tyr 275
280 285 Gly Ser Leu Phe Asp Ala Thr Gln Thr Lys Val Met Glu Val Asp
Gly 290 295 300 Ser Gln Leu Val Lys Val Val Ser Trp Tyr Asp Asn Glu
Met Ser Tyr 305 310 315 320 Thr Ala Gln Leu Val Arg Thr Leu Glu Tyr
Phe Ala Lys Ile Ala Lys 325 330 335 27 934 DNA Bos taurus 27
cgcatcgggc gcctggtcac cagggctgct tttaattctg gcaaagtgga catcgtcgcc
60 atcaatgacc ccttcattga ccttcactac atggtctaca tgttccagta
tgattccacc 120 cacggcaagt tcaacggcac agtcaaggca gagaacggga
agctcgtcat caatggaaag 180 gccatcacca tcttccagga gcgagatcct
gccaacatca agtggggtga tgctggtgct 240 gagtatgtag tggagtccac
tggggtcttc actaccatgg agaaggctgg ggctcacttg 300 aagggtggcg
ccaagagggt catcatctct gcaccttctg ccgatgcccc catgtttgtg 360
atgggcgtga accacgagaa gtataacaac accctcaaga ttgtcagcaa tgcctcctgc
420 accaccaact gcttggcccc cctggccaag gtcatccatg acccatttgg
catcgtggag 480 ggacttatga ccactgtcca cgccatcact gccacccaga
agactgtgga tggcccctcc 540 gggaagctgt ggcgtgacgg ccgaggggct
gcccagaata ttatccctgc ttctactggc 600 gctgccaagg ccgtgggcaa
ggtcatccct gagctcaacg ggaagctcac tggcatggcc 660 ttccgcgtcc
ccactcccaa cgtgtctgtt gtggatctga cctgccgcct ggagaaacct 720
gccaagtatg atgagatcaa gaaggtggtg aagcaggcgt cagagggccg tctcaagggc
780 attctaggct acactgagga ccaggttgtc tcctgcgact tcaacagcga
tactcactct 840 tccaccttcg atgctggggc tggcatagcc ctcaacgccc
actttgtcaa gctcatatcc 900 tggtacgaca atgaatttgg ctacagcaaa cagg 934
28 311 PRT Bos taurus 28 Arg Ile Gly Arg Leu Val Thr Arg Ala Ala
Phe Asn Ser Gly Lys Val 1 5 10 15 Asp Ile Val Ala Ile Asn Asp Pro
Phe Ile Asp Leu His Tyr Met Val 20 25 30 Tyr Met Phe Gln Tyr Asp
Ser Thr His Gly Lys Phe Asn Gly Thr Val 35 40 45 Lys Ala Glu Asn
Gly Lys Leu Val Ile Asn Gly Lys Ala Ile Thr Ile 50 55 60 Phe Gln
Glu Arg Asp Pro Ala Asn Ile Lys Trp Gly Asp Ala Gly Ala 65 70 75 80
Glu Tyr Val Val Glu Ser Thr Gly Val Phe Thr Thr Met Glu Lys Ala 85
90 95 Gly Ala His Leu Lys Gly Gly Ala Lys Arg Val Ile Ile Ser Ala
Pro 100 105 110 Ser Ala Asp Ala Pro Met Phe Val Met Gly Val Asn His
Glu Lys Tyr 115 120 125 Asn Asn Thr Leu Lys Ile Val Ser Asn Ala Ser
Cys Thr Thr Asn Cys 130 135 140 Leu Ala Pro Leu Ala Lys Val Ile His
Asp His Phe Gly Ile Val Glu 145 150 155 160 Gly Leu Met Thr Thr Val
His Ala Ile Thr Ala Thr Gln Lys Thr Val 165 170 175 Asp Gly Pro Ser
Gly Lys Leu Trp Arg Asp Gly Arg Gly Ala Ala Gln 180 185 190 Asn Ile
Ile Pro Ala Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val 195 200 205
Ile Pro Glu Leu Asn Gly Lys Leu Thr Gly Met Ala Phe Arg Val Pro 210
215 220 Thr Pro Asn Val Ser Val Val Asp Leu Thr Cys Arg Leu Glu Lys
Pro 225 230 235 240 Ala Lys Tyr Asp Glu Ile Lys Lys Val Val Lys Gln
Ala Ser Glu Gly 245 250 255 Pro Leu Lys Gly Ile Leu Gly Tyr Thr Glu
Asp Gln Val Val Ser Cys 260 265 270 Asp Phe Asn Ser Asp Thr His Ser
Ser Thr Phe Asp Ala Gly Ala Gly 275 280 285 Ile Ala Leu Asn Asp His
Phe Val Lys Leu Ile Ser Trp Tyr Asp Asn 290 295 300 Glu Phe Gly Tyr
Ser Lys Gln 305 310
* * * * *
References