U.S. patent application number 14/008344 was filed with the patent office on 2014-06-12 for compositions and methods for the detection of anaplasma platys.
The applicant listed for this patent is Yasuko Rikihisa. Invention is credited to Yasuko Rikihisa.
Application Number | 20140162256 14/008344 |
Document ID | / |
Family ID | 46932403 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140162256 |
Kind Code |
A1 |
Rikihisa; Yasuko |
June 12, 2014 |
COMPOSITIONS AND METHODS FOR THE DETECTION OF ANAPLASMA PLATYS
Abstract
Described herein are improved diagnostic tools for veterinary
and human use which can be used for serodiagnosing A. platys in
mammals, particularly in members of the Canidae family and in
humans. The diagnostic tools are a group of outer membrane proteins
of A. platys and variants thereof, referred to hereinafter as the
"OMP proteins", a group of outer membrane proteins of A. platys and
variants thereof referred to hereinafter as the "P44 proteins", and
antibodies to the OMP proteins and the P44 proteins.
Inventors: |
Rikihisa; Yasuko;
(Worthington, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rikihisa; Yasuko |
Worthington |
OH |
US |
|
|
Family ID: |
46932403 |
Appl. No.: |
14/008344 |
Filed: |
March 30, 2012 |
PCT Filed: |
March 30, 2012 |
PCT NO: |
PCT/US12/31580 |
371 Date: |
February 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61470209 |
Mar 31, 2011 |
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Current U.S.
Class: |
435/6.11 ;
435/6.15; 435/7.1 |
Current CPC
Class: |
G01N 2333/29 20130101;
C07K 14/29 20130101; C12Q 2600/16 20130101; C12Q 1/689 20130101;
C12Q 1/6893 20130101; A61K 39/395 20130101; G01N 33/56911 20130101;
G01N 2469/20 20130101; C07K 16/1246 20130101 |
Class at
Publication: |
435/6.11 ;
435/7.1; 435/6.15 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/569 20060101 G01N033/569 |
Goverment Interests
GOVERNMENT RIGHTS
[0001] This work was sponsored in part by a National Institutes of
Health Grant (R01A147885). The United States government may have
certain rights in the invention.
Claims
1-103. (canceled)
104. A method for detecting an infection or exposure to Anaplasma
in a subject, comprising contacting a biological sample from the
subject with a polypeptide, and assaying the biological sample for
the presence of an antibody that specifically binds the
polypeptide, wherein the polypeptide comprises an amino acid
sequence that is sufficiently conserved to detect infection or
exposure to at least two species of Anaplasma.
105. The method of claim 104, wherein the polypeptide is an
immunogenic fragment of an Anaplasma P44 protein.
106. The method of claim 104, wherein the polypeptide is an
immunogenic fragment of an Anaplasma OMP-1X protein.
107. The method of claim 104, wherein the polypeptide is encoded by
one or more of the nucleotides of SEQ ID NOs: 124-132.
108. The method of claim 104, wherein the Anaplasma is selected
from the group consisting of Anaplasma phagocytophilum, Anaplasma
marginale, Anaplasma centrale, Anaplasma mesaeterum, Anaplasma
ovis, and Anaplasma platys.
109. The method of claim 104, further comprising assaying the
biological sample from the subject for infection or exposure to
Ehrlichia canis.
110. A method for detecting an infection or exposure to Anaplasma
in a subject, comprising contacting a biological sample from the
subject with a DNA primer or probe comprising a nucleic acid
sequence that is sufficiently conserved to detect infection or
exposure to at least two species of Anaplasma.
111. The method of claim 110, wherein the DNA primer or probe
comprises a fragment of an Anaplasma P44 gene.
112. The method of claim 110, wherein the DNA primer or probe
comprises a fragment of an Anaplasma OMP-1X gene.
113. The method of claim 110, wherein the DNA primer or probe
comprises a fragment of an Anaplasma Tr1 gene.
114. The method of claim 110, wherein the Anaplasma is selected
from the group consisting of Anaplasma phagocytophilum, Anaplasma
marginale, Anaplasma centrale, Anaplasma mesaeterum, Anaplasma
ovis, and Anaplasma platys.
115. The method of claim 110, wherein the DNA primer or probe
comprises one or more of the nucleotides of SEQ ID NOs:
124-132.
116. The method of claim 110, wherein the DNA primer or probe is
capable of hybridizing to one or more of the nucleotides of SEQ ID
NOs: 124-132 under stringent conditions.
117. The method of claim 110, further comprising assaying the
biological sample from the subject for infection or exposure to
Ehrlichia canis.
Description
BACKGROUND
[0002] Anaplasma platys (formerly Ehrlichia platys), an obligatory
intracellular bacterium, was first described as a rickettsia-like
agent in the platelets of dogs from Florida with infectious canine
cyclic thrombocytopenia (ICCT) in 1978..sup.25 Authors pointed out
morphological and biological similarity of this bacterium to
Ehrlichia canis in infected dogs and Anaplasma marginale in
infected cattle, two members of the family Anaplasmataceae,.sup.25
which were well-known at that time. Clinical signs of ICCT are
fever, depression, appetite loss, anorexia, and bleeding
tendencies..sup.22 Parasitemia and thrombocytopenia occur in cycles
at approximately 10 to 14 day intervals..sup.22 Anaplasma platys
responds well to doxycycline, a tetracycline antibiotic, as the
primary means of treatment.
[0003] Based on indirect fluorescence antibody (IFA) tests using
the platelet-rich plasma from a dog experimentally infected with
ICCT, minimal serologic cross-reaction was found to occur between
A. platys and E. canis, and the researchers proposed the name
"Ehrlichia platys" for this bacterium..sup.22 In 1992, the 16S rRNA
gene sequence of A. platys was reported..sup.3 Subsequently, the
groEL gene sequence of A. platys was disclosed..sup.29, 67
Phylogenetic analysis of these sequences showed that this is a
distinct bacterium closely related to Anaplasma phagocytophilum and
Anaplasma marginale, which led to reclassification of this
bacterium into the genus Anaplasma..sup.17 Later it was reported
that although A. platys does not cross-react with serum antibodies
from dogs infected with E. canis on IFA tests, the A. platys
antigen cross-reacts with anti-Anaplasma phagocytophilum
antibodies..sup.32
[0004] Seropositive dogs have been found in Florida, Pennsylvania,
Texas, Louisiana, Illinois, California, Arkansas, Mississippi,
Idaho, and North Carolina. High rates of A. platys and E. canis
dual positive dogs have also been reported throughout these
areas..sup.22 A. platys DNA has also been detected in dogs
throughout Brazil,.sup.20 Greece,.sup.43 France,.sup.33
Taiwan,.sup.15 Spain,.sup.54 China,.sup.28 Australia,.sup.12
Portugal,.sup.13 the Democratic Republic of Congo,.sup.55
Japan,.sup.61 Thailand,.sup.29 and Venezuela..sup.59 It is believed
that the brown dog tick, Rhipicephalus sanguineus, is the
biological vector which transfers A. platys to potential hosts. In
fact, A. platys has been detected in brown dog ticks in Okinawa,
Japan,.sup.34 Spain,.sup.58 and the Democratic Republic of
Congo..sup.55 However, it has not been experimentally proven that
R. sanguineus is the biological vector responsible for the transfer
of A. platys..sup.56 To date, A. platys has never been culture
isolated. Consequently this bacterium is poorly understood at the
molecular, cellular, or immunologic level, and to date, no antigen
has been identified for this bacterium.
[0005] In A. phagocytophilum and A. marginale, surface-exposed
immunodominant 44 kDa major outer membrane proteins (P44s/Msp2s)
are encoded by the p44 (msp2) polymorphic multigene family..sup.6,
9, 39, 41, 69-71 In A. phagocytophilum, P44 proteins consist of a
single central hypervariable region of approximately 94 amino acid
residues and an N-terminal and C-terminal conserved regions of
approximately 186 and 146 amino acid residues, respectively..sup.41
A single polymorphic p44/msp2 expression locus (p44/msp2ES) is
found in the genome of A. phagocytophilum.sup.10 and A.
marginale,.sup.26 respectively. Both expression loci are found
downstream of tr1 genes encoding putative transcriptional factor
and homologs of Ehrlichia chaffeensis omp-1 genes encoding
polymorphic major outer membrane protein (MOMP)..sup.6, 8, 39 At
p44/msp2ES, p44s and msp2 donor sequences elsewhere in the genome
undergo recombination via RecF pathway to allow variable p44/msp2ES
expression under the same promoter..sup.6, 8, 39, 40 This mechanism
is thought to facilitate P44/Msp2 antigenic variation persistent
infection and for adaptation to new environments such as
transmission between tick and mammalian hosts..sup.7, 11, 38, 40,
65, 71 Purified native P44 from A. phagocytophilum and purified
native OMP-1s (P28 and OMP-1F) of Ehrlichia chaffeensis have porin
activity..sup.30, 37
[0006] Anaplasma platys (Apl) is an obligate intracellular bacteria
that infects platelets and causes a cyclic thrombocytopenia in the
dog. The observation than a dog can be affected by this rickettsial
agent, and the disease is most likely transmitted by the
Rhipicephalus spp of ticks. Anaplasma platys was first reported in
the United States in 1978 and has since been reported in Europe,
Asia, South America, the Middle East, Australia, and Africa.
Because of the common vector, Anaplasma platys infection is often
found as a co-infection with Ehrlichia canis. The ability of the
organism to produce clinical disease in the dog appears to vary
with geography, suggesting that strain differences may contribute
to virulence. Anaplasma platys is related to another Anaplasma
species known to cause clinical disease in the dog, Anaplasma
phagocytophilum (Aph).
[0007] Current diagnostic tests that attempt to distinguish Aph and
Anaplasma platys have limited specificity. PCR for Aph and
Anaplasma platys using 16SrRNA has also had problems with
specificity. Therefore, assays for specific detection of Anaplasma
platys are needed in the art. Additionally, serological tests for
Anaplasma platys are also needed.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0008] FIG. 1 shows the (A) strategy for A. platys p44ES cluster
sequencing. A. phagocytophilum p44ES and A. marginale msp2ES were
aligned to design primer F1 (targeting tr1/orf3 upstream highly
conserved region) and degenerate primers R1 (targeting p44ES/msp2
C-terminal highly conserved region), R2 (targeting conserved
intergenetic region between omp-1X/omp4 and omp-1N/omp3), R3
(targeting p44ES/msp2 N-terminal conserved region) and R4 (based on
p44ES/msp2ES downstream conserved valS gene). Primers F2 and F3
were designed based on the sequence results. (B) The final sequence
(4,009 bp) was assembled with the SeqMan program from the DNASTAR
software suite. Genes are represented as boxes with arrows
indicating their orientation. (C) The entire expression locus
fragment D (arrow) amplified from the dog 2 blood DNA specimen by
primers F1 and R5.
[0009] FIG. 2 depicts the synteny analysis, using the Artemis
comparison tool, of the A. platys (A. pl) p44ES cluster relative to
A. phagocytophilum (A. ph) and A. marginale (A. ma). Each bar
corresponds to a good match. Numbers indicate bp. Score cutoffs:
140.
[0010] FIG. 3 depicts the synteny analysis, using the Artemis
comparison tool, of the A. platys (A. pl) p44ES cluster relative to
A. phagocytophilum (A. ph) and A. marginale (A. ma). Each bar
corresponds to a good match. Numbers indicate bp. Score cutoffs:
140 using the Artemis Comparison Tool.
[0011] FIG. 4 shows the hylogenetic tree of OMP-1X proteins of A.
platys, A. phagocytophilum, A. marginale, E. canis, E. chaffeensis,
E. ewingii, E. ruminantium. The tree was constructed using DNASTAR
MegAlign Clustal W method.
[0012] FIG. 5 shows the Tr1 sequence alignment of A. platys, A.
phagocytophilum, and A. marginale.
[0013] FIG. 6A shows the phylogram of Tr1 proteins of A. platys, A.
phagocytophilum, and A. marginale. The tree was constructed using
DNASTAR MegAlign Clustal W method. FIG. 6B shows the amino acid
sequences identity between A. platys Tr1 and A. phagocytophilum Tr1
(YP.sub.--505749) or A. marginale AM1138 (YP.sub.--154239) were
86.4% and 73.1%, respectively.
[0014] FIG. 7 shows the OMP-1X sequence alignment of A. platys, A.
phagocytophilum, and A. marginale.
[0015] FIG. 8A shows the phylogram of OMP-1X proteins of A. platys,
A. phagocytophilum, and A. marginale. The tree was constructed
using DNASTAR MegAlign Clustal W method. FIG. 8B shows the amino
acid sequence identities between A. platys OMP-1X and A.
phagocytophilum OMP-1X (YP.sub.--505750) or A. marginale outer
membrane protein 1 (YP.sub.--154240) were 42.3% and 39.5%,
respectively.
[0016] FIG. 9 shows the P44 sequence alignment of A. platys, A.
phagocytophilum, and A. marginale.
[0017] FIG. 10A shows the phylogram of P44 proteins of A. platys,
A. phagocytophilum, and A. marginale. The tree was constructed
using DNASTAR MegAlign Clustal W method. FIG. 10B shows the amino
acid sequences identity between A. platys P44ES and A.
phagocytophilum P44-18ES (YP.sub.--505752) or A. marginale msp2
(YP.sub.--154245) were 55.0.about.56.9% and 41.5.about.42.1%,
respectively.
[0018] FIGS. 11A and 11B show P44ES/Msp2 proteins of A. platys, A.
phagocytophilum, and A. marginale. A total of 24 P44/Msp2s were
segregated into 3 clusters. The tree was constructed using the
Neighbor-Joining (NEIGHBOR program from PHYLIP) method based on the
alignment generated with Clustal V; 1,000 bootstrap replications
were performed. The nodes supported by bootstrap values greater
than 60% are labeled.
[0019] FIG. 13 shows the sequence alignment was completed using a
DNASTAR SeqMan program. Alignment of A. platys (A. pl) OMP-1X
protein with related proteins from A. phagocytophilum (A. ph), A.
marginale (A. ma), E. canis (E. ca), E. chaffeensis (E. ch), E.
ewingii (E. ew), and E. ruminantium (E. ru) using the Clustal W
method revealed a unique region in A. platys (AVQEKKPPEA (SEQ ID
NO: 98), box lined by dashed bar). The antigenic index and surface
probability profile suggest that this region is both antigenic and
surface-exposed.
[0020] FIG. 14 shows the ELISA analysis of samples from A. platys
PCR-positive dogs (No. 1-3), A. platys PCR-negative dogs (No. 4-6),
and A. phagocytophilum seropositive horse serum samples (No. 7-9)
with the A. platys specific peptide. The y axis shows the
OD.sub.415-OD.sub.492 values. A reaction was considered positive
when the plasmas from infected dogs yielded an
OD.sub.415-OD.sub.492 value greater than the mean
OD.sub.415-OD.sub.492 value for negative control plasma plus 3
standard deviations (dashed line). Representative data from
triplicate assays are shown.
[0021] FIG. 15 shows the alignment of A. platys, A.
phagocytophilum, and A. marginale p44/msp2 DNA. A. platys-species
specific primers useful for species-specific PCR diagnosis are
underlined in bold.
[0022] FIG. 16 shows the A. platys, A. phagocytophilum, and A.
marginale amino acid sequence alignment.
[0023] FIG. 17 shows the complete sequence assembly for the A.
platys expression locus.
[0024] FIG. 18 shows the A. platys P44 alignment (DNA and
protein).
[0025] FIGS. 19A and 19B show the A. platys-specific P44 sequence,
distinct from A. phagocytophilum and A. marginale or IDEXX P44
partial sequences useful for species-specific serodiagnosis.
[0026] FIG. 20 shows the comparison of the A. platys-specific P44
sequence to the sequences of A. phagocytophilum and A.
marginale.
[0027] FIG. 21 shows the A. platys-specific primer regions and
sequences from dog 2.
[0028] FIG. 22 shows the sequence alignment was completed using a
DNASTAR SeqMan program. Alignment of A. platys (A. pl) OMP-1X
protein with related proteins from A. phagocytophilum (A. ph), A.
marginale (A. ma), E. canis (E. ca), E. chaffeensis (E. ch), E.
ewingii (E. ew), and E. ruminantium (E. ru) using the Clustal W
method revealed a unique region in A. platys (AVQEKKPPEA (SEQ ID
NO: 98). The antigenic index and surface probability profile
suggest that this region is both antigenic and surface-exposed, and
distinct from those of other species. FIG. 22 also shows the
identification of two regions in A. platys that are both antigenic
and surface-exposed, and distinct from those of other species.
These two regions are identified in boxes as "1" and "2".
[0029] FIG. 23 shows the sequence alignment was completed using a
DNASTAR SeqMan program. Alignment of A. platys (A. pl) p44ES
protein with related proteins from A. phagocytophilum (A. ph), A.
marginale (A. ma), E. canis (E. ca), E. chaffeensis (E. ch), E.
ewingii (E. ew), and E. ruminantium (E. ru) using the Clustal W
method revealed regions in A. platys wherein the antigenic index
and surface probability profile suggest that these regions are both
antigenic and surface-exposed, and distinct from other species.
These regions are identified in boxes as "1" through "6" as well as
in brackets under the antigenic index and surface probability
profile.
DETAILED DESCRIPTION
[0030] Described herein are improved diagnostic tools for
veterinary and human use which can be used for serodiagnosing A.
platys in mammals, particularly in members of the Canidae family
and in humans. The diagnostic tools are a group of outer membrane
proteins of A. platys and variants thereof, referred to hereinafter
as the "OMP proteins", a group of outer membrane proteins of A.
platys and variants thereof referred to hereinafter as the "P44
proteins", and antibodies to the OMP proteins and the P44
proteins.
[0031] The present invention can be understood more readily by
reference to the following detailed description of the invention
and the Examples included therein.
[0032] All patents, patent applications, and publications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entireties into this application in order to
more fully describe the state of the art as known to those skilled
therein as of the date of the invention described and claimed
herein.
[0033] Unless otherwise expressly stated, it is in no way intended
that any method or aspect set forth herein be construed as
requiring that its steps be performed in a specific order.
Accordingly, where a method claim does not specifically state in
the claims or descriptions that the steps are to be limited to a
specific order, it is in no way intended that an order be inferred,
in any respect. This holds for any possible non-express basis for
interpretation, including matters of logic with respect to
arrangement of steps or operational flow, plain meaning derived
from grammatical organization or punctuation, or the number or type
of aspects described in the specification.
DEFINITIONS
[0034] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs. The
terminology used in the description of the embodiments herein is
for describing particular embodiments only and is not intended to
be limiting of the embodiments disclosed. As used in the
description, the singular forms "a," "an," and "the" are intended
to include the plural forms as well, unless the context clearly
indicates otherwise. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety.
[0035] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in this disclosure are to be understood as being modified in all
instances by the term "about." Accordingly, unless indicated to the
contrary, the numerical parameters set forth in this disclosure are
approximations that may vary depending upon the desired properties
sought to be obtained by the present disclosure. At the very least,
and not as an attempt to limit the application of the doctrine of
equivalents to the scope of any claims, each numerical parameter
should be construed in light of the number of significant digits
and ordinary rounding approaches.
[0036] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values described herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
throughout the application, data are provided in a number of
different formats, and that these data, represent endpoints,
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units is also
disclosed. For example, if 10 and 15 are disclosed, then 11, 12,
13, and 14 are also disclosed.
[0037] The term "subject" means an individual. In one aspect, a
subject is a mammal such as a primate, and, more preferably, a
human. Non-human primates include marmosets, monkeys, chimpanzees,
gorillas, orangutans, and gibbons, to name a few. The term
"subject" also includes domesticated animals, such as cats, dogs,
etc., livestock (for example, cattle (cows), horses, pigs, sheep,
goats, etc.), laboratory animals (for example, ferret, chinchilla,
mouse, rabbit, rat, gerbil, guinea pig, etc.) and avian species
(for example, chickens, turkeys, ducks, pheasants, pigeons, doves,
parrots, cockatoos, geese, etc.). Subjects can also include, but
are not limited to fish (for example, zebrafish, goldfish, tilapia,
salmon, and trout), amphibians and reptiles. As used herein, a
"subject" is the same as a "patient," and the terms can be used
interchangeably.
[0038] As used herein, the term "amino acid sequence" refers to a
list of abbreviations, letters, characters or words representing
amino acid residues. The amino acid abbreviations used herein are
conventional one letter codes for the amino acids and are expressed
as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic
acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K,
lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,
glutamine; R, arginine; S, serine; T, threonine; V, valine; W,
tryptophan; Y, tyrosine.
[0039] "Polypeptide" as used herein refers to any peptide,
oligopeptide, polypeptide, gene product, expression product, or
protein. A polypeptide is comprised of consecutive amino acids. The
term "polypeptide" encompasses naturally occurring or synthetic
molecules. The terms "polypeptide," "peptide," and "protein" can be
used interchangeably.
[0040] In addition, as used herein, the term "polypeptide" refers
to amino acids joined to each other by peptide bonds or modified
peptide bonds, e.g., peptide isosteres, etc. and may contain
modified amino acids other than the 20 gene-encoded amino acids.
The polypeptides can be modified by either natural processes, such
as post-translational processing, or by chemical modification
techniques which are well known in the art. Modifications can occur
anywhere in the polypeptide, including the peptide backbone, the
amino acid side-chains and the amino or carboxyl termini. The same
type of modification can be present in the same or varying degrees
at several sites in a given polypeptide. Also, a given polypeptide
can have many types of modifications. Modifications include,
without limitation, acetylation, acylation, ADP-ribosylation,
amidation, covalent cross-linking or cyclization, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of a phosphytidylinositol, disulfide bond formation,
demethylation, formation of cysteine or pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristolyation, oxidation,
pergylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, and transfer-RNA mediated
addition of amino acids to protein such as arginylation. (See
Proteins--Structure and Molecular Properties 2nd Ed., T. E.
Creighton, W.H. Freeman and Company, New York (1993);
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York, pp. 1-12 (1983)).
[0041] As used herein, "isolated polypeptide" or "purified
polypeptide" is meant to mean a polypeptide (or a fragment thereof)
that is substantially free from the materials with which the
polypeptide is normally associated in nature. The polypeptides of
the invention, or fragments thereof, can be obtained, for example,
by extraction from a natural source (for example, a mammalian
cell), by expression of a recombinant nucleic acid encoding the
polypeptide (for example, in a cell or in a cell-free translation
system), or by chemically synthesizing the polypeptide. In
addition, polypeptide fragments may be obtained by any of these
methods, or by cleaving full length proteins and/or
polypeptides.
[0042] As used herein, "peptidomimetic" means a mimetic of a
function of a protein which includes some alteration of the normal
peptide chemistry. Peptidomimetics typically are short sequences of
amino acids that in biological properties, mimic the function(s) of
a particular protein. Peptide analogs enhance some property of the
original peptide, such as increase stability, increased efficacy,
enhanced delivery, increased half life, etc. Methods of making
peptidomimetics based upon a known polypeptide sequence is
described, for example, in U.S. Pat. Nos. 5,631,280; 5,612,895; and
5,579,250. Use of peptidomimetics can involve the incorporation of
a non-amino acid residue with non-amide linkages at a given
position. One embodiment of the present invention is a
peptidomimetic wherein the compound has a bond, a peptide backbone
or an amino acid component replaced with a suitable mimic. Some
non-limiting examples of unnatural L- or D-amino acids which may be
suitable amino acid mimics include .beta.-alanine, L-.alpha.-amino
butyric acid, L-.gamma.-amino butyric acid, L-.alpha.-amino
isobutyric acid, L-.epsilon.-amino caproic acid, 7-amino heptanoic
acid, L-aspartic acid, L-glutamic acid,
N-.epsilon.-Boc-N-.alpha.-CBZ-L-lysine,
N-.epsilon.-Boc-N-.alpha.-Fmoc-L-lysine, L-methionine sulfone,
L-norleucine, L-norvaline, N-.alpha.-Boc-N-.delta.BZ-L-ornithine,
N-.delta.-Boc-N-.alpha.-CBZ-L-ornithine,
Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and
Boc-L-thioproline.
[0043] The word "or" as used herein means any one member of a
particular list and also includes any combination of members of
that list.
[0044] The phrase "nucleic acid" as used herein refers to a
naturally occurring or synthetic oligonucleotide or polynucleotide,
whether DNA or RNA or DNA-RNA hybrid, single-stranded or
double-stranded, sense or antisense, which is capable of
hybridization to a complementary nucleic acid by Watson-Crick
base-pairing. Nucleic acids of the invention can also include
nucleotide analogs (e.g., BrdU), and non-phosphodiester
internucleoside linkages (e.g., peptide nucleic acid (PNA) or
thiodiester linkages). In particular, nucleic acids can include,
without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any
combination thereof
[0045] As used herein, "isolated nucleic acid" or "purified nucleic
acid" is meant to mean DNA that is free of the genes that, in the
naturally-occurring genome of the organism from which the DNA of
the invention is derived, flank the gene. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector, such as an autonomously replicating plasmid or virus; or
incorporated into the genomic DNA of a prokaryote or eukaryote
(e.g., a transgene); or which exists as a separate molecule (for
example, a cDNA or a genomic or cDNA fragment produced by PCR,
restriction endonuclease digestion, or chemical or in vitro
synthesis). It also includes a recombinant DNA which is part of a
hybrid gene encoding additional polypeptide sequence. The term
"isolated nucleic acid" also refers to RNA, e.g., an mRNA molecule
that is encoded by an isolated DNA molecule, or that is chemically
synthesized, or that is separated or substantially free from at
least some cellular components, for example, other types of RNA
molecules or polypeptide molecules.
[0046] As used herein, "sample" is meant to mean an animal; a
tissue or organ from an animal; a cell (either within a subject,
taken directly from a subject, or a cell maintained in culture or
from a cultured cell line); a cell lysate (or lysate fraction) or
cell extract; or a solution containing one or more molecules
derived from a cell or cellular material (e.g. a polypeptide or
nucleic acid), which is assayed as described herein. A sample may
also be any body fluid or excretion (for example, but not limited
to, blood, urine, stool, saliva, tears, bile) that contains cells
or cell components.
[0047] As used herein, "modulate" is meant to mean to alter, by
increasing or decreasing.
[0048] As used herein, "effective amount" of a compound is meant to
mean a sufficient amount of the compound to provide the desired
effect. The exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of disease (or underlying genetic defect)
that is being treated, the particular compound used, its mode of
administration, and the like. Thus, it is not possible to specify
an exact "effective amount." However, an appropriate "effective
amount" may be determined by one of ordinary skill in the art using
only routine experimentation.
[0049] As used herein, "prevent" is meant to mean minimize the
chance that a subject who has an increased susceptibility for
developing A. platys infection will develop A. platys
infection.
[0050] As used herein, "specifically binds" is meant that an
antibody recognizes and physically interacts with its cognate
antigen (for example, the disclosed A. platys peptides) and does
not significantly recognize and interact with other antigens; such
an antibody may be a polyclonal antibody or a monoclonal antibody,
which are generated by techniques that are well known in the
art.
[0051] As used herein, "probe," "primer," or oligonucleotide is
meant to mean a single-stranded DNA or RNA molecule of defined
sequence that can base-pair to a second DNA or RNA molecule that
contains a complementary sequence (the "target"). The stability of
the resulting hybrid depends upon the extent of the base-pairing
that occurs. The extent of base-pairing is affected by parameters
such as the degree of complementarity between the probe and target
molecules and the degree of stringency of the hybridization
conditions. The degree of hybridization stringency is affected by
parameters such as temperature, salt concentration, and the
concentration of organic molecules such as formamide, and is
determined by methods known to one skilled in the art. Probes or
primers specific for nucleic acids capable of encoding the
disclosed A. platys peptides (for example, genes and/or mRNAs) have
at least 80%-90% sequence complementarity, preferably at least
91%-95% sequence complementarity, more preferably at least 96%-99%
sequence complementarity, and most preferably 100% sequence
complementarity to the region of the nucleic acid capable of
encoding the disclosed A. platys peptides to which they hybridize.
Probes, primers, and oligonucleotides may be detectably-labeled,
either radioactively, or non-radioactively, by methods well-known
to those skilled in the art. Probes, primers, and oligonucleotides
are used for methods involving nucleic acid hybridization, such as:
nucleic acid sequencing, reverse transcription and/or nucleic acid
amplification by the polymerase chain reaction, single stranded
conformational polymorphism (SSCP) analysis, restriction fragment
polymorphism (RFLP) analysis, Southern hybridization, Northern
hybridization, in situ hybridization, and electrophoretic mobility
shift assay (EMSA).
[0052] As used herein, "specifically hybridizes" is meant to mean
that a probe, primer, or oligonucleotide recognizes and physically
interacts (that is, base-pairs) with a substantially complementary
nucleic acid (for example, a nucleic acid capable of encoding the
disclosed A. platys peptides) under high stringency conditions, and
does not substantially base pair with other nucleic acids.
[0053] As used herein, "high stringency conditions" is meant to
mean conditions that allow hybridization comparable with that
resulting from the use of a DNA probe of at least 40 nucleotides in
length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM
EDTA, and 1% BSA (Fraction V), at a temperature of 65.degree. C.,
or a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH
7.6, 1X Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at
a temperature of 42.degree. C. Other conditions for high stringency
hybridization, such as for PCR, Northern, Southern, or in situ
hybridization, DNA sequencing, etc., are well-known by those
skilled in the art of molecular biology. (See, for example, F.
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York, N.Y., 1998).
[0054] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the disclosure are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Every numerical range given throughout this specification will
include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0055] In addition, where features or aspects of the inventions are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
Compositions
[0056] Described herein are the components to be used to prepare
the disclosed compositions as well as the compositions themselves
to be used within the methods disclosed herein. These and other
materials are disclosed herein, and it is understood that when
combinations, subsets, interactions, groups, etc. of these
materials are disclosed that while specific reference of each
various individual and collective combinations and permutation of
these compounds may not be explicitly disclosed, each is
specifically contemplated and described herein. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an example of a combination molecule, A-D is
disclosed, then even if each is not individually recited each is
individually and collectively contemplated meaning combinations,
A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C--F are considered
disclosed. Likewise, any subset or combination of these is also
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
would be considered disclosed. This concept applies to all aspects
of this application including, but not limited to, steps in methods
of making and using the disclosed compositions. Thus, if there are
a variety of additional steps that can be performed it is
understood that each of these additional steps can be performed
with any specific embodiment or combination of embodiments of the
disclosed methods.
[0057] Also disclosed are the components to be used to prepare the
disclosed compositions as well as the compositions themselves to be
used within the methods disclosed herein. These and other materials
are disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein.
[0058] Described herein are compositions and methods for the
detection of Anaplasma platys in a sample obtained from an animal,
particularly a member of the Canidae family. One embodiment of the
invention provides a PCR-based method for the amplification of
minute amounts of A. platys DNA isolated from canines. For example,
and not to be limiting, amplification of DNA can be carried out
with a high fidelity Taq polymerase.
Polynucleotides
[0059] Described herein are isolated or purified nucleotides. For
example, disclosed herein are Anaplasma platys nucleotides. The
disclosed Anaplasma platys nucleotides can be used in one or more
of the methods disclosed herein.
[0060] As used herein, "Anaplasma platys nucleotides" or "Anaplasma
platy polynucleotides" refers to the P44 or the OMP-1X nucleotide
sequences as well as combinations or fragments thereof described
herein. For example, Anaplasma platys nucleotides include, but are
not limited to, the P44 nucleotide sequences provided in the
Figures as well as the sequences provided in SEQ ID NOs: 30-38, SEQ
ID NOs: 46-51, combinations thereof as well as fragments thereof.
Such sequences can also be referred to as Anaplasma platys P44
nucleotides or Anaplasma platys P44 polynucleotides. Additional
examples of Anaplasma platys P44 nucleotides include, but are not
limited to Genebank Accession Nos: GQ868750, GU357491, GU357492,
GU357493, GU357494, GU357495, GU357496, GU357497, and.
HQ738571.
[0061] Also disclosed herein are regions of the Anaplasma platys
P44 and OMP-1X peptides that have been identified as being highly
antigenic as identified through the Jameson-Wolf method as well
through a surface probability plot analysis. These regions are
herein referred to as a "Box" regions. For example, six regions of
the Anaplasma platys P44 protein sequence have been identified in
FIGS. 19 and 23. These six regions, from the N-terminal to the
C-terminal regions are herein referred to as "P44 Box 1", "P44 Box
2", "P44 Box 3", "P44 Box 4", "P44 Box 5", and "P44 Box 6",
respectively. In addition, two regions of the Anaplasma platys
OMP-1X protein sequence have been identified in FIG. 22. These two
regions, from the N-terminal to the C-terminal regions are herein
referred to as "OMP-1X Box 1" and "OMP-1X Box 2", respectively.
[0062] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "P44 Box 1".
P44 Box 1 includes, but is not limited to, SEQ ID NO: 46. Also
disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "P44 Box 1". For
example, P44 Box 1 primers can include, but are not limited to: SEQ
ID NOs: 52 and 53.
[0063] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "P44 Box 2".
P44 Box 2 includes, but is not limited to, SEQ ID NO: 47. Also
disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "P44 Box 2". For
example, P44 Box 1 primers can include, but are not limited to: SEQ
ID NOs: 54 and 55.
[0064] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "P44 Box 3".
P44 Box 3 includes, but is not limited to, SEQ ID NO: 48. Also
disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "P44 Box 3". For
example, P44 Box 1 primers can include, but are not limited to: SEQ
ID NOs: 56 and 57.
[0065] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "P44 Box 4".
P44 Box 4 includes, but is not limited to, SEQ ID NO: 49. Also
disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "P44 Box 4". For
example, P44 Box 1 primers can include, but are not limited to: SEQ
ID NOs: 58 and 59.
[0066] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "P44 Box 5".
P44 Box 5 includes, but is not limited to, SEQ ID NO: 50. Also
disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "P44 Box 5". For
example, P44 Box 1 primers can include, but are not limited to: SEQ
ID NOs: 60 and 61.
[0067] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "P44 Box 6".
P44 Box 6 includes, but is not limited to, SEQ ID NO: 51. Also
disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "P44 Box 6". For
example, P44 Box 1 primers can include, but are not limited to: SEQ
ID NOs: 62 and 63.
[0068] Also disclosed are primers that can be used to amplify one
or more Anaplasma platys P44 nucleotides. Examples include, but are
not limited to SEQ ID NOs: 82-91.
[0069] Anaplasma platys nucleotides include, but are not limited
to, the OMP-1X nucleotide sequences provided in the Figures as well
as the sequences provided in SEQ ID NOs: 11-17, combinations
thereof as well as fragments thereof. Such sequences can also be
referred to as Anaplasma platys OMP-1X nucleotides or Anaplasma
platys OMP-1X polynucleotides. Other examples of Anaplasma platys
OMP-1X nucleotides include, but are not limited to the sequences
provided in GenBank Accession Nos: GQ868750, HQ738571,
GU357491.
[0070] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "OMP-1X Box
1". OMP-1X Box 1 includes, but is not limited to, SEQ ID NO: 14.
Also disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "OMP-1X Box 1".
[0071] Disclosed herein are isolated or purified polynucleotides
that consist of or comprise the nucleotide sequence of "OMP-1X Box
2". OMP-1X Box 2 includes, but is not limited to, SEQ ID NO: 15.
Also disclosed herein are isolated or purified polynucleotides that
consist of or comprise a polynucleotide sequence capable of
hybridizing to or amplifying the sequence of "OMP-1X Box 2".
[0072] The polynucleotides described herein can contain less than
an entire microbial genome and can be RNA, DNA, or combinations
thereof. Polynucleotides described herein can be isolated. An
isolated polynucleotide is a naturally-occurring polynucleotide
that is not immediately contiguous with one or both of the 5' and
3' flanking genomic sequences that it is naturally associated with.
Isolated polynucleotides can also include non-naturally occurring
nucleic acid molecules. In some aspects, polynucleotides can also
comprise fragments that encode immunogenic polypeptides.
[0073] In some aspects, polynucleotides described herein can be
probes or primers, for example, PCR primers, to detect the presence
of A. platys polynucleotides in a biological sample. Probes are
molecules capable of interacting with a target nucleic acid,
typically in a sequence specific manner, for example, through
hybridization. Primers are a subset of probes that can support an
enzymatic manipulation and that can hybridize with a target nucleic
acid sequence. A primer can be made from any combination of
nucleotides or nucleotide derivatives or analogs available in the
art that do not interfere with the manipulation of peptides,
enzymes, or proteins. In one aspect, the primers disclosed herein
can comprise any of the isolated polynucleotides described herein.
For example, and not to be limiting, the isolated polynucleotide
can be SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,
SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID
NO: 90, SEQ ID NO: 91, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,
SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID
NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,
SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO: 20, or SEQ ID NO: 99.
[0074] The hybridization of nucleic acids is well understood in the
art and hence need not be discussed herein. Typically a primer can
be made from any combination of nucleotides, nucleotide
derivatives, and analogs available in the art. The ability of such
probes and primers to specifically hybridize to A. platys
polynucleotide sequences can enable the primer to be used for the
detection of the presence of complementary sequences. In some
embodiments, polynucleotide primers and probes of the invention
described herein can hybridize to complementary sequences in a
sample, including saliva, blood, plasma, serum, cerebrospinal
fluid, and tissue. In some embodiments, the polynucleotides from
the sample can be subjected to gel electrophoresis, size separation
techniques, immobilization without size separation, and labeling.
Suitable labels and methods for labeling primers are known in the
art and include radioactive labels, biotin labels, fluorescent
labels, bioluminescent labels, and enzyme labels.
[0075] When referring to a nucleotide sequence "N" represents any
of the four common nucleotides (e.g., A, C, G, or T), "M"
represents either an A or C nucleotide, "S" will be defined to mean
C or G, and "Y" will be defined henceforth as C or T. For example,
SEQ ID NO: 85 (GCAAACCTAACACCMAAYTCMCCACC) includes an "M" at
positions 15 and 22. As such, position 15 or 22 of SEQ ID NO: 85
can be an A or C nucleotide. In addition, SEQ ID NO: 85 includes a
"Y" at position 19. As such, position 9 of SEQ ID NO: 85 can be a C
or T.
Primers
[0076] Disclosed herein are P44 primer sets comprising F1 through
F3 and R1 through R5. Each p44 primer set comprises a first primer,
i.e., forward, and a second primer, i.e., reverse, both of which
can be about 10 to about 35 nucleotides in length or a primer of
alternant length (e.g., 10-15, 10-20, 10-25, 10-30, 15-20, 15-25,
15-30, 15-35, 20-25, 20-30, 20-35, 25-30, 25-35, 30-35). The first
primer comprises a sequence that is complementary to a consecutive
sequence of at least 10 nucleotides in length, within the following
sequences: ATTATGTATGATTTATCCTAAGTTATCTGAG (SEQ ID NO: 82),
GGGATATCGGCGTTGATAGGG (SEQ ID NO: 83), and
GGTTTGTGTTGCTGGTGATTGGAGG (SEQ ID NO: 84). The second primer
comprises a sequence which is complementary to the inverse
complement of a consecutive sequence of at least 10 nucleotides in
length, within the following sequences: GCAAACCTAACACCMAAYTCMCCACC
(SEQ ID NO: 85), TATACTAAAAAAGAATTAAGTCAAGAG (SEQ ID NO: 86),
ATGGTAGAAASCCCCAGCAAA (SEQ ID NO: 87), CACGTNTTTAGTTACTGCCA (SEQ ID
NO: 88), and GTACTAGTCAGCGCCACTAACATCAA (SEQ ID NO: 89). As used
herein, "N" represents any of the four common nucleotides (e.g., A,
C, G, or T), "M" represents either an A or C nucleotide, "S" will
be defined to mean C or G, and "Y" will be defined henceforth as C
or T. Such primers can be useful for detecting the presence of A.
platys in members of the Anaplasmataceae family. HVF and HVR (Table
1) are the A. platys-specific primers (FIG. 16). Using the nested
PCR (genus-specific primer Pair F3 and R1 during the first PCR and
species-specific primer pair HVF and HVR in second PCR) sensitive
and A. platys-specific PCR can be performed. Such primers can also
useful for detecting the presence of A. platys DNA in samples
obtained from ticks or other invertebrate carriers that feed on the
vertebrate hosts.
[0077] Also disclosed herein are primers that can be used to
synthesize one or more of the A. platys polypeptides described
herein. For example, disclosed herein are primers that can be used
to produce an A. platys polypeptide comprising a sequence that is
capable of encoding a multimeric A. platys polypeptide wherein the
intervening sequence present in the full A. platys P44 or OMP-1X
nucleotide or peptide sequence are removed. For example, disclosed
herein are primers that can be used to add a restriction site into
a nucleic acid sequence described herein through inverse PCR. The
nucleotide can then be digested and self-ligated to remove a
specific intervening sequence. For example, SEQ ID NO: 66 (OMP-1X
box 1 and box 2 Forward primer: AACATATGAATCTTGTGAGCGCGG) can be
used to introduce an NdeI site in combination with SEQ ID NO: 67
(OMP-1X box 1 and box 2 Reverse primer: GGGGATCCGGCTGGGGGAGCAGAAG)
which can introduce a BamHI site.
[0078] Also disclosed are primers that can be used to remove an
intervening sequence between OMP-1X Box 1 and Box 2. For example,
the primer pair of SEQ ID NO: 68 can be used in combination with
SEQ ID NO: 69.
[0079] Also disclosed are primers that can be used to remove an
intervening sequence between P44 Box 1 and Box 2. For example, the
primer pair of SEQ ID NO: 70 can be used in combination with SEQ ID
NO: 71.
[0080] Also disclosed are primers that can be used to remove the
intervening sequence of P44 Box 1 and Box 2. For example, the
primer pair of SEQ ID NO: 72 can be used in combination with SEQ ID
NO: 73.
[0081] Also disclosed are primers that can be used to remove the
intervening sequence of P44 Box 3 and Box 4. For example, the
primer pair of SEQ ID NO: 74 can be used in combination with SEQ ID
NO: 75 or SEQ ID NO: 76 can be used in combination with SEQ ID NO:
77
[0082] Also disclosed are primers that can be used to remove the
intervening sequence of P44 Box 5 and Box 6. For example, the
primer pair of SEQ ID NO: 78 can be used in combination with SEQ ID
NO: 79 or SEQ ID NO: 80 can be used in combination with SEQ ID NO:
81
[0083] Also disclosed are methods for detecting A. platys provides
a p44 primer set comprising a first primer sequence which can be
complementary to a sequence of the A. platys p44 gene sense strand
and a second primer which can be complementary to the sequence of
the A. platys p44 gene antisense strand, amplifying the DNA in the
sample using a polymerase chain reaction (PCR) and the p44 primer
set, and determining the length which corresponds to the sequence
or length of that portion to which the first p44 primer and the
second p44 primer bind is indicative of the presence of A. platys
in the DNA sample.
[0084] Also disclosed herein are aspects related to primers in the
p44 primer set. The first p44 and the second p44 primers can be
from about 10 to about 35 nucleotides in length or a primer of
alternant length (e.g., 10-15, 10-20, 10-25, 10-30, 15-20, 15-25,
15-30, 15-35, 20-25, 20-30, 20-35, 25-30, 25-35, 30-35). The first
p44 primer, comprises a sequence which is substantially identical
to the complement of a consecutive sequence of at least 10
nucleotides in length, within the following sequence:
GAAGAATACGAAAGCGGCGG (SEQ ID NO: 90). In some embodiments, the
primer can be capable of hybridizing to a target sequence.
[0085] The second primer comprises a sequence which can be
complementary to the inverse complement of a consecutive sequence
of at least 10 nucleotides in length, within the following
sequence: TACTTAGGTCTTCCGCTTTCGC (SEQ ID NO: 91).
[0086] HVF and HVR (Table 1 and FIG. 16) can be useful for
detecting the presence of A. platys in samples obtained from
vertebrate animals such as humans or dogs, or from the invertebrate
vectors such as brown ticks, which can transmit this pathogen from
one vertebrate animal to another.
[0087] Also disclosed herein are compositions and methods for
detecting the presence of A. platys in samples obtained from a
vertebrate or invertebrate animal. The method comprises amplifying
the DNA contained within the sample using a primer set comprising
primers which comprise sequences that can be complementary to
select regions of the p44 gene of A. platys and a polymerase chain
reaction (PCR) to provide a pool of PCR products, and then assaying
the pool for the presence or absence of a PCR product whose length
or sequence indicates that PCR product corresponds to the region of
the p44 gene that is flanked by the nucleotide sequences which are
complementary to the first and second members of the p44 primer
set. The tools are the members of the p44 primer sets. Multiple A.
platys p44 gene sequences are set forth in GenBank under accession
No. GQ868750 and GU357491, respectively. Additional p44ES and p44
sequences were set forth in GenBank under accession No. GU357492,
GU357493, GU357494, GU357495, GU357496 and GU357497.
[0088] In some embodiments, the primers in the p44 primer set can
be based upon select sequences in the p44 gene of A. platys. The
p44 gene encodes a major outer membrane protein of A. platys. The
sequences of the first and second primers in the p44 primer set are
distinct from sequences found in the closely related p44 gene
homologs in A. phagocytophilum or A. marginale. The first primer in
the p44 primer set can be an oligonucleotide of various lengths,
including but not limited to 10-15, 10-20, 10-25, 10-30, 15-20,
15-25, 15-30, 15-35, 20-25, 20-30, 20-35, 25-30, 25-35, 30-35, and
10 to 35 nucleotides in length. In some embodiments, the first
primer can be at least 10 nucleotides in length. The second p44
primer in the A. platys primer set can be an oligonucleotide of
10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 15-35, 20-25,
20-30, 20-35, 25-30, 25-35, 30-35, or 10 to 35 nucleotides in
length. In one embodiment, the second p44 primer can be at least
about 10 nucleotides in length. The first p44 primer can comprise a
sequence which is substantially identical to the complement of
consecutive sequence located between nucleotide positions 540-559
of the sense strand of the open reading frame sequence of the p44
gene of A. platys.
[0089] As used herein the term "substantially identical" means that
the sequence is at least 90% identical, at least 95% identical, or
100% identical to a particular reference sequence (nucleotides
540-559 or 812-849) within FIG. 22.
[0090] The second p44 primer, comprises a sequence which is
substantially identical to and the inverse of a consecutive
sequence located between nucleotides 812-849 of the sense strand of
the p44 gene of A. platys. The sequence of the second p44 primer is
substantially identical to the complement of the inverse complement
of a consecutive sequence contained within FIG. 22. In some
embodiments, the primers can be capable of hybridizing to target
sequences.
[0091] In specific embodiments, the first and second primers in the
p44 primer set can comprise the sequences shown in Table 1. The
first and second primers can also comprise sequences which are
shorter by one to ten nucleotides than the sequences shown in Table
1 below. The first and second primers of the A. platys primer set
can also comprise a sequence which is longer than the sequences
shown in Table 1 below. Such sequences can have one to ten
additional nucleotides attached to the 5' end of the above-listed
sequences. The additional nucleotides can be selected from the
group consisting of adenine, cytosine, guanine, thymine, adenylic
acid, guanylic acid, and combinations thereof.
[0092] In another embodiment, the sequence of the nine first and
second p44 primer sets shown in Table 1 can be based upon a
comparison of the open-reading frame sequences of nine A. platys
isolates. Such primer sets can specifically amplify the target
sequence of multiple A. platys isolates, but not A. phagocytophilum
or A. marginale isolates. The primers shown in Table 1 are both
species-universal (e.g., F1, F2, F3, R1, R2, R3, R4, and R5) and
species-specific (e.g., HVF and HVR) for A. platys.
[0093] Disclosed herein are isolated polynucleotides encoding an
outer membrane protein of Anaplasma platys, or a fragment thereof.
In one aspect, the outer membrane protein can be P44 or OMP-1X
protein. In a further aspect, disclosed herein are isolated
polynucleotides comprising any of the sequences described herein,
or a fragment thereof. For example, and not to be limiting, the
polynucleotide sequence can be SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,
SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, or a fragment thereof. In yet a further
aspect, the isolated polynucleotide sequence can be a
polynucleotide capable of encoding any peptide sequence described
herein or a fragment thereof. For example and not to be limiting,
the polynucleotide sequence can be a polynucleotide capable of
encoding SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 1, SEQ
ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3,
SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, or
a fragment thereof. By way of further example, the polynucleotide
sequences disclosed herein can also be polynucleotides capable of
encoding the amino acid sequence comprising the amino acid from
about position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, a combination or a fragment thereof. In still a
further aspect, disclosed herein are isolated polynucleotides that
encode the peptide sequences described herein. For example, and not
to be limiting, the polynucleotide sequence can encode P44 Box 1,
P44, Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, OMP-1X Box
1, OMP-1X Box 2, OMP-1X Box 1 and OMP-1X Box 2, SEQ ID NO: 2, SEQ
ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8,
or a fragment thereof. By way of further example, the
polynucleotide sequences disclosed herein can also be
polynucleotides that can encode the amino acid sequence comprising
the amino acid from about position 66 to about position 192 of FIG.
22, the amino acid from about position 70 to about position 180 of
FIG. 22, a combination of the amino acid from about position 66 to
about position 192 and the amino acid from about position 70 to
about position 180 of FIG. 22, the amino acid from about position
240 to about position 312 of FIG. 22, the amino acid from about
position 230 to about position 300 of FIG. 22, a combination of the
amino acid from about position 240 to about position 312 and the
amino acid from about position 230 to about position 300 of FIG.
22, or a fragment thereof.
[0094] Also disclosed herein are isolated polynucleotides that
encode variants of the proteins described herein. In one aspect,
disclosed herein are isolated polynucleotides that can encode a
variant of an outer membrane protein of Anaplasma platys, or a
fragment thereof. For example, and not to be limiting, the outer
membrane protein can be P44 or OMP-1X protein. In a further aspect,
the polynucleotide can encode a variant that can have at least 95%
identity to, for example, and not to be limiting, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39,
SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID
NO: 44, SEQ ID NO: 45, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10,
SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO:
4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID
NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42,
SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID
NO: 97, the amino acid sequence comprising the amino acid from
about position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7,
SEQ ID NO: 4, SEQ ID NO: 8, the amino acid from about position 66
to about position 192 of FIG. 22, the amino acid from about
position 70 to about position 180 of FIG. 22, a combination of the
amino acid from about position 66 to about position 192 and the
amino acid from about position 70 to about position 180 of FIG. 22,
the amino acid from about position 240 to about position 312 of
FIG. 22, the amino acid from about position 230 to about position
300 of FIG. 22, a combination of the amino acid from about position
240 to about position 312 and the amino acid from about position
230 to about position 300 of FIG. 22, the amino acid from about
position 1 to about position 41 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 78 to about position
85 of FIG. 19, the amino acid from about position 174 to about
position 192 of FIG. 19, the amino acid sequence comprising the
amino acid from about position 227 to about position 234 of FIG. 19
the amino acid from about position 276 to about position 294 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 416 to about position 433 of FIG. 19, or a fragment
thereof. In a further aspect, the variant can be immunoreactive
with at least one antibody that binds to P44 protein, P44 Box 1,
P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, OMP-1X
protein, OMP-1X Box 1, OMP-1X Box 2, OMP-1X Box 1 and OMP-1X Box 2,
or a fragment thereof.
[0095] Disclosed herein are polynucleotides that contain less than
an entire microbial genome and can be single- or double-stranded
nucleic acids. A polynucleotide can be RNA, DNA, cDNA, genomic DNA,
chemically synthesized RNA or DNA or combinations thereof. The
polynucleotides can be purified free of other components, such as
proteins, lipids and other polynucleotides. For example, the
polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% purified. The polynucleotides described herein can encode one
or more of the polypeptides described elsewhere herein. For
example, disclosed herein are polynucleotides capable of encoding
the peptides described herein, for example: an Anaplasma platys P44
or OMP-1X protein; a variant of the Anaplasma platys P44 or OMP-1X
protein; or an antigenic fragment of the Anaplasma platys P44 or
OMP-1X protein, or fragments thereof. Polynucleotides can comprise
other nucleotide sequences, such as sequences coding for linkers,
signal sequences, TMR stop transfer sequences, transmembrane
domains, or ligands useful in protein purification such as
glutathione-S-transferase, histidine tag, and Staphylococcal
protein A.
[0096] The polynucleotides disclosed herein can be isolated. An
isolated polynucleotide is a naturally-occurring polynucleotide
that is not immediately contiguous with one or both of the 5' and
3' flanking genomic sequences that it is naturally associated with.
An isolated polynucleotide can be, for example, a recombinant DNA
molecule of any length, provided that the nucleic acid sequences
naturally found immediately flanking the recombinant DNA molecule
in a naturally-occurring genome is removed or absent. Isolated
polynucleotides can also include non-naturally occurring nucleic
acid molecules. A nucleic acid molecule existing among hundreds to
millions of other nucleic acid molecules within, for example, cDNA
or genomic libraries, or gel slices containing a genomic DNA
restriction digest are not to be considered an isolated
polynucleotide.
[0097] The polynucleotides disclosed herein can also comprise
fragments that encode immunogenic polypeptides. The polynucleotides
disclosed herein can encode full-length polypeptides, polypeptide
fragments, and variant or fusion polypeptides.
[0098] The polynucleotides disclosed herein can be degenerate
nucleotide sequences encoding one or more of the polypeptides
disclosed herein, as well as homologous nucleotide sequences that
are at least about 80, 85, 90, 95, 96, 97, 98, 99% or 100%
identical to the polynucleotide sequences disclosed herein and the
complements thereof are also disclosed herein. Percent sequence
identity can be calculated as described elsewhere herein.
Degenerate nucleotide sequences are polynucleotides that encode a
polypeptide of the invention or fragments thereof, but differ in
nucleic acid sequence from the wild-type polynucleotide sequence,
due to the degeneracy of the genetic code. Complementary DNA (cDNA)
molecules, species homologs, and variants of Anaplasma platys
polynucleotides that encode biologically functional Anaplasma
platys polypeptides also are Anaplasma platys polynucleotides.
[0099] The polynucleotides described herein can be isolated from
nucleic acid sequences present in, for example, a biological
sample, such as blood, serum, saliva, or tissue from an infected
individual. Polynucleotides can also be synthesized in the
laboratory, for example, using an automatic synthesizer. An
amplification method such as PCR can be used to amplify
polynucleotides from either genomic DNA or cDNA encoding the
polypeptides.
[0100] The polynucleotides disclosed herein can be used, for
example, as probes or primers, for example, PCR primers, to detect
the presence of Anaplasma platys polynucleotides in a test sample,
such as a biological sample. Probes are molecules capable of
interacting with a target nucleic acid, typically in a sequence
specific manner, for example, through hybridization. Primers are a
subset of probes that can support an enzymatic manipulation and
that can hybridize with a target nucleic acid such that the
enzymatic manipulation occurs. A primer can be made from any
combination of nucleotides or nucleotide derivatives or analogs
available in the art that do not interfere with the enzymatic
manipulation.
[0101] The hybridization of nucleic acids is well understood in the
art and discussed herein. Typically a probe can be made from any
combination of nucleotides or nucleotide derivatives or analogs
available in the art. The ability of such probes and primers to
specifically hybridize to Anaplasma platys P44 or Anaplasma platys
OMP-lx polynucleotide sequences will enable them to be of use in
detecting the presence of complementary sequences in a given test
sample. Polynucleotide probes and primers can hybridize to
complementary sequences in a test sample such as a biological
sample, including, but not limited to, saliva, sputum, blood,
plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid,
wound exudate, or tissue. Polynucleotides from the sample can be,
for example, subjected to gel electrophoresis or other size
separation techniques or can be immobilized without size
separation. The polynucleotide probes or primers can be labeled.
Suitable labels and methods for labeling probes and primers are
known in the art, and include, for example, radioactive labels
incorporated by nick translation or by kinase, biotin labels,
fluorescent labels, chemiluminescent labels, bioluminescent labels,
metal chelator labels and enzyme labels. The polynucleotides from
the sample are contacted with the probes or primers under
hybridization conditions of suitable stringencies.
[0102] Depending on the application, varying conditions of
hybridization can be used to achieve varying degrees of selectivity
of the probe or primer towards the target sequence. For
applications requiring high selectivity, relatively stringent
conditions can be used, such as low salt and/or high temperature
conditions, such as provided by a salt concentration of from about
0.02 M to about 0.15 M salt at temperatures of from about
50.degree. C. to about 70.degree. C. For applications requiring
less selectivity, less stringent hybridization conditions can be
used. For example, salt conditions from about 0.14 M to about 0.9M
salt, at temperatures ranging from about 20.degree. C. to about
55.degree. C. The presence of a hybridized complex comprising the
probe or primer and a complementary polynucleotide from the test
sample indicates the presence of Anaplasma platys or an Anaplasma
platys polynucleotide sequence in the sample.
Polypeptides
[0103] Described herein are isolated or purified polypeptides. For
example, disclosed herein are isolated or purified Anaplasma platys
polypeptides. The disclosed isolated or purified Anaplasma platys
polypeptides can be used in one or more of the methods disclosed
herein.
[0104] A polypeptide can be a polymer of three or more amino acids
covalently linked by amide bonds. A polypeptide can be
post-translationally modified. A purified polypeptide can be a
polypeptide preparation that is substantially free of cellular
material, other peptides and polypeptides, chemical precursors,
synthetic chemicals, or combinations thereof.
[0105] As used herein, "Anaplasma platys peptides" or "Anaplasma
platy proteins" refers to the P44 or the OMP-1X peptide sequences
as well as combinations or fragments thereof described herein. For
example, Anaplasma platys peptides include, but are not limited to,
the P44 amino acid sequences provided in the Figures as well as the
sequences provided in SEQ ID NOs: 21-29, SEQ ID NOs: 39-45, SEQ ID
NOs: 92-98, combinations thereof as well as fragments thereof. Such
sequences can also be referred to as Anaplasma platys P44 peptides
or Anaplasma platys P44 proteins. Other examples of Anaplasma
platys P44 peptides include, but are not limited to the sequences
provided in GenBank Accession Nos: GQ868750, GU357491, GU357492,
GU357493, HQ738571, GU357494, GU357495, GU357496, and GU357497.
[0106] Anaplasma platys peptides also include, but are not limited
to, the OMP-1X amino acid sequences provided in the Figures as well
as the sequences provided in SEQ ID NOs: 1-11, combinations thereof
as well as fragments thereof. Such sequences can also be referred
to as Anaplasma platys OMP-1X proteins. Other examples of Anaplasma
platys OMP-1X peptides include, but are not limited to the
sequences provided in GenBank Accession Nos: GQ868750, HQ738571,
and GU357491.
[0107] Also disclosed herein are regions of the Anaplasma platys
P44 and OMP-1X peptides that have been identified as being highly
antigenic as identified through the Jameson-Wolf method as well
through a surface probability plot analysis. These regions are
herein referred to as a "Box" regions. For example, six regions of
the Anaplasma platys P44 protein sequence have been identified in
FIGS. 19 and 23. These six regions, from the N-terminal to the
C-terminal regions are herein referred to as "P44 Box 1", "P44 Box
2", "P44 Box 3", "P44 Box 4", "P44 Box 5", and "P44 Box 6",
respectively. In addition, two regions of the Anaplasma platys
OMP-1X protein sequence have been identified in FIG. 22. These two
regions, from the N-terminal to the C-terminal regions are herein
referred to as "OMP-1X Box 1" and "OMP-1X Box 2", respectively.
[0108] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "P44 Box 1". P44
Box 1 includes, but is not limited to, the P44 Box 1 amino acid
sequences identified in FIG. 19 or 23, for example the sequence
from about position 22 to about position 39 of FIG. 19 or the
sequence from about position 40 to about position 64 of FIG. 23. In
addition, P44 Box 1 includes, but is not limited to, the amino acid
sequences of SEQ ID NO: 39, SEQ ID NO: 92, SEQ ID NOs: 100-103, the
amino acid sequences from about position 22 to about position 39 of
SEQ ID NOs: 21-24, or the amino acid sequence encoded by SEQ ID NO:
46. Also disclosed herein are isolated or purified polynucleotides
that consist of or comprise a polynucleotide sequence capable of
encoding the amino acid sequence of "P44 Box 1".
[0109] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "P44 Box 2". P44
Box 2 includes, but is not limited to, the P44 Box 2 amino acid
sequences identified in FIG. 19 or 23, for example the sequence
from about position 77 to about position 85 of FIG. 19 or the
sequence from about position 102 to about position 111 of FIG. 23.
In addition, P44 Box 2 includes, but is not limited to, the amino
acid sequences of SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NOs:
104-107, the amino acid sequences from about position 77 to about
position 85 of SEQ ID NOs: 21-24, or the amino acid sequence
encoded by SEQ ID NO: 47. Also disclosed herein are isolated or
purified polynucleotides that consist of or comprise a
polynucleotide sequence capable of encoding the amino acid sequence
of "P44 Box 2".
[0110] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "P44 Box 3". P44
Box 3 includes, but is not limited to, the P44 Box 3 amino acid
sequences identified in FIG. 19 or 23, for example the sequence
from about position 151 to about position 190 of FIG. 19 or the
sequence from about position 178 to about position 222 of FIG. 23.
In addition, P44 Box 3 includes, but is not limited to, the amino
acid sequences of SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NOs:
108-111, the amino acid sequences from about position 151 to about
position 191 of SEQ ID NOs: 21-24, or the amino acid sequence
encoded by SEQ ID NO: 48. Also disclosed herein are isolated or
purified polynucleotides that consist of or comprise a
polynucleotide sequence capable of encoding the amino acid sequence
of "P44 Box 3".
[0111] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "P44 Box 4". P44
Box 4 includes, but is not limited to, the P44 Box 4 amino acid
sequences identified in FIG. 19 or 23, for example the sequence
from about position 227 to about position 234 of FIG. 19 or the
sequence from about position 259 to about position 266 of FIG. 23.
In addition, P44 Box 4 includes, but is not limited to, the amino
acid sequences of SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NOs:
112-115, the amino acid sequences from about position 207 to about
position 214 of SEQ ID NOs: 21-24, or the amino acid sequence
encoded by SEQ ID NO: 49. Also disclosed herein are isolated or
purified polynucleotides that consist of or comprise a
polynucleotide sequence capable of encoding the amino acid sequence
of "P44 Box 4".
[0112] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "P44 Box 5". P44
Box 5 includes, but is not limited to, the P44 Box 5 amino acid
sequences identified in FIG. 19 or 23, for example the sequence
from about position 276 to about position 308 of FIG. 19 or the
sequence from about position 319 to about position 340 of FIG. 23.
In addition, P44 Box 5 includes, but is not limited to, the amino
acid sequences of SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NOs:
116-119, the amino acid sequences from about position 248 to about
position 269 of SEQ ID NOs: 21-24, or the amino acid sequence
encoded by SEQ ID NO: 50. Also disclosed herein are isolated or
purified polynucleotides that consist of or comprise a
polynucleotide sequence capable of encoding the amino acid sequence
of "P44 Box 5".
[0113] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "P44 Box 6". P44
Box 6 includes, but is not limited to, the P44 Box 6 amino acid
sequences identified in FIG. 19 or 23, for example the sequence
from about position 417 to about position 426 of FIG. 19 or the
sequence from about position 451 to about position 460 of FIG. 23.
In addition, P44 Box 6 includes, but is not limited to, the amino
acid sequences of SEQ ID NO: 44, SEQ ID NO: 97, SEQ ID NOs:
120-123, the amino acid sequences from about position 378 to about
position 386 of SEQ ID NOs: 21-24, or the amino acid sequence
encoded by SEQ ID NO: 51. Also disclosed herein are isolated or
purified polynucleotides that consist of or comprise a
polynucleotide sequence capable of encoding the amino acid sequence
of "P44 Box 6".
[0114] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "OMP-1X Box 1".
OMP-1X Box 1 includes, but is not limited to, the OMP-1X Box 1
amino acid sequences identified in FIG. 22, for example the
sequence from about position 66 to about position 192 of FIG. 22.
In addition, OMP-1X Box 1 includes, but is not limited to, the
amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO:
14. Also disclosed herein are isolated or purified polynucleotides
that consist of or comprise a polynucleotide sequence capable of
encoding the amino acid sequence of "OMP-1X Box 1".
[0115] Disclosed herein are isolated or purified polypeptides that
consist of or comprise the amino acid sequence of "OMP-1X Box 2".
OMP-1X Box 2 includes, but is not limited to, the OMP-1X Box 2
amino acid sequences identified in FIG. 22, for example the
sequence from about position 241 to about position 309 of FIG. 22.
In addition, OMP-1X Box 2 includes, but is not limited to, the
amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO:
15. Also disclosed herein are isolated or purified polynucleotides
that consist of or comprise a polynucleotide sequence capable of
encoding the amino acid sequence of "OMP-1X Box 2".
[0116] Also described herein are purified polypeptides comprising
the sequences outlined in FIG. 17, or at least about 10 contiguous
amino acids of the sequence from FIG. 17 wherein the at least 10
contiguous amino acids are chosen from amino acids 300-410. In some
aspects, the polypeptides described herein also inherently disclose
the nucleotide sequence as related to the amino acids sequence
300-410 from FIG. 17.
[0117] In one aspect, described herein are purified polypeptides
comprising at least about 8, 10, 15, 20, 30, 40, 50, or more
contiguous amino acids, wherein the contiguous amino acids can be
chosen from amino acids 300-410 from FIG. 17.
[0118] Disclosed herein are purified polypeptides that can either
be full-length polypeptides or fragments of polypeptides. For
example, fragments of polypeptides disclosed herein can comprise
about 8, 10, 15, 20, 30, 40, 50, or more amino acids of
polypeptides of the aspects described herein. Variant polypeptides
can be at least about 90, 96, 98, or 99% identical to the
polypeptide sequences shown in FIGS. 17 and 22. Variant
polypeptides can have one or more conservative amino acid
variations or other minor modifications and retain biological
activity, i.e., are biologically functional equivalents. A
biologically active equivalent can have substantially equivalent
function when compared to the corresponding wild-type
polypeptide.
[0119] Described herein are isolated or purified polypeptides
comprising a sequence chosen from the following: a Anaplasma platys
P44 protein, a variant of an Anaplasma platys P44 protein, or an
antigenic fragment of an Anaplasma platys P44 protein. In one
aspect, the Anaplasma platys P44 protein can comprise or consist
of: P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box
6, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ
ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ
ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO:
42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ
ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO:
44, SEQ ID NO: 97, the amino acid sequence comprising the amino
acid from about position 20 to about position 40 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
40 to about position 64 of FIG. 23, the amino acid from about
position 75 to about position 85 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 102 to about
position 111 of FIG. 23, the amino acid from about position 170 to
about position 190 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 178 to about position 222 of
FIG. 23, the amino acid from about position 205 to about position
215 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 259 to about position 266 of FIG. 23, the amino
acid from about position 270 to about position 290 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
319 to about position 340 of FIG. 23, the amino acid from about
position 365 to about position 380 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 451 to about
position 460 of FIG. 23, the amino acid from about position 1 to
about position 41 of FIG. 19, the amino acid sequence comprising
the amino acid from about position 78 to about position 85 of FIG.
19, the amino acid from about position 174 to about position 192 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 227 to about position 234 of FIG. 19 the amino acid
from about position 276 to about position 294 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 416 to
about position 433 of FIG. 19, or a fragment thereof.
[0120] In a further aspect, the Anaplasma platys P44 protein can
comprise or consist of a variant of: P44 Box 1, P44, Box 2, P44 Box
3, P44 Box 4, P44 Box 5, P44 Box 6, SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40,
SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID
NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93,
SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID
NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, the amino acid
sequence comprising the amino acid from about position 20 to about
position 40 of FIG. 23, the amino acid sequence comprising the
amino acid from about position 40 to about position 64 of FIG. 23,
the amino acid from about position 75 to about position 85 of FIG.
23, the amino acid sequence comprising the amino acid from about
position 102 to about position 111 of FIG. 23, the amino acid from
about position 170 to about position 190 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 178 to about
position 222 of FIG. 23, the amino acid from about position 205 to
about position 215 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 259 to about position 266 of
FIG. 23, the amino acid from about position 270 to about position
290 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 319 to about position 340 of FIG. 23, the amino
acid from about position 365 to about position 380 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
451 to about position 460 of FIG. 23, the amino acid from about
position 1 to about position 41 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 78 to about position
85 of FIG. 19, the amino acid from about position 174 to about
position 192 of FIG. 19, the amino acid sequence comprising the
amino acid from about position 227 to about position 234 of FIG. 19
the amino acid from about position 276 to about position 294 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 416 to about position 433 of FIG. 19, or a fragment
thereof.
[0121] It is understood that one way to define the variants and
derivatives of the disclosed proteins herein is to define them in
terms of homology/identity to specific known sequences.
Specifically disclosed are variants of A. platys peptides and other
proteins or peptides herein disclosed which have at least, 70% or
at least 75% or at least 80% or at least 85% or at least 90% or at
least 95% homology to the A. platys peptides specifically recited
herein. Those of skill in the art readily understand how to
determine the homology of two proteins.
[0122] As this specification discusses various polypeptides and
polypeptide sequences it is understood that the nucleic acids that
can encode those polypeptide sequences are also disclosed. This
would include all degenerate sequences related to a specific
polypeptide sequence, i.e. all nucleic acids having a sequence that
encodes one particular polypeptide sequence as well as all nucleic
acids, including degenerate nucleic acids, encoding the disclosed
variants and derivatives of the protein sequences. Thus, while each
particular nucleic acid sequence may not be written out herein, it
is understood that each and every sequence is in fact disclosed and
described herein through the disclosed polypeptide sequences.
[0123] In yet a further aspect, the Anaplasma platys P44 protein
can comprise or consist of an antigenic fragment of: P44 Box 1,
P44, Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ
ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO:
40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ
ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO:
97, the amino acid sequence comprising the amino acid from about
position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, or the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19. In still a further aspect, the Anaplasma platys P44
proteins described herein can comprise or consist of a combination
of one or more of the sequences described herein.
[0124] In one aspect, the variants or antigenic fragments of the
Anaplasma platys P44 proteins described herein can be
immunoreactive with at least one antibody that binds to their
corresponding peptide sequence.
[0125] Also described herein are isolated or purified polypeptides
that can comprise a sequence that is at least 95% identical to an
Anaplasma platys P44 protein, a variant of the Anaplasma platys P44
protein, or an antigenic fragment of the Anaplasma platys P44
protein. Thus, in one aspect, the polypeptides described herein can
be at least 95% identical to: P44 Box 1, P44 Box 2, P44 Box 3, P44
Box 4, P44 Box 5, P44 Box 6, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID
NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID
NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,
SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID
NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43,
SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, the amino acid
sequence comprising the amino acid from about position 20 to about
position 40 of FIG. 23, the amino acid sequence comprising the
amino acid from about position 40 to about position 64 of FIG. 23,
the amino acid from about position 75 to about position 85 of FIG.
23, the amino acid sequence comprising the amino acid from about
position 102 to about position 111 of FIG. 23, the amino acid from
about position 170 to about position 190 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 178 to about
position 222 of FIG. 23, the amino acid from about position 205 to
about position 215 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 259 to about position 266 of
FIG. 23, the amino acid from about position 270 to about position
290 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 319 to about position 340 of FIG. 23, the amino
acid from about position 365 to about position 380 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
451 to about position 460 of FIG. 23, the amino acid from about
position 1 to about position 41 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 78 to about position
85 of FIG. 19, the amino acid from about position 174 to about
position 192 of FIG. 19, the amino acid sequence comprising the
amino acid from about position 227 to about position 234 of FIG. 19
the amino acid from about position 276 to about position 294 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 416 to about position 433 of FIG. 19, or a fragment
thereof.
[0126] In a further aspect, the polypeptides described herein can
be at least 95% identical to a variant of: P44 Box 1, P44, Box 2,
P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, SEQ ID NO: 21, SEQ ID
NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,
SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID
NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,
SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID
NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95,
SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, the
amino acid sequence comprising the amino acid from about position
20 to about position 40 of FIG. 23, the amino acid sequence
comprising the amino acid from about position 40 to about position
64 of FIG. 23, the amino acid from about position 75 to about
position 85 of FIG. 23, the amino acid sequence comprising the
amino acid from about position 102 to about position 111 of FIG.
23, the amino acid from about position 170 to about position 190 of
FIG. 23, the amino acid sequence comprising the amino acid from
about position 178 to about position 222 of FIG. 23, the amino acid
from about position 205 to about position 215 of FIG. 23, the amino
acid sequence comprising the amino acid from about position 259 to
about position 266 of FIG. 23, the amino acid from about position
270 to about position 290 of FIG. 23, the amino acid sequence
comprising the amino acid from about position 319 to about position
340 of FIG. 23, the amino acid from about position 365 to about
position 380 of FIG. 23, the amino acid sequence comprising the
amino acid from about position 451 to about position 460 of FIG.
23, the amino acid from about position 1 to about position 41 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, or a fragment thereof.
[0127] In yet a further aspect, the polypeptides described herein
can be at least 95% identical to an antigenic fragment of: P44 Box
1, P44, Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID
NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID
NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42,
SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID
NO: 97, the amino acid sequence comprising the amino acid from
about position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, or the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19. In still a further aspect, the polypeptides
described herein can be at least 95% identical to one or more of
the peptide sequences described herein. In still a further aspect,
the Anaplasma platys P44 proteins described herein can comprise or
consist of a combination of one or more of the sequences described
herein.
[0128] In one aspect, the variants or antigenic fragments can be at
least 95% identical to the Anaplasma platys P44 proteins described
herein can be immunoreactive with at least one antibody that binds
to their corresponding peptide sequence.
[0129] In one aspect, the isolated polypeptides described herein
can be: the P44 Box 1 protein, the P44 Box 2 protein, the P44 Box 3
protein, the P44 Box 4 protein, the P44 Box 5 protein, the P44 Box
6 protein; a variant of the P44 Box 1 protein, the P44 Box 2
protein, the P44 Box 3 protein, the P44 Box 4 protein, the P44 Box
5 protein, the P44 Box 6 protein; or an antigenic fragment of the
P44 Box 1 protein, the P44 Box 2 protein, the P44 Box 3 protein,
the P44 Box 4 protein, the P44 Box 5 protein, the P44 Box 6
protein.
[0130] Furthermore, described herein are isolated or purified
polypeptides comprising a sequence chosen from the following: a
Anaplasma platys OMP-1X protein, a variant of the Anaplasma platys
OMP-1X protein, or an antigenic fragment of the Anaplasma platys
OMP-1X protein. In one aspect, the Anaplasma platys OMP-1X protein
can comprise or consist of: OMP-1X Box 1, OMP-1X Box 2, SEQ ID NO:
1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID
NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ
ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4,
SEQ ID NO: 8, the amino acid from about position 66 to about
position 192 of FIG. 22, the amino acid from about position 70 to
about position 180 of FIG. 22, a combination of the amino acid from
about position 66 to about position 192 and the amino acid from
about position 70 to about position 180 of FIG. 22, the amino acid
from about position 240 to about position 312 of FIG. 22, the amino
acid from about position 230 to about position 300 of FIG. 22, a
combination of the amino acid from about position 240 to about
position 312 and the amino acid from about position 230 to about
position 300 of FIG. 22, or a fragment thereof.
[0131] In a further aspect, the Anaplasma platys OMP-1X protein can
comprise or consist of a variant of: OMP-1X Box 1, OMP-1X Box 2,
SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO:
6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ
ID NO: 4, SEQ ID NO: 8, the amino acid from about position 66 to
about position 192 of FIG. 22, the amino acid from about position
70 to about position 180 of FIG. 22, a combination of the amino
acid from about position 66 to about position 192 and the amino
acid from about position 70 to about position 180 of FIG. 22, the
amino acid from about position 240 to about position 312 of FIG.
22, the amino acid from about position 230 to about position 300 of
FIG. 22, a combination of the amino acid from about position 240 to
about position 312 and the amino acid from about position 230 to
about position 300 of FIG. 22, or a fragment thereof.
[0132] In yet a further aspect, the Anaplasma platys OMP-1X protein
can comprise or consist of an antigenic fragment of: OMP-1X Box 1,
OMP-1X Box 2, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO:
2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ
ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, the amino acid from about
position 66 to about position 192 of FIG. 22, the amino acid from
about position 70 to about position 180 of FIG. 22, a combination
of the amino acid from about position 66 to about position 192 and
the amino acid from about position 70 to about position 180 of FIG.
22, the amino acid from about position 240 to about position 312 of
FIG. 22, the amino acid from about position 230 to about position
300 of FIG. 22, or a combination of the amino acid from about
position 240 to about position 312 and the amino acid from about
position 230 to about position 300 of FIG. 22. In still a further
aspect, the Anaplasma platys OMP-1X proteins described herein can
comprise or consist of a combination of one or more of the
sequences described herein.
[0133] In one aspect, the variants or antigenic fragments of the
Anaplasma platys OMP-1X proteins described herein can be
immunoreactive with at least one antibody that binds to their
corresponding peptide sequence.
[0134] Also described herein are isolated or purified polypeptides
that can comprise a sequence that is at least 95% identical to an
Anaplasma platys OMP-1X protein, a variant of the Anaplasma platys
OMP-1X protein, or an antigenic fragment of the Anaplasma platys
OMP-1X protein. Thus, in one aspect, the polypeptides described
herein can be at least 95% identical to: OMP-1X Box 1, OMP-1X Box
2, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID
NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7,
SEQ ID NO: 4, SEQ ID NO: 8, the amino acid from about position 66
to about position 192 of FIG. 22, the amino acid from about
position 70 to about position 180 of FIG. 22, a combination of the
amino acid from about position 66 to about position 192 and the
amino acid from about position 70 to about position 180 of FIG. 22,
the amino acid from about position 240 to about position 312 of
FIG. 22, the amino acid from about position 230 to about position
300 of FIG. 22, a combination of the amino acid from about position
240 to about position 312 and the amino acid from about position
230 to about position 300 of FIG. 22, or a fragment thereof.
[0135] In a further aspect, the polypeptides described herein can
be at least 95% identical to a variant of: OMP-1X Box 1, OMP-1X Box
2, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID
NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7,
SEQ ID NO: 4, SEQ ID NO: 8, the amino acid from about position 66
to about position 192 of FIG. 22, the amino acid from about
position 70 to about position 180 of FIG. 22, a combination of the
amino acid from about position 66 to about position 192 and the
amino acid from about position 70 to about position 180 of FIG. 22,
the amino acid from about position 240 to about position 312 of
FIG. 22, the amino acid from about position 230 to about position
300 of FIG. 22, a combination of the amino acid from about position
240 to about position 312 and the amino acid from about position
230 to about position 300 of FIG. 22, or a fragment thereof.
[0136] In yet a further aspect, the polypeptides described herein
can be at least 95% identical to an antigenic fragment of: OMP-1X
Box 1, OMP-1X Box 2, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ
ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4,
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO:
3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, the amino acid from
about position 66 to about position 192 of FIG. 22, the amino acid
from about position 70 to about position 180 of FIG. 22, a
combination of the amino acid from about position 66 to about
position 192 and the amino acid from about position 70 to about
position 180 of FIG. 22, the amino acid from about position 240 to
about position 312 of FIG. 22, the amino acid from about position
230 to about position 300 of FIG. 22, or a combination of the amino
acid from about position 240 to about position 312 and the amino
acid from about position 230 to about position 300 of FIG. 22. In
still a further aspect, the Anaplasma platys OMP-1X proteins
described herein can comprise or consist of a combination of one or
more of the sequences described herein. In still a further aspect,
the polypeptides described herein can be at least 95% identical to
one or more of the peptide sequences described herein. In still a
further aspect, the Anaplasma platys OMP-1X proteins described
herein can comprise or consist of a combination of one or more of
the sequences described herein.
[0137] In one aspect, the variants or antigenic fragments can be at
least 95% identical to the Anaplasma platys OMP-1X proteins
described herein can be immunoreactive with at least one antibody
that binds to their corresponding peptide sequence.
[0138] In one aspect, the isolated polypeptides described herein
can be: the OMP-1X protein, the OMP-1X Box 1 protein, or the OMP-1X
Box 2 protein; a variant of the OMP-1X protein, the OMP-1X Box 1
protein, or the OMP-1X Box 2 protein; or an antigenic fragment of
the OMP-1X protein, the OMP-1X Box 1 protein, or the OMP-1X Box 2
protein.
[0139] Also disclosed herein are isolated polynucleotides that
encode the polypeptides described herein. A purified polypeptide
can further comprising a carrier. A purified polypeptide can be in
a multimeric form. A purified polypeptide can be linked to an
indicator reagent, an amino acid spacer, an amino acid linker, a
signal sequence, a stop transfer sequence, a transmembrane domain,
a protein purification ligand, a heterologous polypeptide or a
combination thereof.
[0140] Purified polypeptides described herein can either be
full-length polypeptides or fragments of polypeptides. For example,
fragments of polypeptides described herein can comprise about 10,
15, 20, 50, 75, 100, 150, 200, 250 or more amino acids of
polypeptides of the invention. For example, and not to be limiting,
variant polypeptides can be at least about 80, or about 90, 96, 98,
or 99% identical to the polypeptide sequences shown in SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ
ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO:
40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ
ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO:
97, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID
NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7,
SEQ ID NO: 4, SEQ ID NO: 8, or a fragment thereof, and are also
polypeptides of the invention. Variant polypeptides have one or
more conservative amino acid variations or other minor
modifications and retain biological activity, i.e., are
biologically functional equivalents. A biologically active
equivalent has substantially equivalent function when compared to
the corresponding wild-type polypeptide.
[0141] Percent sequence identity has an art recognized meaning and
there are a number of methods to measure identity between two
polypeptide or polynucleotide sequences. See, e.g., Lesk, Ed.,
Computational Molecular Biology, Oxford University Press, New York,
(1988); Smith, Ed., Biocomputing: Informatics And Genome Projects,
Academic Press, New York, (1993); Griffin & Griffin, Eds.,
Computer Analysis Of Sequence Data, Part I, Humana Press, New
Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology,
Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence
Analysis Primer, M Stockton Press, New York, (1991). Methods for
aligning polynucleotides or polypeptides are codified in computer
programs, including the GCG program package (Devereux et al., Nuc.
Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al.,
J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, Wis.
53711) which uses the local homology algorithm of Smith and
Waterman (Adv. App. Math., 2:482-489 (1981)). For example, the
computer program ALIGN which employs the FASTA algorithm can be
used, with an affine gap search with a gap open penalty of -12 and
a gap extension penalty of -2.
[0142] When using any of the sequence alignment programs to
determine whether a particular sequence is, for instance, about 95%
identical to a reference sequence, the parameters are set such that
the percentage of identity is calculated over the full length of
the reference polynucleotide or polypeptide and that gaps in
identity of up to 5% of the total number of nucleotides or amino
acids in the reference polynucleotide or polypeptide are
allowed.
[0143] Variants can generally be identified by modifying one of the
polypeptide sequences of the invention, and evaluating the
properties of the modified polypeptide to determine if it is a
biological equivalent. A variant is a biological equivalent if it
reacts substantially the same as a polypeptide of the invention in
an assay such as an immunohistochemical assay, an enzyme-linked
immunosorbent Assay (ELISA), a radioimmunoassay (RIA), immunoenzyme
assay or a western blot assay, e.g. has 90-110% of the activity of
the original polypeptide. In one embodiment, the assay is a
competition assay wherein the biologically equivalent polypeptide
is capable of reducing binding of the polypeptide of the invention
to a corresponding reactive antigen or antibody by about 80, 95,
99, or 100%. An antibody that specifically binds a corresponding
wild-type polypeptide also specifically binds the variant
polypeptide. Variant polypeptides of the invention can comprise
about 1, 2, 3, 4, 5, 10, or 20 conservative amino acid
substitutions.
[0144] A conservative substitution is one in which an amino acid is
substituted for another amino acid that has similar properties,
such that one skilled in the art of peptide chemistry would expect
the secondary structure and hydropathic nature of the polypeptide
to be substantially unchanged. In general, the following groups of
amino acids represent conservative changes: (1) ala, pro, gly, glu,
asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu,
met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, tip, his.
[0145] The polypeptides described herein can further comprise a
signal (or leader) sequence that co-translationally or
post-translationally directs transfer of the protein. The
polypeptide can also comprise a linker or other sequence for ease
of synthesis, purification or identification of the polypeptide
(e.g., poly-His), or to enhance binding of the polypeptide to a
solid support. For example, a polypeptide can be conjugated to an
immunoglobulin Fc region or bovine serum albumin.
[0146] The polypeptides described herein can be covalently or
non-covalently linked to an amino acid sequence to which the
polypeptide is not normally associated with in nature, i.e., a
heterologous amino acid sequence. A heterologous amino acid
sequence can be from a non-Anaplasma platys organism (e.g., an
Anaplasma phagocytophilum organism), a synthetic sequence, or an
Anaplasma platys sequence not usually located at the carboxy or
amino terminus of a polypeptide of the invention. Additionally, a
polypeptide can be covalently or non-covalently linked to compounds
or molecules other than amino acids. For example, a polypeptide can
be linked to an indicator reagent, an amino acid spacer, an amino
acid linker, a signal sequence, a stop transfer sequence, a
transmembrane domain, a protein purification ligand, or a
combination thereof. In one embodiment of the invention a protein
purification ligand can be one or more C amino acid residues at,
for example, the amino terminus or carboxy terminus of a
polypeptide of the invention. An amino acid spacer is a sequence of
amino acids that are not usually associated with a polypeptide of
the invention in nature. An amino acid spacer can comprise about 1,
5, 10, 20, 100, or 1,000 amino acids.
[0147] If desired, a polypeptide can be a fusion protein, which can
also contain other amino acid sequences, such as amino acid
linkers, amino acid spacers, signal sequences, TMR stop transfer
sequences, transmembrane domains, as well as ligands useful in
protein purification, such as glutathione-S-transferase, histidine
tag, and Staphylococcal protein A, or combinations thereof. More
than one polypeptide of the invention can be present in a fusion
protein. Fragments of polypeptides of the invention can be present
in a fusion protein of the invention. A fusion protein of the
invention can comprise one or more of Anaplasma platys polypeptides
described herein, fragments thereof, or combinations thereof. A
fusion protein can also comprise multiple copies of a same
Anaplasma platys polypeptide or combination of different Anaplasma
platys polypeptides described herein.
[0148] Polypeptides of the invention can be in a multimeric form.
That is, a polypeptide can comprise one or more copies of an
Anaplasma platys polypeptide of the invention or a combination
thereof. A multimeric polypeptide can be a multiple antigen peptide
(MAP). See e.g., Tam, J. Immunol. Methods, 196:17-32 (1996).
[0149] Polypeptides of the invention can comprise an antigen that
is recognized by an antibody specific for Anaplasma platys P44 or
Anaplasma platys OMP-1X. The antigen can comprise one or more
epitopes (i.e., antigenic determinants). An epitope can be a linear
epitope, sequential epitope or a conformational epitope. Epitopes
within a polypeptide of the invention can be identified by several
methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf,
CABIOS 4:181-186 (1988). For example, a polypeptide of the
invention can be isolated and screened. A series of short peptides,
which together span an entire polypeptide sequence, can be prepared
by proteolytic cleavage. By starting with, for example, 100-mer
polypeptide fragments, each fragment can be tested for the presence
of epitopes recognized in an ELISA. For example, in an ELISA assay
an Anaplasma platys polypeptide, such as a 100-mer polypeptide
fragment, is attached to a solid support, such as the wells of a
plastic multi-well plate. A population of antibodies are labeled,
added to the solid support and allowed to bind to the unlabeled
antigen, under conditions where non-specific absorption is blocked,
and any unbound antibody and other proteins are washed away.
Antibody binding is detected by, for example, a reaction that
converts a colorless substrate into a colored reaction product.
Progressively smaller and overlapping fragments can then be tested
from an identified 100-mer to map the epitope of interest.
[0150] The polypeptides described herein can be produced
recombinantly. A polynucleotide encoding a polypeptide described
herein can be introduced into a recombinant expression vector,
which can be expressed in a suitable expression host cell system
using techniques well known in the art. A variety of bacterial,
yeast, plant, mammalian, and insect expression systems are
available in the art and any such expression system can be used.
Optionally, a polynucleotide encoding a polypeptide can be
translated in a cell-free translation system. A polypeptide can
also be chemically synthesized or obtained from Anaplasma platys
cells.
[0151] For example, and not to be limiting, an immunogenic
polypeptide of the invention can comprise an amino acid sequence
shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ
ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO:
94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ
ID NO: 44, SEQ ID NO: 97, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO:
10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID
NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ
ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, or fragments
thereof. An immunogenic polypeptide can elicit antibodies or other
immune responses (e.g., T-cell responses of the immune system) that
recognize epitopes of a polypeptide having SEQ ID NO: 21, SEQ ID
NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,
SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID
NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,
SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID
NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95,
SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, SEQ ID
NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ
ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO:
4, SEQ ID NO: 8, or fragments thereof. An immunogenic polypeptide
of the invention can also be a fragment of a polypeptide that has
an amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:
27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ
ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:
45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ
ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO:
43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, SEQ ID NO: 1, SEQ
ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3,
SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID
NO: 8. An immunogenic polypeptide fragment of the invention can be
about 10, 15, 20, 25, 30, 40, 50 or more amino acids in length.
Polypeptide Production
[0152] Polypeptides that can be used in the disclosed methods can
be produced by any method known in the art. One method of producing
the disclosed polypeptides is to link two or more amino acid
residues, peptides or polypeptides together by protein chemistry
techniques. For example, peptides or polypeptides are chemically
synthesized using currently available laboratory equipment using
either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc.,
Foster City, Calif.). A peptide or polypeptide can be synthesized
and not cleaved from its synthesis resin, whereas the other
fragment of a peptide or protein can be synthesized and
subsequently cleaved from the resin, thereby exposing a terminal
group, which is functionally blocked on the other fragment. By
peptide condensation reactions, these two fragments can be
covalently joined via a peptide bond at their carboxyl and amino
termini, respectively, (Grant G A (1992) Synthetic Peptides: A User
Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B.,
Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc.,
NY). Alternatively, the peptide or polypeptide is independently
synthesized in vivo. Once isolated, these independent peptides or
polypeptides can be linked to form a peptide or fragment thereof
via similar peptide condensation reactions.
[0153] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two-step chemical reaction (Dawson et al. Science, 266:776-779
(1994)). The first step is the chemoselective reaction of an
unprotected synthetic peptide-thioester with another unprotected
peptide segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site (Baggiolim M et al. (1992)
FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochem., 30:3128 (1991);
Rajarathnam K et al., Biochem. 33:6623-30 (1994)).
[0154] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
[0155] Also disclosed are the components to be used to prepare the
disclosed A. platys peptides that can be used in the disclosed
methods as well as the compositions themselves to be used within
the methods disclosed herein. These and other materials are
disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular polynucleotide is
disclosed and discussed and a number of modifications that can be
made to a number of molecules including the polynucleotide are
discussed, specifically contemplated is each and every combination
and permutation of polynucleotide and the modifications that are
possible unless specifically indicated to the contrary. Thus, if a
class of molecules A, B, and C are disclosed as well as a class of
molecules D, E, and F and an example of a combination molecule, A-D
is disclosed, then even if each is not individually recited each is
individually and collectively contemplated meaning combinations,
A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C--F are considered
disclosed. Likewise, any subset or combination of these is also
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
would be considered disclosed. This concept applies to all aspects
of this application including, but not limited to, steps in methods
of making and using the disclosed compositions. Thus, if there are
a variety of additional steps that can be performed it is
understood that each of these additional steps can be performed
with any specific embodiment or combination of embodiments of the
disclosed methods.
[0156] It is understood that one way to define any known variants
and derivatives or those that might arise, of the disclosed genes
and proteins herein is through defining the variants and
derivatives in terms of homology to specific known sequences.
Specifically disclosed are variants of the genes and proteins
herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99 percent homology to the stated sequence.
Those of skill in the art readily understand how to determine the
homology of two proteins or nucleic acids, such as genes. For
example, the homology can be calculated after aligning the two
sequences so that the homology is at its highest level.
Antibodies
[0157] Described herein are isolated or purified antibodies that
selectively hybridize to a peptide chosen from: Anaplasma platys
P44 protein, Anaplasma platys OMP-1X protein, P44 Box 1, P44, Box
2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, OMP-1X Box 1, OMP-1X
Box 2, or a fragment thereof. In one aspect, the antibodies
described herein can hybridize to a peptide chosen from one or more
of: P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box
6, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ
ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ
ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO:
42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ
ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO:
44, SEQ ID NO: 97, the amino acid sequence comprising the amino
acid from about position 20 to about position 40 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
40 to about position 64 of FIG. 23, the amino acid from about
position 75 to about position 85 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 102 to about
position 111 of FIG. 23, the amino acid from about position 170 to
about position 190 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 178 to about position 222 of
FIG. 23, the amino acid from about position 205 to about position
215 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 259 to about position 266 of FIG. 23, the amino
acid from about position 270 to about position 290 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
319 to about position 340 of FIG. 23, the amino acid from about
position 365 to about position 380 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 451 to about
position 460 of FIG. 23, the amino acid from about position 1 to
about position 41 of FIG. 19, the amino acid sequence comprising
the amino acid from about position 78 to about position 85 of FIG.
19, the amino acid from about position 174 to about position 192 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 227 to about position 234 of FIG. 19 the amino acid
from about position 276 to about position 294 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 416 to
about position 433 of FIG. 19, OMP-1X Box 1, OMP-1X Box 2, SEQ ID
NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ
ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO:
4, SEQ ID NO: 8, the amino acid from about position 66 to about
position 192 of FIG. 22, the amino acid from about position 70 to
about position 180 of FIG. 22, a combination of the amino acid from
about position 66 to about position 192 and the amino acid from
about position 70 to about position 180 of FIG. 22, the amino acid
from about position 240 to about position 312 of FIG. 22, the amino
acid from about position 230 to about position 300 of FIG. 22, a
combination of the amino acid from about position 240 to about
position 312 and the amino acid from about position 230 to about
position 300 of FIG. 22, or a fragment thereof. In a further
aspect, the antibodies described herein can hybridize to one or
more of a peptide that is at least 95% identical to the P44 or
OMP-1X peptide sequences described herein. In a further aspect, the
antibodies described herein can hybridize to one or more of a
variant or antigenic fragment of the P44 or OMP-1X peptide
sequences described herein. In still a further aspect, the
antibodies described herein can hybridize to one or more of a
variant or antigenic fragment of a peptide that is at least 95%
identical to the P44 or OMP-1X peptide sequences described
herein.
[0158] Disclosed herein are antibodies that specifically and stably
bind to an Anaplasma platys p44 polypeptide, an Anaplasma platys
OMP-1X peptide, or fragment thereof. Antibodies can also
specifically and stably bind to an Anaplasma platys P44 Box 1, P44
Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6 polypeptide or
fragment thereof. Antibodies can also specifically and stably bind
to an Anaplasma platys OMP-1X Box 1 polypeptide, an OMP-1X Box 2
polypeptide or fragment thereof. One of skill in the art can easily
determine if an antibody is specific for an Anaplasma platys
polypeptide using assays described herein. An antibody can be a
polyclonal antibody, a monoclonal antibody, a single chain antibody
(scFv), or an antigen binding fragment of an antibody.
Antigen-binding fragments of antibodies are a portion of an intact
antibody comprising the antigen binding site or variable region of
an intact antibody, wherein the portion is free of the constant
heavy chain domains of the Fc region of the intact antibody.
Examples of antibody fragments include Fab, Fab', Fab'-SH,
F(ab').sub.2 and F.sub.v fragments.
[0159] The antibodies described herein can be any antibody class,
including for example, IgG, IgM, IgA, IgD and IgE. An antibody or
fragment thereof binds to an epitope of a polypeptide of the
invention. An antibody can be made in vivo in suitable laboratory
animals or in vitro using recombinant DNA techniques. Means for
preparing and characterizing antibodies are well know in the art.
See, e.g., Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods
Mol. Biol. 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88
(1994); Gullick, Methods Mol. Biol. 32:389-99 (1994); Drenckhahn et
al. Methods Cell. Biol. 37:7-56 (1993); Morrison, Ann. Rev.
Immunol. 10:239-65 (1992); Wright et al. Crit. Rev. Immunol.
12:125-68 (1992). For example, polyclonal antibodies can be
produced by administering a polypeptide of the invention to an
animal, such as a human or other primate, mouse, rat, rabbit,
guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serum
from the immunized animal is collected and the antibodies are
purified from the plasma by, for example, precipitation with
ammonium sulfate, followed by chromatography, such as affinity
chromatography. Techniques for producing and processing polyclonal
antibodies are known in the art.
[0160] "Specifically binds" or "specific for" means that a first
antigen, e.g., an Anaplasma platys polypeptide, recognizes and
binds to an antibody of the invention with greater affinity than
other, non-specific molecules. A non-specific molecule is an
antigen that shares no common epitope with the first antigen. In
this case, Anaplasma platys polypeptides would not generally be
desirable choices for non-specific control molecules. For example,
an antibody raised against a first antigen (e.g., a polypeptide) to
which it binds more efficiently than to a non-specific antigen can
be described as specifically binding to the first antigen. In a
preferred embodiment, an antibody or antigen-binding portion
thereof specifically binds to a polypeptide of SEQ ID NO: 21, SEQ
ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ
ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:
44, SEQ ID NO: 45, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ
ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO:
95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, SEQ
ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6,
SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID
NO: 4, SEQ ID NO: 8, or fragments thereof. Specific binding can be
tested using, for example, an enzyme-linked immunosorbant assay
(ELISA), a radioimmunoassay (RIA), or a western blot assay using
methodology well known in the art.
[0161] Additionally, monoclonal antibodies directed against
epitopes present on a polypeptide of the invention can also be
readily produced. For example, normal B cells from a mammal, such
as a mouse, which was immunized with a polypeptide of the invention
can be fused with, for example, HAT-sensitive mouse myeloma cells
to produce hybridomas. Hybridomas producing Anaplasma
platys-specific antibodies can be identified using RIA or ELISA and
isolated by cloning in semi-solid agar or by limiting dilution.
Clones producing Anaplasma platys-specific antibodies are isolated
by another round of screening. Monoclonal antibodies can be
screened for specificity using standard techniques, for example, by
binding a polypeptide of the invention to a microtiter plate and
measuring binding of the monoclonal antibody by an ELISA assay.
Techniques for producing and processing monoclonal antibodies are
known in the art. See e.g., Kohler & Milstein, Nature, 256:495
(1975). Particular isotypes of a monoclonal antibody can be
prepared directly, by selecting from the initial fusion, or
prepared secondarily, from a parental hybridoma secreting a
monoclonal antibody of a different isotype by using a sib selection
technique to isolate class-switch variants. See Steplewski et al.,
P.N.A.S. U.S.A. 82:8653 1985; Spria et al., J. Immunolog. Meth.
74:307, 1984. Monoclonal antibodies of the invention can also be
recombinant monoclonal antibodies. See, e.g., U.S. Pat. No.
4,474,893; U.S. Pat. No. 4,816,567. Antibodies of the invention can
also be chemically constructed. See, e.g., U.S. Pat. No.
4,676,980.
[0162] Antibodies can be chimeric (see, e.g., U.S. Pat. No.
5,482,856), humanized (see, e.g., Jones et al., Nature 321:522
(1986); Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op.
Struct. Biol. 2:593 (1992)), caninized, canine, or human
antibodies. Human antibodies can be made by, for example, direct
immortilization, phage display, transgenic mice, or a Trimera
methodology, see e.g., Reisener et al., Trends Biotechnol.
16:242-246 (1998).
[0163] Antibodies that specifically bind Anaplasma platys antigens
(e.g., Anaplasma platys polypeptides), are particularly useful for
detecting the presence of Apl or Apl antigens in a sample, such as
a serum, blood, plasma, urine, fecal, or saliva sample from an Apl-
or Aph-infected animal. An immunoassay for Anaplasma platys antigen
can utilize one antibody or several antibodies. An immunoassay for
Anaplasma platys antigen can use, for example, a monoclonal
antibody specific for an Anaplasma platys epitope, a combination of
monoclonal antibodies specific for epitopes of one Anaplasma platys
polypeptide, monoclonal antibodies specific for epitopes of
different Anaplasma platys polypeptides, polyclonal antibodies
specific for the same Anaplasma platys antigen, polyclonal
antibodies specific for different Anaplasma platys antigens, or a
combination of monoclonal and polyclonal antibodies. Immunoassay
protocols can be based upon, for example, competition, direct
reaction, or sandwich type assays using, for example, labeled
antibody. Antibodies can be labeled with any type of label known in
the art, including, for example, fluorescent, chemiluminescent,
radioactive, enzyme, colloidal metal, radioisotope and
bioluminescent labels.
[0164] Antibodies or fragments thereof can be bound to a support
and used to detect the presence of Anaplasma platys antigen.
Supports include, for example, glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, agaroses and magletite.
[0165] Antibodies can further be used to isolate Anaplasma platys
organisms or Anaplasma platys antigens by immunoaffinity columns.
The antibodies can be affixed to a solid support by, for example,
adsorbtion or by covalent linkage so that the antibodies retain
their immunoselective activity. Optionally, spacer groups can be
included so that the antigen binding site of the antibody remains
accessible. The immobilized antibodies can then be used to bind
Anaplasma platys organisms or Anaplasma platys antigens from a
sample, such as a biological sample including saliva, serum,
sputum, blood, urine, feces, cerebrospinal fluid, amniotic fluid,
wound exudate, or tissue. The bound Anaplasma platys organisms or
Anaplasma platys antigens are recovered from the column matrix by,
for example, a change in pH.
[0166] Antibodies can also be used in immunolocalization studies to
analyze the presence and distribution of a polypeptide of the
invention during various cellular events or physiological
conditions. Antibodies can also be used to identify molecules
involved in passive immunization and to identify molecules involved
in the biosynthesis of non-protein antigens. Identification of such
molecules can be useful in vaccine development. Antibodies,
including, for example, monoclonal antibodies and single chain
antibodies, can be used to monitor the course of amelioration of a
disease caused by Anaplasma platys. By measuring the increase or
decrease of Anaplasma platys antibodies to Anaplasma platys
antigens in a test sample from an animal, it can be determined
whether a particular therapeutic regiment aimed at ameliorating the
disorder is effective. Antibodies can be detected and/or quantified
using for example, direct binding assays such as RIA, ELISA, or
western blot assays.
[0167] In one aspect, the antibodies can be immunoglobulin
molecules that specifically and stably bind to A. platys P44 or
OMP-1X polypeptide or fragment thereof. In a further aspect, the
antibody can be monoclonal, polyclonal, or a single chain antibody.
In yet a further aspect, an antibody can be an antigen-binding
fragments, which is a portion of an intact antibody comprising the
antigen binding site or variable region of an intact antibody,
wherein the portion is free of the constant heavy chain domains of
the Fc region of the intact antibody.
[0168] In one aspect, monoclonal antibodies directed against
epitopes present on a polypeptide discussed herein can be produced.
In a further aspect, clones producing A. platys-specific antibodies
can be isolated via additional screening. In yet a further aspect,
monoclonal antibodies can also be recombinant monoclonal
antibodies. Monoclonal antibodies can be screened for specificity
using standard techniques known in the art.
[0169] In one aspect, an antibody can belong to any antibody class.
In a further aspect, an antibody or fragment thereof can bind to an
epitope of a polypeptide disclosed herein. An antibody can be made
in vivo in suitable laboratory animals or in vitro via recombinant
DNA techniques known in the art.
[0170] Means for preparing and characterizing antibodies are well
known in the art. For example, polyclonal antibodies can be
produced by administering a polypeptide described herein to an
animal, such as a human or other primate, mouse, rat, rabbit, dog,
cow, sheep, or horse. Serum from the immunized animal can be
collected and the antibodies can be purified from the plasma.
[0171] In one aspect, antibodies can be chimeric, canine, or human
antibodies. In a further aspect, antibodies or fragments thereof
can be bound to a support. Supports can include, glass,
polystyrene, polypropylene, polyethylene, nylon, celluloses, or
polyacrylamides.
Vaccines
[0172] In one aspect, described herein are Anaplasma platys P44
protein based vaccines. Thus, described herein are peptides
comprising one or more amino-acid sequences selected from the group
consisting of P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box
5, P44 Box 6, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID
NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,
SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 39,
SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID
NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96,
SEQ ID NO: 44, SEQ ID NO: 97, the amino acid sequence comprising
the amino acid from about position 20 to about position 40 of FIG.
23, the amino acid sequence comprising the amino acid from about
position 40 to about position 64 of FIG. 23, the amino acid from
about position 75 to about position 85 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 102 to about
position 111 of FIG. 23, the amino acid from about position 170 to
about position 190 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 178 to about position 222 of
FIG. 23, the amino acid from about position 205 to about position
215 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 259 to about position 266 of FIG. 23, the amino
acid from about position 270 to about position 290 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
319 to about position 340 of FIG. 23, the amino acid from about
position 365 to about position 380 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 451 to about
position 460 of FIG. 23, the amino acid from about position 1 to
about position 41 of FIG. 19, the amino acid sequence comprising
the amino acid from about position 78 to about position 85 of FIG.
19, the amino acid from about position 174 to about position 192 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 227 to about position 234 of FIG. 19 the amino acid
from about position 276 to about position 294 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 416 to
about position 433 of FIG. 19, or a fragment thereof. In a further
aspect, the peptides disclosed herein can comprise one or more
amino-acid sequences selected from the group consisting of a
combination of any P44 peptide sequences described herein.
[0173] In yet a further aspect, the vaccines described herein can
comprise one or more amino-acid sequences selected from the group
consisting of a variant of the P44 proteins described herein, or an
antigenic fragment of the P44 proteins described herein. In still a
further aspect, the vaccines described herein can comprise one or
more amino-acid sequences selected from the group consisting of a
sequence that is at least 95% identical to a P44 protein, a variant
of the P44 protein, or an antigenic fragment of the P44 protein
sequences described herein. The peptides described herein can also
be any antigenically related variant of the peptide sequences which
have an identity of 95% and are capable of immunologically
mimicking the corresponding antigenic determinant site of the P44
protein of Anaplasma platys.
[0174] In one aspect, the vaccines described herein can be
Anaplasma platys OMP-1X protein based vaccines. Thus, described
herein are peptides comprising one or more amino-acid sequences
selected from the group consisting of OMP-1X Box 1, OMP-1X Box 2,
SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO:
6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ
ID NO: 4, SEQ ID NO: 8, the amino acid from about position 66 to
about position 192 of FIG. 22, the amino acid from about position
70 to about position 180 of FIG. 22, a combination of the amino
acid from about position 66 to about position 192 and the amino
acid from about position 70 to about position 180 of FIG. 22, the
amino acid from about position 240 to about position 312 of FIG.
22, the amino acid from about position 230 to about position 300 of
FIG. 22, a combination of the amino acid from about position 240 to
about position 312 and the amino acid from about position 230 to
about position 300 of FIG. 22, or a fragment thereof. In a further
aspect, the peptides disclosed herein can comprise one or more
amino-acid sequences selected from the group consisting of a
combination of any OMP-1X peptide sequences described herein.
[0175] In yet a further aspect, the vaccines described herein can
comprise one or more amino-acid sequences selected from the group
consisting of a variant of the OMP-1X proteins described herein, or
an antigenic fragment of the OMP-1X proteins described herein. In
still a further aspect, the vaccines described herein can comprise
one or more amino-acid sequences selected from the group consisting
of a sequence that is at least 95% identical to a OMP-1X protein, a
variant of the OMP-1X protein, or an antigenic fragment of the
OMP-1X protein sequences described herein. The peptides described
herein can also be any antigenically related variant of the peptide
sequences which have an identity of 95% and are capable of
immunologically mimicking the corresponding antigenic determinant
site of the OMP-1X protein of Anaplasma platys. Antigenically
related variants can have amino acids added, inserted, substituted
or deleted.
[0176] Furthermore, described herein are chimeric peptides
comprising: one or more Anaplasma platys P44 proteins or Anaplasma
platys OMP-1X proteins described herein; one or more variants of
the Anaplasma platys P44 proteins or Anaplasma platys OMP-1X
proteins described herein; one or more antigenic fragments of the
Anaplasma platys P44 proteins or Anaplasma platys OMP-1X proteins
described herein; or one or more proteins that are at least 95%
identical to the Anaplasma platys P44 proteins or Anaplasma platys
OMP-1X proteins described herein, linked to a carrier polypeptide
that can comprise at least one T-cell epitope. In one aspect, the
chimeric peptides described herein can further comprise a
purification tag peptide sequence. For example, and not to be
limiting, the purification tag sequence can be a Histidine-tag
sequence. Also disclosed herein are purified antibodies that are
immunospecific to the chimeric peptides described herein. In one
aspect, a purification tag peptide sequence (such as a Histidine
tag or a Glutathione-S-transferase tag) can be used in order to aid
subsequent purification of the polypeptide. Optional short peptide
spacer sequences can be introduced between elements of the chimeric
polypeptide. When one is required a Histidine tag sequence can be
located at the C-terminus of the polypeptide.
[0177] Further described herein are vaccine compositions comprising
an immunogenic amount of at least: one Anaplasma platys P44 protein
or Anaplasma platys OMP-1X protein described herein; one variant of
the Anaplasma platys P44 proteins or Anaplasma platys OMP-1X
proteins described herein; one antigenic fragment of the Anaplasma
platys P44 proteins or Anaplasma platys OMP-1X proteins described
herein; or one protein that is at least 95% identical to the
Anaplasma platys P44 proteins or Anaplasma platys OMP-1X proteins
described herein, wherein the protein or peptide can be in a
pharmaceutically acceptable excipient, and an optional adjuvant.
Vaccine preparation is generally described in Vaccine Design ("The
subunit and adjuvant approach" (eds. Powell M. F. & Newman M.
J). (1995) Plenum Press New York), which is hereby incorporated in
its entirety by this reference. Suitable adjuvants include, but are
not limited to an aluminium salt such as aluminium hydroxide gel
(alum) or aluminium phosphate, but can also be a salt of calcium,
iron or zinc, or can be an insoluble suspension of acylated
tyrosine, or acylated sugars, cationically or anionically
derivatised polysaccharides, or polyphosphazenes. Other known
adjuvants include CpG containing oligonucleotides. The
oligonucleotides can be characterized in that the CpG dinucleotide
is unmethylated. Such oligonucleotides are well known in the art
and are described in, for example WO96/02555. In one aspect, the
adjuvants can induce an immune response, for example, of the TH1
type. High levels of Th1-type cytokines can favor the induction of
cell mediated immune responses to the given antigen, while high
levels of Th2-type cytokines can favor the induction of humoral
immune responses to the antigen. Suitable adjuvant systems can
include, for example, monophosphoryl lipid A, 3-de-O-acylated
monophosphoryl lipid A (3D-MPL), or a combination of 3D-MPL
together with an aluminium salt. CpG oligonucleotides can also
induce a TH1 response. An enhanced system can involve the
combination of a monophosphoryl lipid A and a saponin derivative,
for example, the combination of QS21 and 3D-MPL as disclosed in WO
94/00153, or a less reactogenic composition where the QS21 can be
quenched with cholesterol as described in WO 96/33739. Another
adjuvant formulation involving QS21 3D-MPL & tocopherol in an
oil in water emulsion is described in WO 95/17210.
[0178] Also described herein are methods of inducing an immune
response in a mammal susceptible to Anaplasma platys infection
comprising administering to the mammal an effective amount of the
vaccine compositions described herein. As used herein, "infection"
can also mean "exposure," and the terms can be used
interchangeably.
[0179] Additionally, described herein are methods of preventing
Anaplasma platys infection comprising administering to a mammal an
effective amount of the vaccine compositions described herein.
Vectors
[0180] Also described herein are vectors for transformation of a
host cell comprising an isolated polynucleotide that can encode an
outer membrane protein of Anaplasma platys, a variant of said outer
membrane protein, or an immunogenic fragment of said outer membrane
protein. In one aspect, the outer membrane protein can be the P44
protein, P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44
Box 6, OMP-1X protein, OMP-1X Box 1, OMP-1X Box 2, OMP-1X Box 1 and
OMP-1X Box 2, or a fragment thereof. The vectors disclosed herein
can comprise any of the isolated polynucleotide sequences disclosed
or described herein.
[0181] The polynucleotides described herein can comprise coding
sequences for naturally occurring polypeptides or can encode
altered sequences that do not occur in nature. If desired,
polynucleotides can be cloned into an expression vectors comprising
expression control elements, including for example, origins of
replication, promoters, enhancers, or other regulatory elements
that drive expression of the polynucleotides of the invention in
host cells. An expression vector can be, for example, a plasmid,
such as pBR322, pUC, or ColE1, or an adenovirus vector, such as an
adenovirus Type 2 vector or Type 5 vector. Optionally, other
vectors can be used, including but not limited to Sindbis virus,
simian virus 40, alphavirus vectors, poxvirus vectors, and
cytomegalovirus and retroviral vectors, such as murine sarcoma
virus, mouse mammary tumor virus, Moloney murine leukemia virus,
and Rous sarcoma virus. Minichromosomes such as MC and MC1,
bacteriophages, phagemids, yeast artificial chromosomes, bacterial
artificial chromosomes, virus particles, virus-like particles,
cosmids (plasmids into which phage lambda cos sites have been
inserted) and replicons (genetic elements that are capable of
replication under their own control in a cell) can also be
used.
[0182] Methods for preparing polynucleotides operably linked to an
expression control sequence and expressing them in a host cell are
well-known in the art. See, e.g., U.S. Pat. No. 4,366,246. A
polynucleotide of the invention is operably linked when it is
positioned adjacent to or close to one or more expression control
elements, which direct transcription and/or translation of the
polynucleotide.
[0183] In a further aspect, the vectors described herein can be
used in a process for making a corresponding outer membrane protein
of Anaplasma platys, a variant of said outer membrane protein, or
an immunogenic fragment of said outer membrane protein. For
example, and not to be limiting, the process can comprise
transfecting host cells with any of the vectors described herein
and inducing expression of the outer membrane protein or the
variant or immunogenic fragment thereof in any of the host cells
described herein.
[0184] Expression vectors for production of proteins and peptides
are well known in the art (see Ausubel et al., 2004, Current
Protocols In Molecular Biology, Greene Publishing and
Wiley-Interscience, New York). Such expression vectors can include
the nucleic acid sequence encoding the Anaplasma platys
polypeptides linked to regulatory elements, such as a promoter,
which drives transcription of the DNA and can be adapted for
expression in prokaryotic (e.g., E. coli) and eukaryotic (e.g.,
yeast, insect or mammalian cells) hosts. A variant Anaplasma platys
polypeptide can also be expressed in an expression vector in which
a variant Anaplasma platys gene is operably linked to a promoter.
The promoter can be a eukaryotic promoter for expression in a
mammalian cell. The transcription regulatory sequences can comprise
a heterologous promoter and optionally an enhancer, which is
recognized by the host cell. Commercially available expression
vectors can also be used. Expression vectors can include
host-recognized replication systems, amplifiable genes, selectable
markers, host sequences useful for insertion into the host genome,
and the like.
Host Cells
[0185] Also disclosed herein are host cells comprising any of the
vectors disclosed or described herein. Suitable host cells can
include, but are not limited to, bacteria such as E. coli, yeast,
filamentous fungi, mollusk cells, snail cells, insect cells, and
mammalian cells, which are typically immortalized, including mouse,
hamster, human, and monkey cell lines, and derivatives thereof.
Host cells may be able to process the Anaplasma platys gene product
to produce an appropriately processed, mature polypeptide. Such
processing can include glycosylation, ubiquitination, disulfide
bond formation, and the like.
Kits
[0186] Described herein are kits for diagnosing Anaplasma platys in
a subject, wherein the kit can comprise the Anaplasma platys P44
protein, an antigenic fragment of the Anaplasma platys P44 protein,
or both. In one aspect, in the kits disclosed herein, the protein
can comprise: one or more Anaplasma platys P44 proteins described
herein; one or more variants of the Anaplasma platys P44 proteins
described herein; one or more antigenic fragments of the Anaplasma
platys P44 proteins described herein; or one or more proteins that
are at least 95% identical to the Anaplasma platys P44 proteins
described herein.
[0187] Also described herein are kits for diagnosing Anaplasma
platys in a subject, wherein the kit can comprise the Anaplasma
platys OMP-1X protein, an antigenic fragment of the Anaplasma
platys OMP-1X protein, or both. In one aspect, in the kits
disclosed herein, the protein can comprise: one or more Anaplasma
platys OMP-1X proteins described herein; one or more variants of
the Anaplasma platys OMP-1X proteins described herein; one or more
antigenic fragments of the Anaplasma platys OMP-1X proteins
described herein; or one or more proteins that are at least 95%
identical to the Anaplasma platys OMP-1X proteins described
herein.
[0188] Further described herein are kits for diagnosing Anaplasma
platys in a subject comprising one or more of the antibodies
described herein. In one aspect, the kits described herein can
further comprise a biomolecule for detecting an interaction between
the reagent and antibodies in a sample from an animal.
[0189] Also described herein are reagent kits for diagnosing
infection or exposure with Anaplasma platys in a subject comprising
a DNA probe or primer constructed to correspond to the P44 protein,
P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5, or P44 Box 6
of Anaplasma platys, characterized in that the probe or primer
comprises one or more of the nucleotides or polynucleotides
described herein.
[0190] Also described herein are reagent kits for diagnosing
infection or exposure with Anaplasma platys in a subject comprising
a DNA probe or primer constructed to correspond to the OMP-1X
protein, OMP-1X Box 1, or OMP-1X Box 2 of Anaplasma platys,
characterized in that the probe or primer comprises one or more of
the nucleotides or polynucleotides described herein.
[0191] The kits described herein can comprise one or more of the
polypeptides described herein and means for determining binding of
the polypeptide to anti-Anaplasma platys or antibody fragments in
the sample. A kit or article of manufacture can also comprise one
or more antibodies or antibody fragments described herein and means
for determining binding of the antibodies or antibody fragments to
Anaplasma platys and/or Anaplasma platys polypeptides in the
sample. A kit can comprise a device containing one or more
polypeptides or antibodies described herein and instructions for
use of the one or more polypeptides or antibodies for, e.g., the
identification of an Anaplasma platys infection in a mammal. The
kit can also comprise packaging material comprising a label that
indicates that the one or more polypeptides or antibodies of the
kit can be used for the identification of Anaplasma platys
infection. Other components such as buffers, controls, and the
like, known to those of ordinary skill in art, can be included in
such test kits. The polypeptides, antibodies, assays, and kits of
the invention are useful, for example, in the diagnosis of
individual cases of Anaplasma platys infection in a subject, as
well as epidemiological studies of Anaplasma platys outbreaks.
Exposure to Anaplasma platys can also be detected. Exposure would
include the presence of Anaplasma platys organisms without clinical
symptoms and prior infection with Anaplasma platys.
Samples
[0192] Vertebrate host samples are collected from body tissue or
bodily fluid, such as for example, blood, plasma, saliva, and
peripheral blood mononuclear cells. For the invertebrate vectors
which can transmit the pathogen from one vertebrate host to
another, the sample can be from dissected ticks (e.g., midgut,
salivary glands, and hemolymph), tick pieces, and frozen and
smashed ticks in preparation for PCR assays. Further preparation of
tick tissues can involve heating the sample, digesting the samples
with proteases, and isolating pure DNA from the tick tissues. Other
suitable samples include, but are not limited to, saliva, cheek
scrapings, biopsies of retina, kidney or liver or other organs or
tissues; skin biopsies; amniotic fluid; or CNS samples; and the
like.
Methods
PCR Based Diagnostics
[0193] Described herein are methods for detecting Anaplasma platys
in a sample obtained from a subject, comprising (a) providing a
primer set comprising: (i) one or more forward primers comprising
the sequence of: SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO:84, or SEQ
ID NO: 90 and (ii) one or more reverse primers comprising the
sequence of: SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID
NO:88, SEQ ID NO: 89 or SEQ ID NO: 91; (b) amplifying DNA in the
sample with the said primer set and a polymerase chain reaction,
and (c) determining the length or sequence of the PCR products of
step (b), wherein the presence of a PCR product having a length or
sequence which corresponds to the length or sequence, respectively,
of that region of the Anaplasma platys p44 gene which is located
between the regions to which the one or more forward primers and
the one or more reverse primers bind is indicative of the presence
of Anaplasma platys in the sample. For example, and not to be
limiting, the one or more forward or reverse primers can be from 15
to 35 nucleotides in length. In one aspect, the forward and the
reverse primer can comprise SEQ ID NO: 90 and SEQ ID NO: 91,
respectively.
[0194] Further described herein are primer sets for detecting
Anaplasma platys in a sample, the primer set comprising: (a) one or
more forward primers comprising the sequence of: SEQ ID NO: 82, SEQ
ID NO: 83, SEQ ID NO:84, or SEQ ID NO: 90 and (ii) one or more
reverse primers comprising the sequence of: SEQ ID NO: 85, SEQ ID
NO: 86, SEQ ID NO: 87, SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO:
91. For example, and not to be limiting, the one or more forward or
reverse primers can be from 15 to 35 nucleotides in length. In one
aspect, the forward and the reverse primer can comprise SEQ ID NO:
90 and SEQ ID NO: 91, respectively.
[0195] Also described herein are methods for detecting Anaplasma
platys in a sample obtained from a subject, comprising (a)
providing a primer set comprising: (i) one or more forward primers
comprising the sequence of: SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID
NO:56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62 and (ii) one
or more reverse primers comprising the sequence of: SEQ ID NO: 53,
SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ
ID NO: 63; (b) amplifying DNA in the sample with the said primer
set and a polymerase chain reaction, and (c) determining the length
or sequence of the PCR products of step (b), wherein the presence
of a PCR product having a length or sequence which corresponds to
the length or sequence, respectively, of that region of the
Anaplasma platys p44 gene which is located between the regions to
which the one or more forward primers and the one or more reverse
primers bind is indicative of the presence of Anaplasma platys in
the sample. For example, and not to be limiting, the one or more
forward or reverse primers can be from 15 to 35 nucleotides in
length. In one aspect, the forward and the reverse primer can
comprise one or more of pairs of sequences described herein,
including, but not limited to: PAIR 1: SEQ ID NO: 52 and SEQ ID NO:
53; PAIR 2: SEQ ID NO: 54 and SEQ ID NO: 55; PAIR 3: SEQ ID NO: 56
and SEQ ID NO: 57; PAIR 4: SEQ ID NO: 58 and SEQ ID NO: 59; PAIR 5:
SEQ ID NO: 60 and SEQ ID NO: 61; or PAIR 6: SEQ ID NO: 62 and SEQ
ID NO: 63.
[0196] Further described herein are primer sets for detecting
Anaplasma platys in a sample, the primer set comprising: (a) one or
more forward primers comprising the sequence of: SEQ ID NO: 82, SEQ
ID NO: 83, SEQ ID NO:84, or SEQ ID NO: 90 and (ii) one or more
reverse primers comprising the sequence of: SEQ ID NO: 85, SEQ ID
NO: 86, SEQ ID NO: 87, SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO:
91. For example, and not to be limiting, the one or more forward or
reverse primers can be from 15 to 35 nucleotides in length.
[0197] Described herein are methods for detecting Anaplasma platys
in a sample obtained from a subject, comprising (a) providing a
primer set comprising: (i) one or more forward primers comprising
the sequence of: SEQ ID NO: 64, SEQ ID NO: 18, or SEQ ID NO: 20 and
(ii) one or more reverse primers comprising the sequence of: SEQ ID
NO: 65, SEQ ID NO: 19, or SEQ ID NO: 99; (b) amplifying DNA in the
sample with the said primer set and a polymerase chain reaction,
and (c) determining the length or sequence of the PCR products of
step (b), wherein the presence of a PCR product having a length or
sequence which corresponds to the length or sequence, respectively,
of that region of the Anaplasma platys OMP-1X gene which is located
between the regions to which the one or more forward primers and
the one or more reverse primers bind is indicative of the presence
of Anaplasma platys in the sample. For example, and not to be
limiting, the one or more forward or reverse primers can be from 15
to 35 nucleotides in length. In one aspect, the forward and reverse
primers can comprise one or more pairs of sequences described
herein, including, but not limited to: PAIR 1: SEQ ID NO: 64 and
SEQ ID NO: 65; PAIR 2: SEQ ID NO: 18 and SEQ ID NO: 19; or PAIR 3:
SEQ ID NO: 20 and SEQ ID NO: 99, respectively.
[0198] Further described herein are primer sets for detecting
Anaplasma platys in a sample, the primer set comprising: (a) one or
more forward primers comprising the sequence of: SEQ ID NO: 64, SEQ
ID NO: 18, or SEQ ID NO: 20 and (ii) one or more reverse primers
comprising the sequence of: SEQ ID NO: 65, SEQ ID NO: 19, or SEQ ID
NO: 99. For example, and not to be limiting, the one or more
forward or reverse primers can be from 15 to 35 nucleotides in
length.
[0199] Also described herein are methods of detecting the presence
of Anaplasma platys in a sample by contacting the sample with a DNA
probe or primer constructed to correspond to the P44 protein of
Anaplasma platys, characterized in that the probe or primer
comprises one or more of the nucleotides or polynucleotides
described herein.
[0200] Further described herein are methods of detecting the
presence of Anaplasma platys in a sample by contacting the sample
with a DNA probe or primer constructed to correspond to the OMP-1X
protein, OMP-1X Box 1, or OMP-1X Box 2 of Anaplasma platys,
characterized in that the probe or primer comprises one or more of
the nucleotides or polynucleotides described herein.
[0201] PCR assays are well known in the art, including, for
example, U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202;U.S. Pat.
No. 4,965,188. Generally, polynucleotide primers are annealed to
denatured strands of a target nucleic acid. Primer extension
products are formed by polymerization of deoxynucleoside
triphosphates by a polymerase. PCR then involves repetitive cycles
of template nucleic acid denaturation, primer annealing and
extension of the annealed primers by the action of a thermostable
polymerase. The process results in exponential amplification of the
target Anaplasma platys nucleic acids in the test sample, which
allows for the detection of target polynucleotides existing in very
low concentrations in a sample.
[0202] Real-time PCR assays are based on the detection of a signal,
e.g., a fluorescent reporter signal. This signal increases in
direct proportion to the amount of PCR product in a reaction.
Real-time PCR is any amplification technique that makes it possible
to monitor the evolution of an ongoing amplification reaction. See,
Quantitation of DNA/RNA Using Real-Time PCR Detection, Perkin Elmer
Applied Biosystems (1999); PCR Protocols (Academic Press New York,
1989). By recording the amount of fluorescence emission at each
cycle, it is possible to monitor the PCR reaction during
exponential phase where the first significant increase in the
amount of PCR product correlates to the initial amount of target
template. The higher the starting copy number of the nucleic acid
target, the sooner a significant increase in fluorescence is
observed.
[0203] Described herein are methods for detecting and/or
quantifying Anaplasma platys polynucleotides in a test sample.
Sense primers and antisense primers can be added to a test sample
under conditions suitable for a polymerase chain reaction. The
primers hybridize with Anaplasma platys P44 or OMP-1X
polynucleotides such that an amplification product is formed if
Anaplasma platys P44 or OMP-1X polynucleotides are present in the
test sample. In one aspect, the primers can be SEQ ID NOs: 90 and
91. Amplification products are detected and the presence and/or
quantity of Anaplasma platys P44 or OMP-1X polynucleotides is
determined. Amplification products can be detected with a
polynucleotide probe that hybridizes, under conditions suitable for
a polymerase chain reaction, with an Anaplasma platys P44 or OMP-1X
polynucleotide sequence. Examples of probes include SEQ ID NOs: 17
which can be used to identify the presence of an OMP-1X
polynucleotide. The amplification product can be quantified by
measuring a detection signal from the probe and comparing said
detection signal to a second probe detection signal from a
quantification standard. The quantification standard can be
extracted in parallel with the test sample.
[0204] Also disclosed are methods wherein the PCR primers can be
selected from the variable regions of an Anaplasma platys P44 or
OMP-1X polynucleotide. For example, primers of 10, 15, 20, 25, 30,
or 40 contiguous nucleotides can be selected from the regions of
P44 Boxes 1-6 or OMP-1X Boxes 1 or 2.
[0205] The polynucleotides described herein can be used to detect
the presence of Anaplasma platys polynucleotides in a sample. The
polynucleotides can be used to detect Anaplasma platys
polynucleotides in a sample by a simple hybridization reaction and
can also be used in, e.g., polymerase chain reactions (PCR) such as
a real-time PCR reaction. The methods and compositions described
herein can also be used to differentially detect the presence
Anaplasma platys from Anaplasma phagocytophilum or other Anaplasma
species.
Antibody Based Diagnostics
[0206] Also described herein are methods for detecting Anaplasma
platys in a sample by contacting the sample with one or more of the
antibodies described herein. In one aspect, to the Anaplasma platys
in a sample of a bodily fluid from a patient. The method comprises
providing an isolated outer membrane protein of Anaplasma platys,
for example, a recombinant form of the isolated protein, contacting
the outer membrane protein or polypeptide with a sample taken from
the patient; and assaying for the formation of a complex between
the outer membrane protein or polypeptide and antibodies in the
sample. In one aspect, the isolated protein or polypeptide be
attached to a substrate such as a column, plastic dish, matrix, or
membrane, preferably nitrocellulose. The sample can be a tissue or
a biological fluid, including urine, whole blood, exudate, or
serum. The sample can be untreated, subjected to precipitation,
fractionation, separation, or purification before combining with
the isolated protein or peptide. Interactions between antibodies in
the sample and the isolated protein or peptide can be detected by
radiometric, colorimetric, or fluorometric means, size-separation,
or precipitation. In one aspect, detection of the antibody-outer
membrane protein complex can be by addition of a secondary antibody
that can be coupled to a detectable tag, such as for example, an
enzyme, fluorophore, or chromophore. Formation of the complex is
indicative of the presence of anti-Anaplasma platys antibodies,
either IgM or IgG, in the patient. Thus, the method can be used to
determine whether a subject is infected with Anaplasma platys.
[0207] In one aspect, the method can employ an enzyme-linked
immunosorbent assay (ELISA) or a Western immunoblot procedure. Such
methods can be relatively simple to perform and do not require
special equipment as long as membrane strips are coated with a high
quality antigen. Accordingly, in one aspect, it can be advantageous
to use a recombinant form of the outer membrane protein of
Anaplasma platys since such proteins, typically, are more pure and
consistent in quality than a purified form of such protein.
Peptide Based Diagnostics
[0208] Described herein are methods of detecting antibodies that
specifically bind an Anaplasma platys polypeptide, comprising: (a)
contacting a purified polypeptide comprising the amino acid
sequence of one or more of the following: (i) P44 Box 1, P44 Box 2,
P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, or a fragment thereof;
(ii) a variant of P44 Box 1, P44, Box 2, P44 Box 3, P44 Box 4, P44
Box 5, P44 Box 6, or a fragment thereof; (iii) an antigenic
fragment of P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5,
P44 Box 6, or a fragment thereof; (iv) SEQ ID NO: 21, SEQ ID NO:
22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ
ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO:
40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ
ID NO: 45, or a fragment thereof; (v) a variant of SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39,
SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID
NO: 44, SEQ ID NO: 45, or a fragment thereof; (vi) an antigenic
fragment of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45; (vii) SEQ ID
NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41,
SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID
NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, or a fragment thereof; (viii)
a variant of SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID
NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95,
SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, or a
fragment thereof; (ix) an antigenic fragment of SEQ ID NO: 39, SEQ
ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO:
94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ
ID NO: 44, or SEQ ID NO: 97; (x) the amino acid sequence comprising
the amino acid from about position 20 to about position 40 of FIG.
23, the amino acid sequence comprising the amino acid from about
position 40 to about position 64 of FIG. 23, the amino acid from
about position 75 to about position 85 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 102 to about
position 111 of FIG. 23, the amino acid from about position 170 to
about position 190 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 178 to about position 222 of
FIG. 23, the amino acid from about position 205 to about position
215 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 259 to about position 266 of FIG. 23, the amino
acid from about position 270 to about position 290 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
319 to about position 340 of FIG. 23, the amino acid from about
position 365 to about position 380 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 451 to about
position 460 of FIG. 23, the amino acid from about position 1 to
about position 41 of FIG. 19, the amino acid sequence comprising
the amino acid from about position 78 to about position 85 of FIG.
19, the amino acid from about position 174 to about position 192 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 227 to about position 234 of FIG. 19 the amino acid
from about position 276 to about position 294 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 416 to
about position 433 of FIG. 19, or a fragment thereof; (xi) a
variant of the amino acid sequence comprising the amino acid from
about position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, or a fragment thereof; (xii) an antigenic fragment
of the amino acid sequence comprising the amino acid from about
position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, or a fragment thereof; (xii) a combination of one
or more of the sequences in (i)-(xii); (xiv) OMP-1X Box 1, OMP-1X
Box 2, or a fragment thereof; (xv) a variant of OMP-1X Box 1,
OMP-1X Box 2, or a fragment thereof; (xvi) an antigenic fragment of
OMP-1X Box 1, OMP-1X Box 2, or a fragment thereof; (xvii) SEQ ID
NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ
ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9,
or a fragment thereof; (xviii) a variant of SEQ ID NO: 1, SEQ ID
NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ
ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, or a fragment
thereof; (xix) an antigenic fragment of SEQ ID NO: 1, SEQ ID NO: 5,
SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO:
7, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 9; (xx) SEQ ID NO: 2,
SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO:
8, or a fragment thereof; (xxi) a variant of SEQ ID NO: 2, SEQ ID
NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, or a
fragment thereof; (xxii) an antigenic fragment of SEQ ID NO: 2, SEQ
ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, or SEQ ID NO:
8; (xxiii) the amino acid from about position 66 to about position
192 of FIG. 22, the amino acid from about position 70 to about
position 180 of FIG. 22, a combination of the amino acid from about
position 66 to about position 192 and the amino acid from about
position 70 to about position 180 of FIG. 22, the amino acid from
about position 240 to about position 312 of FIG. 22, the amino acid
from about position 230 to about position 300 of FIG. 22, a
combination of the amino acid from about position 240 to about
position 312 and the amino acid from about position 230 to about
position 300 of FIG. 22, or a fragment thereof; (xxiv) a variant of
the amino acid from about position 66 to about position 192 of FIG.
22, the amino acid from about position 70 to about position 180 of
FIG. 22, a combination of the amino acid from about position 66 to
about position 192 and the amino acid from about position 70 to
about position 180 of FIG. 22, the amino acid from about position
240 to about position 312 of FIG. 22, the amino acid from about
position 230 to about position 300 of FIG. 22, a combination of the
amino acid from about position 240 to about position 312 and the
amino acid from about position 230 to about position 300 of FIG.
22, or a fragment thereof; (xxv) an antigenic fragment of the amino
acid from about position 66 to about position 192 of FIG. 22, the
amino acid from about position 70 to about position 180 of FIG. 22,
a combination of the amino acid from about position 66 to about
position 192 and the amino acid from about position 70 to about
position 180 of FIG. 22, the amino acid from about position 240 to
about position 312 of FIG. 22, the amino acid from about position
230 to about position 300 of FIG. 22, a combination of the amino
acid from about position 240 to about position 312 and the amino
acid from about position 230 to about position 300 of FIG. 22, or a
fragment thereof; or (xxvi) a combination of one or more of the
sequences in (xiv)-(xxv); with a test sample, under conditions that
allow polypeptide/antibody complex to form; (b) detecting
polypeptide/antibody complexes; wherein the detection of
polypeptide/antibody complexes is an indication that antibodies
specific for an Anaplasma platys polypeptide is present in the test
sample. For example, and not to be limiting, the test sample can be
a biological sample from a subject, and the detection of
polypeptide/antibody complexes can be an indication that the
subject has an Anaplasma platys infection or has been exposed to
Anaplasma platys.
[0209] In one aspect, the methods of detecting antibodies that
specifically bind an Anaplasma platys polypeptide described herein
can further comprise determining the amount of antibody in the test
sample. In yet a further aspect, the purified polypeptide can be
attached to a substrate. In still a further aspect, the purified
protein can be a fusion protein. For example, and not to be
limiting, the purified polypeptide can be fused to an indicator
reagent, an amino acid spacer, an amino acid linker, a signal
sequence, a stop transfer sequence, a transmembrane domain, a
protein purification ligand, a heterologous protein, or a
combination thereof. In a further aspect, the purified polypeptide
can be in multimeric form.
[0210] In yet a further aspect, the methods of detecting antibodies
that specifically bind an Anaplasma platys polypeptide described
herein can further comprise a microtiter plate assay, reversible
flow chromatographic binding assay, an enzyme linked immunosorbent
assay, a radioimmunoassay, a hemagglutination assay, a western blot
assay, a fluorescence polarization immunoassay, or an indirect
immunofluorescence assay.
[0211] Also described herein are methods of detecting an Anaplasma
platys infection or exposure to Anaplasma platys in a subject
comprising: (a) obtaining a biological sample from the subject; (b)
contacting a purified polypeptide comprising the amino acid
sequence of one or more of the following: (i) P44 Box 1, P44 Box 2,
P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, or a fragment thereof;
(ii) a variant of P44 Box 1, P44, Box 2, P44 Box 3, P44 Box 4, P44
Box 5, P44 Box 6, or a fragment thereof; (iii) an antigenic
fragment of P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4, P44 Box 5,
P44 Box 6, or a fragment thereof; (iv) SEQ ID NO: 21, SEQ ID NO:
22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ
ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO:
40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ
ID NO: 45, or a fragment thereof; (v) a variant of SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39,
SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID
NO: 44, SEQ ID NO: 45, or a fragment thereof; (vi) an antigenic
fragment of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45; (vii) SEQ ID
NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41,
SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID
NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, or a fragment thereof; (viii)
a variant of SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID
NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95,
SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ ID NO: 97, or a
fragment thereof; (ix) an antigenic fragment of SEQ ID NO: 39, SEQ
ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO:
94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ
ID NO: 44, or SEQ ID NO: 97; (x) the amino acid sequence comprising
the amino acid from about position 20 to about position 40 of FIG.
23, the amino acid sequence comprising the amino acid from about
position 40 to about position 64 of FIG. 23, the amino acid from
about position 75 to about position 85 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 102 to about
position 111 of FIG. 23, the amino acid from about position 170 to
about position 190 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 178 to about position 222 of
FIG. 23, the amino acid from about position 205 to about position
215 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 259 to about position 266 of FIG. 23, the amino
acid from about position 270 to about position 290 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
319 to about position 340 of FIG. 23, the amino acid from about
position 365 to about position 380 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 451 to about
position 460 of FIG. 23, the amino acid from about position 1 to
about position 41 of FIG. 19, the amino acid sequence comprising
the amino acid from about position 78 to about position 85 of FIG.
19, the amino acid from about position 174 to about position 192 of
FIG. 19, the amino acid sequence comprising the amino acid from
about position 227 to about position 234 of FIG. 19 the amino acid
from about position 276 to about position 294 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 416 to
about position 433 of FIG. 19, or a fragment thereof; (xi) a
variant of the amino acid sequence comprising the amino acid from
about position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, or a fragment thereof; (xii) an antigenic fragment
of the amino acid sequence comprising the amino acid from about
position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, or a fragment thereof; (xiii) a combination of one
or more of the sequences in (i)-(xii); (xiv) OMP-1X Box 1, OMP-1X
Box 2, or a fragment thereof; (xv) a variant of OMP-1X Box 1,
OMP-1X Box 2, or a fragment thereof; ((xvi) an antigenic fragment
of OMP-1X Box 1, OMP-1X Box 2, or a fragment thereof; (xvii) SEQ ID
NO: 1, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ
ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9,
or a fragment thereof; (xviii) a variant of SEQ ID NO: 1, SEQ ID
NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ
ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, or a fragment
thereof; (xix) an antigenic fragment of SEQ ID NO: 1, SEQ ID NO: 5,
SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO:
7, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 9; (xx) SEQ ID NO: 2,
SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO:
8, or a fragment thereof; (xxi) a variant of SEQ ID NO: 2, SEQ ID
NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, or a
fragment thereof; (xxii) an antigenic fragment of SEQ ID NO: 2, SEQ
ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, or SEQ ID NO:
8; (xxiii) the amino acid from about position 66 to about position
192 of FIG. 22, the amino acid from about position 70 to about
position 180 of FIG. 22, a combination of the amino acid from about
position 66 to about position 192 and the amino acid from about
position 70 to about position 180 of FIG. 22, the amino acid from
about position 240 to about position 312 of FIG. 22, the amino acid
from about position 230 to about position 300 of FIG. 22, a
combination of the amino acid from about position 240 to about
position 312 and the amino acid from about position 230 to about
position 300 of FIG. 22, or a fragment thereof; (xxiv) a variant of
the amino acid from about position 66 to about position 192 of FIG.
22, the amino acid from about position 70 to about position 180 of
FIG. 22, a combination of the amino acid from about position 66 to
about position 192 and the amino acid from about position 70 to
about position 180 of FIG. 22, the amino acid from about position
240 to about position 312 of FIG. 22, the amino acid from about
position 230 to about position 300 of FIG. 22, a combination of the
amino acid from about position 240 to about position 312 and the
amino acid from about position 230 to about position 300 of FIG.
22, or a fragment thereof; (xxv) an antigenic fragment of the amino
acid from about position 66 to about position 192 of FIG. 22, the
amino acid from about position 70 to about position 180 of FIG. 22,
a combination of the amino acid from about position 66 to about
position 192 and the amino acid from about position 70 to about
position 180 of FIG. 22, the amino acid from about position 240 to
about position 312 of FIG. 22, the amino acid from about position
230 to about position 300 of FIG. 22, a combination of the amino
acid from about position 240 to about position 312 and the amino
acid from about position 230 to about position 300 of FIG. 22, or a
fragment thereof; or (xxvi) a combination of one or more of the
sequences in (xiv)-(xxv); with the biological sample under
conditions that allow polypeptide/antibody complexes to form; and
(c) detecting polypeptide/antibody complexes; wherein the detection
of polypeptide/antibody complexes is an indication that the subject
has an Anaplasma platys infection or exposure to Anaplasma platys.
In one aspect, the methods of detecting an Anaplasma platys
infection or exposure to Anaplasma platys in a subject can further
comprise contacting the polypeptide/antibody complexes of step (b)
with an indicator reagent that generates a measurable signal prior
to the performance of step (c).
[0212] In a further aspect, the purified protein can be a fusion
protein. For example, and not to be limiting, the purified
polypeptide can be fused to an indicator reagent, an amino acid
spacer, an amino acid linker, a signal sequence, a stop transfer
sequence, a transmembrane domain, a protein purification ligand, a
heterologous protein, or a combination thereof. In yet a further
aspect, the polypeptide/antibody complexes can be detected at about
10 days after exposure or infection of the subject by Anaplasma
platys.
[0213] Further described herein are methods of detecting Anaplasma
platys polypeptides in a test sample comprising: (a) contacting one
or more antibodies that specifically bind to a Anaplasma platys
polypeptide with the test sample under conditions that allow
polypeptide/antibody complexes to form; wherein the Anaplasma
platys polypeptide comprises the amino acid sequence of one or more
of the following: (i) P44 Box 1, P44 Box 2, P44 Box 3, P44 Box 4,
P44 Box 5, P44 Box 6, or a fragment thereof; (ii) a variant of P44
Box 1, P44, Box 2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, or a
fragment thereof; (iii) an antigenic fragment of P44 Box 1, P44 Box
2, P44 Box 3, P44 Box 4, P44 Box 5, P44 Box 6, or a fragment
thereof; (iv) SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID
NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,
SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or a fragment
thereof; (v) a variant of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ
ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:
41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or
a fragment thereof; (vi) an antigenic fragment of SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39,
SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID
NO: 44, or SEQ ID NO: 45; (vii) SEQ ID NO: 39, SEQ ID NO: 42, SEQ
ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO:
42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, SEQ
ID NO: 97, or a fragment thereof; (viii) a variant of SEQ ID NO:
39, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 93, SEQ ID NO: 41, SEQ
ID NO: 94, SEQ ID NO: 42, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO:
96, SEQ ID NO: 44, SEQ ID NO: 97, or a fragment thereof; (ix) an
antigenic fragment of SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 40,
SEQ ID NO: 93, SEQ ID NO: 41, SEQ ID NO: 94, SEQ ID NO: 42, SEQ ID
NO: 95, SEQ ID NO: 43, SEQ ID NO: 96, SEQ ID NO: 44, or SEQ ID NO:
97; (x) the amino acid sequence comprising the amino acid from
about position 20 to about position 40 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 40 to about
position 64 of FIG. 23, the amino acid from about position 75 to
about position 85 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 102 to about position 111 of
FIG. 23, the amino acid from about position 170 to about position
190 of FIG. 23, the amino acid sequence comprising the amino acid
from about position 178 to about position 222 of FIG. 23, the amino
acid from about position 205 to about position 215 of FIG. 23, the
amino acid sequence comprising the amino acid from about position
259 to about position 266 of FIG. 23, the amino acid from about
position 270 to about position 290 of FIG. 23, the amino acid
sequence comprising the amino acid from about position 319 to about
position 340 of FIG. 23, the amino acid from about position 365 to
about position 380 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 451 to about position 460 of
FIG. 23, the amino acid from about position 1 to about position 41
of FIG. 19, the amino acid sequence comprising the amino acid from
about position 78 to about position 85 of FIG. 19, the amino acid
from about position 174 to about position 192 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 227 to
about position 234 of FIG. 19 the amino acid from about position
276 to about position 294 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 416 to about position
433 of FIG. 19, or a fragment thereof; (xi) a variant of the amino
acid sequence comprising the amino acid from about position 20 to
about position 40 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 40 to about position 64 of FIG.
23, the amino acid from about position 75 to about position 85 of
FIG. 23, the amino acid sequence comprising the amino acid from
about position 102 to about position 111 of FIG. 23, the amino acid
from about position 170 to about position 190 of FIG. 23, the amino
acid sequence comprising the amino acid from about position 178 to
about position 222 of FIG. 23, the amino acid from about position
205 to about position 215 of FIG. 23, the amino acid sequence
comprising the amino acid from about position 259 to about position
266 of FIG. 23, the amino acid from about position 270 to about
position 290 of FIG. 23, the amino acid sequence comprising the
amino acid from about position 319 to about position 340 of FIG.
23, the amino acid from about position 365 to about position 380 of
FIG. 23, the amino acid sequence comprising the amino acid from
about position 451 to about position 460 of FIG. 23, the amino acid
from about position 1 to about position 41 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 78 to
about position 85 of FIG. 19, the amino acid from about position
174 to about position 192 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 227 to about position
234 of FIG. 19 the amino acid from about position 276 to about
position 294 of FIG. 19, the amino acid sequence comprising the
amino acid from about position 416 to about position 433 of FIG.
19, or a fragment thereof; (xii) an antigenic fragment of the amino
acid sequence comprising the amino acid from about position 20 to
about position 40 of FIG. 23, the amino acid sequence comprising
the amino acid from about position 40 to about position 64 of FIG.
23, the amino acid from about position 75 to about position 85 of
FIG. 23, the amino acid sequence comprising the amino acid from
about position 102 to about position 111 of FIG. 23, the amino acid
from about position 170 to about position 190 of FIG. 23, the amino
acid sequence comprising the amino acid from about position 178 to
about position 222 of FIG. 23, the amino acid from about position
205 to about position 215 of FIG. 23, the amino acid sequence
comprising the amino acid from about position 259 to about position
266 of FIG. 23, the amino acid from about position 270 to about
position 290 of FIG. 23, the amino acid sequence comprising the
amino acid from about position 319 to about position 340 of FIG.
23, the amino acid from about position 365 to about position 380 of
FIG. 23, the amino acid sequence comprising the amino acid from
about position 451 to about position 460 of FIG. 23, the amino acid
from about position 1 to about position 41 of FIG. 19, the amino
acid sequence comprising the amino acid from about position 78 to
about position 85 of FIG. 19, the amino acid from about position
174 to about position 192 of FIG. 19, the amino acid sequence
comprising the amino acid from about position 227 to about position
234 of FIG. 19 the amino acid from about position 276 to about
position 294 of FIG. 19, the amino acid sequence comprising the
amino acid from about position 416 to about position 433 of FIG.
19, or a fragment thereof; (xiii) a combination of one or more of
the sequences in (i)-(xii); (xiv) OMP-1X Box 1, OMP-1X Box 2, or a
fragment thereof; (xv) a variant of OMP-1X Box 1, OMP-1X Box 2, or
a fragment thereof; ((xvi) an antigenic fragment of OMP-1X Box 1,
OMP-1X Box 2, or a fragment thereof; (xvii) SEQ ID NO: 1, SEQ ID
NO: 5, SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ
ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, or a fragment
thereof; (xviii) a variant of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 10, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ
ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, or a fragment thereof; (xix)
an antigenic fragment of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10,
SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO:
4, SEQ ID NO: 8, or SEQ ID NO: 9; (xx) SEQ ID NO: 2, SEQ ID NO: 6,
SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, or a
fragment thereof; (xxi) a variant of SEQ ID NO: 2, SEQ ID NO: 6,
SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8, or a
fragment thereof; (xxii) an antigenic fragment of SEQ ID NO: 2, SEQ
ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, or SEQ ID NO:
8; (xxiii) the amino acid from about position 66 to about position
192 of FIG. 22, the amino acid from about position 70 to about
position 180 of FIG. 22, a combination of the amino acid from about
position 66 to about position 192 and the amino acid from about
position 70 to about position 180 of FIG. 22, the amino acid from
about position 240 to about position 312 of FIG. 22, the amino acid
from about position 230 to about position 300 of FIG. 22, a
combination of the amino acid from about position 240 to about
position 312 and the amino acid from about position 230 to about
position 300 of FIG. 22, or a fragment thereof; (xxiv) a variant of
the amino acid from about position 66 to about position 192 of FIG.
22, the amino acid from about position 70 to about position 180 of
FIG. 22, a combination of the amino acid from about position 66 to
about position 192 and the amino acid from about position 70 to
about position 180 of FIG. 22, the amino acid from about position
240 to about position 312 of FIG. 22, the amino acid from about
position 230 to about position 300 of FIG. 22, a combination of the
amino acid from about position 240 to about position 312 and the
amino acid from about position 230 to about position 300 of FIG.
22, or a fragment thereof; (xxv) an antigenic fragment of the amino
acid from about position 66 to about position 192 of FIG. 22, the
amino acid from about position 70 to about position 180 of FIG. 22,
a combination of the amino acid from about position 66 to about
position 192 and the amino acid from about position 70 to about
position 180 of FIG. 22, the amino acid from about position 240 to
about position 312 of FIG. 22, the amino acid from about position
230 to about position 300 of FIG. 22, a combination of the amino
acid from about position 240 to about position 312 and the amino
acid from about position 230 to about position 300 of FIG. 22, or a
fragment thereof; or (xxvi) a combination of one or more of the
sequences in (xiv)-(xxv); (b) detecting polypeptide/antibody
complexes; wherein the detection of polypeptide/antibody complexes
is an indication that an Anaplasma platys polypeptide is present in
the test sample. For example, and not to be limiting, the one or
more antibodies can be monoclonal antibodies, polyclonal
antibodies, Fab fragments, Fab' fragments, Fab'-SH fragments,
F(ab').sub.2 fragments, Fv fragments, or single chain antibodies.
In one aspect, the complexes of step (a) can be contacted with an
indicator reagent prior to the performance of step (b).
[0214] In one aspect, the methods of detecting Anaplasma platys
polypeptides in a test sample can further comprise determining the
amount of Anaplasma platys polypeptides in the test sample. In yet
a further aspect, the one or more antibodies can be attached to a
substrate.
[0215] In a further aspect, the methods of detecting Anaplasma
platys polypeptides in a test sample can further comprise a
microtiter plate assay, reversible flow chromatographic binding
assay, an enzyme linked immunosorbent assay, a radioimmunoassay, a
hemagglutination assay, a western blot assay, a fluorescence
polarization immunoassay, or an indirect immunofluorescence
assay.
[0216] The methods described herein can be used to detect
antibodies or antibody fragments specific for Anaplasma platys
polypeptides, Anaplasma platys polynucleotides, or a combination
thereof in a test sample, such as a biological sample, an
environmental sample, or a laboratory sample. A test sample can
potentially comprise Anaplasma platys polynucleotides, Anaplasma
platys polypeptides, or antibodies specific for Anaplasma platys. A
biological sample can include, for example, sera, blood, cells,
plasma, or tissue from a mammal such as a horse, cat, dog or human.
The test sample can be untreated, precipitated, fractionated,
separated, diluted, concentrated, or purified.
[0217] Disclosed herein are methods that comprise contacting an
Anaplasma platys polypeptide with a test sample under conditions
that allow a polypeptide/antibody complex, i.e., an immunocomplex,
to form. That is, one or more of the polypeptides described herein
specifically binds to an antibody specific for Anaplasma platys
antigens located in the sample. One of skill in the art is familiar
with assays and conditions that are used to detect
antibody/polypeptide complex binding. The formation of a complex
between polypeptides and anti-Anaplasma platys in the sample is
detected. In one embodiment of the invention antibody-polypeptide
complexes can be detected at about 10, 15, 20, 25, 30 or less days
after exposure or infection of the subject by Anaplasma platys.
[0218] The antibodies described herein can be used in a method of
the diagnosis of Anaplasma platys infection by obtaining a test
sample from, e.g., a human or animal suspected of having an
Anaplasma platys infection. Exposure to Anaplasma platys can also
be detected. Exposure would include the presence of Anaplasma
platys organisms without clinical symptoms and prior infection with
Anaplasma platys. The test sample is contacted with antibodies of
the invention under conditions enabling the formation of
antibody-antigen complexes (i.e., immunocomplexes). The amount of
antibody-antigen complexes can be determined by methodology known
in the art. A level that is higher than that formed in a control
sample indicates an Anaplasma platys infection. A control sample is
a sample that does not comprise any Anaplasma platys polypeptides
or antibodies specific for Anaplasma platys. In one embodiment of
the invention an antibody is specific for Anaplasma platys P44 or
Anaplasma platys OMP-1X antigens only. Alternatively, a polypeptide
of the invention can be contacted with a test sample. Anaplasma
platys antibodies in a positive body sample will form an
antigen-antibody complex under suitable conditions. The amount of
antibody-antigen complexes can be determined by methods known in
the art.
[0219] Also disclosed herein are methods wherein the
polypeptide/antibody complex is detected when an indicator reagent,
such as an enzyme conjugate, which is bound to the antibody,
catalyzes a detectable reaction. Optionally, an indicator reagent
comprising a signal generating compound can be applied to the
polypeptide/antibody complex under conditions that allow formation
of a polypeptide/antibody/indicator complex. The
polypeptide/antibody/indicator complex is detected. Optionally, the
polypeptide or antibody can be labeled with an indicator reagent
prior to the formation of a polypeptide/antibody complex. The
method can optionally comprise a positive or negative control.
[0220] Disclosed herein are methods wherein one or more of the
antibodies disclosed herein are attached to a solid phase or
substrate. A test sample potentially comprising a protein
comprising one or more of the polypeptides described herein is
added to the substrate. Antibodies that specifically bind to one or
more of the polypeptides described herein are added. The antibodies
can be the same antibodies used on the solid phase or can be from a
different source or species and can be linked to an indicator
reagent, such as an enzyme conjugate. Wash steps can be performed
prior to each addition. A chromophore or enzyme substrate can be
added and color is allowed to develop. The color reaction can be
stopped and the color can be quantified using, for example, a
spectrophotometer.
[0221] Also disclosed herein are methods wherein one or more of the
antibodies described herein are attached to a solid phase or
substrate. A test sample potentially comprising a protein
comprising one or more of the polypeptides described herein is
added to the substrate. Second anti-species antibodies that
specifically bind one or more of the polypeptides described herein
are added. These second antibodies can be from a different species
than the solid phase antibodies. Third anti-species antibodies can
also be added that specifically bind the second antibodies and that
do not specifically bind the solid phase antibodies are added. The
third antibodies can comprise an indicator reagent such as an
enzyme conjugate. Wash steps can be performed prior to each
addition. A chromophore or enzyme substrate is added and color is
allowed to develop. The color reaction is stopped and the color can
be quantified using, for example, a spectrophotometer.
[0222] Disclosed herein are methods of detecting antibodies that
specifically bind an Anaplasma platys polypeptide or both. The
method comprises contacting one or more of the purified
polypeptides described herein with a test sample, under conditions
that allow polypeptide/antibody complexes to form and detecting
polypeptide/antibody complexes. The detection of
polypeptide/antibody complexes is an indication that antibodies
specific for Anaplasma platys are present in the test sample, and
the absence of polypeptide/antibody complexes is an indication that
antibodies specific for Anaplasma platys are not present in the
test sample. The complexes can be contacted with an indicator
reagent prior to the detection step. The amount of antibody in the
test sample can be determined. The purified polypeptide can be
attached to a substrate. The purified polypeptide can be a fusion
protein wherein the purified polypeptide is fused to an indicator
reagent, an amino acid spacer, an amino acid linker, a signal
sequence, a stop transfer sequence, a transmembrane domain, a
protein purification ligand, a heterologous protein, or a
combination thereof. The purified polypeptide can be in multimeric
form. The method can comprise a microtiter plate assay, reversible
flow chromatographic binding assay, an enzyme linked immunosorbent
assay, a radioimmunoassay, a hemagglutination assay a western blot
assay, a fluorescence polarization immunoassay, or an indirect
immunofluorescence assay.
[0223] Disclosed herein are methods of detecting an Anaplasma
platys infection and/or exposure to Anaplasma platys in a subject.
The method can comprise obtaining a biological sample from the
subject; contacting a purified polypeptide of the invention with
the biological sample under conditions that allow
polypeptide/antibody complexes to form; and detecting
polypeptide/antibody complexes. The detection of
polypeptide/antibody complexes is an indication that the subject
has an Anaplasma platys infection and/or exposure to Anaplasma
platys. The absence of polypeptide/antibody complexes is an
indication that the mammal has not had an Anaplasma platys
infection and/or exposure to Anaplasma platys. The
polypeptide/antibody complexes can be contacted with an indicator
reagent that generates a measurable signal prior to the performance
of the detection step. The purified polypeptide can be a fusion
protein wherein the purified polypeptide is fused to an indicator
reagent, an amino acid spacer, an amino acid linker, a signal
sequence, a stop transfer sequence, a transmembrane domain, a
protein purification ligand, a heterologous protein or a
combination thereof. The polypeptide/antibody complexes can
detected at about 10 days after exposure or infection of subject by
Anaplasma platys.
[0224] Also described herein are assays that include, but are not
limited to those based on competition, direct reaction or
sandwich-type assays, including, but not limited to enzyme linked
immunosorbent assay (ELISA), western blot, IFA, radioimmunoassay
(RIA), hemagglutination (HA), fluorescence polarization immunoassay
(FPIA), and microtiter plate assays (any assay done in one or more
wells of a microtiter plate). One assay comprises a reversible flow
chromatographic binding assay. For example, described herein are
assays similar to those described in U.S. Pat. No. 5,726,010.
[0225] Assays can use solid phases or substrates or can be
performed by immunoprecipitation or any other methods that do not
utilize solid phases. Where a solid phase or substrate is used, one
or more of the polypeptides described herein can be directly or
indirectly attached to a solid support or a substrate such as a
microtiter well, magnetic bead, non-magnetic bead, column, matrix,
membrane, fibrous mat composed of synthetic or natural fibers
(e.g., glass or cellulose-based materials or thermoplastic
polymers, such as, polyethylene, polypropylene, or polyester),
sintered structure composed of particulate materials (e.g., glass
or various thermoplastic polymers), or cast membrane film composed
of nitrocellulose, nylon, polysulfone or the like (generally
synthetic in nature). For example, the substrate can be sintered,
fine particles of polyethylene, commonly known as porous
polyethylene, for example, 10-15 micron porous polyethylene from
Chromex Corporation (Albuquerque, N. Mex.). All substrate materials
can be used in suitable shapes, such as films, sheets, or plates,
or they can be coated onto or bonded or laminated to appropriate
inert carriers, such as paper, glass, plastic films, or fabrics.
Suitable methods for immobilizing peptides on solid phases include
ionic, hydrophobic, covalent interactions and the like.
[0226] Disclosed herein is an assay format, wherein one or more
polypeptides can be coated on a solid phase or substrate. A test
sample suspected of containing an anti-Anaplasma platys antibody or
fragment thereof is incubated with an indicator reagent comprising
a signal generating compound conjugated to an antibody or antibody
fragment specific for Anaplasma platys for a time and under
conditions sufficient to form antigen/antibody complexes of either
antibodies of the test sample to the polypeptides of the solid
phase or the indicator reagent compound conjugated to an antibody
specific for Anaplasma platys to the polypeptides of the solid
phase. The reduction in binding of the indicator reagent conjugated
to an anti-Anaplasma platys antibody to the solid phase can be
quantitatively measured. A measurable reduction in the signal
compared to the signal generated from a confirmed negative
Anaplasma platys test sample indicates the presence of
anti-Anaplasma platys antibody in the test sample. This type of
assay can quantitate the amount of anti-Anaplasma platys antibodies
in a test sample.
[0227] Disclosed herein is an assay format, wherein one or more of
the polypeptides disclosed herein are coated onto a support or
substrate. One or more of the polypeptides disclosed herein can be
conjugated to an indicator reagent and added to a test sample. This
mixture can then be applied to the support or substrate. If
Anaplasma platys antibodies are present in the test sample they
will bind the polypeptide conjugated to an indicator reagent and to
the polypeptide immobilized on the support. The
polypeptide/antibody/indicator complex can then be detected. This
type of assay can quantitate the amount of anti-Anaplasma platys
antibodies in a test sample.
[0228] Disclosed herein is an assay format, wherein one or more
polypeptides disclosed herein are coated onto a support or
substrate. The test sample can be applied to the support or
substrate and incubated. Unbound components from the sample can be
washed away by washing the solid support with a wash solution. If
Anaplasma platys specific antibodies are present in the test
sample, they will bind to the polypeptide coated on the solid
phase. This polypeptide/antibody complex can be detected using a
second species-specific antibody that is conjugated to an indicator
reagent. The polypeptide/antibody/anti-species antibody indicator
complex can then be detected. This type of assay can quantitate the
amount of anti-Anaplasma platys antibodies in a test sample.
[0229] The formation of a polypeptide/antibody complex or a
polypeptide/antibody/indicator complex can be detected by
radiometric, colormetric, fluorometric, size-separation, or
precipitation methods. Optionally, detection of a
polypeptide/antibody complex is by the addition of a secondary
antibody that is coupled to an indicator reagent comprising a
signal generating compound. Indicator reagents comprising signal
generating compounds (labels) associated with a
polypeptide/antibody complex can be detected using the methods
described above and include chromogenic agents, catalysts such as
enzyme conjugates fluorescent compounds such as fluorescein and
rhodamine, chemiluminescent compounds such as dioxetanes,
acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive
elements, direct visual labels, as well as cofactors, inhibitors,
magnetic particles, and the like. Examples of enzyme conjugates
include alkaline phosphatase, horseradish peroxidase,
beta-galactosidase, and the like. The selection of a particular
label is not critical, but it will be capable of producing a signal
either by itself or in conjunction with one or more additional
substances.
[0230] Disclosed herein is an assay format wherein the Anaplasma
platys polypeptides, polynucleotides, antibodies or combinations
thereof can be used in conjunction with Raman spectroscopy. Raman
spectroscopy is an analytical technique for chemical and biological
analysis due to the wealth of information on molecular structures,
surface processes, and interface reactions that can be extracted
from experimental data. The Raman technique has been used with gene
probe biosensors. U.S. Pat. No. 5,814,516 ('516 patent) discloses a
gene probe biosensor comprising a support means, a SERS gene probe
having at least one oligonucleotide strand having at least one SERS
label, and a SERS active substrate disposed on the support means.
The support means has at least one SERS gene probe adsorbed
thereon. Biotargets such as bacterial and viral DNA, RNA and PNA
are detected using a SERS gene probe via hybridization to
oligonucleotide strands complementary to the SERS gene probe. U.S.
Pat. No. 5,814,516 is hereby incorporated by reference in its
entirety for it's teaching of the Raman technique.
[0231] The '516 patent does not disclose or suggest operatively
connecting a Raman gene probe with an integrated circuit detection
system to produce a biochip capable of SERS detection. U.S. Pat.
No. 7,267,948 (948 patent) provides another assay format wherein
the Anaplasma platys polypeptides, polynucleotides, antibodies or
combinations thereof can be used. This '948 patent describes Raman
and SERS assay methods and systems including microarrays,
biosensors and biochips for the detection of biotargets such as
DNA, proteins and pathogens using receptor probes. Receptor probes
may include one or more bioreceptors selected from antibodies, DNA,
enzymes, tissues, organelles, as well as other receptor probes, and
combinations thereof described herein. U.S. Pat. No. 7,267,948 is
hereby incorporated by reference in its entirety for it's teaching
of the Raman and SERS assay methods and systems.
[0232] Formation of the complex is indicative of the presence of
anti-Anaplasma platys antibodies in a test sample. Therefore, the
methods of the invention can be used to diagnose Anaplasma platys
infection or exposure in a patient.
[0233] The methods described herein can also indicate the amount or
quantity of anti-Anaplasma platys antibodies in a test sample. With
many indicator reagents, such as enzyme conjugates, the amount of
antibody present is proportional to the signal generated. Depending
upon the type of test sample, it can be diluted with a suitable
buffer reagent, concentrated, or contacted with a solid phase
without any manipulation. For example, serum or plasma samples that
previously have been diluted, or concentrate specimens such as
urine, can be tested in order to determine the presence and/or
amount of antibody present.
[0234] The polypeptides and assays described herein can be combined
with other polypeptides or assays to detect the presence of
Anaplasma platys along with other organisms. For example,
polypeptides and assays of the invention can be combined with
reagents that detect heartworm and/or Borrelia burgdorferi and/or
Anaplasma phagocytophilium and/or Ehrlichia canis.
[0235] Also disclosed herein are methods of detecting an Anaplasma
infection or exposure to Anaplasma in a subject. These methods can
be used as an initial or final method to identify the presence of
one or more species of Anaplasma. Anaplasma is a genus of
rickettsiales bacteria. Anaplasmas can reside in host red blood
cells and lead to the disease anaplasmosis.
Anaplasmas can require intermediate tick hosts for maturation, and
flies may act as mechanical vectors. Species of Anaplasma include,
but are not limited to, Anaplasma marginale, Anaplasma centrale,
Anaplasma mesaeterum, Anaplasma ovis, and Anaplasma platys.
[0236] Disclosed herein are methods of detecting an Anaplasma
infection or exposure to Anaplasma in a subject comprising: (a)
obtaining a biological sample from the subject; (b) contacting a
purified polypeptide encoded by one or more of the nucleotides of
the following: SEQ ID NOs: 124-132 or a combination of one or more
of the sequences of SEQ ID NOs: 124-132; with the biological sample
under conditions that allow polypeptide complexes to form; and (c)
detecting polypeptide complexes; wherein the detection of
polypeptide complexes is an indication that the subject has an
Anaplasma infection or exposure to Anaplasma. In addition, the
methods can further comprise contacting the polypeptide complexes
with an antibody that recognizes the polypeptide or polypeptide
complex.
[0237] Also disclosed are methods of detecting the presence of
Anaplasma in a sample by contacting said sample with a DNA probe or
primer constructed to correspond to an Anaplasma P44 nucleotide
sequence, characterized in that the probe or primer comprises one
or more of the nucleotides of SEQ ID NOs: 124-132.
[0238] Also disclosed are methods of detecting the presence of
Anaplasma in a sample by contacting said sample with a DNA probe or
primer constructed to correspond to an Anaplasma P44 nucleotide
sequence, characterized in that the probe or primer comprises one
or more nucleotides capable of hybridizing to one or more of the
nucleotides of SEQ ID NOs: 124-132.
[0239] A method for detecting Anaplasma in a sample obtained from a
subject, comprising (a) providing a primer set comprising: (i) one
or more forward primers capable of hybridizing to or amplifying:
one or more of the nucleotides of SEQ ID NOs: 124-132 and (ii) one
or more forward primers capable of hybridizing to or amplifying:
one or more of the nucleotides of SEQ ID NOs: 124-132; (b)
amplifying DNA in the sample with the said primer set and a
polymerase chain reaction, and (c) determining the length or
sequence of the PCR products of step (b), wherein the presence of a
PCR product having a length or sequence which corresponds to the
length or sequence, respectively, of that region of the Anaplasma
nucleotide sequence which is located between the regions to which
the one or more forward primers and the one or more reverse primers
bind is indicative of the presence of Anaplasma in the sample.
[0240] Also disclosed herein are isolated or purified
polynucleotides comprising the sequence of one or more of the
polynucleotide sequences of SEQ ID NOs: 124-132, or a fragment
thereof. Also disclosed herein are isolated or purified
polynucleotides comprising 80%, 85%, 90%, 95%, or 100% sequence
identity to the sequence of one or more of the polynucleotide
sequences of SEQ ID NOs: 124-132, or a fragment thereof.
[0241] Also disclosed are vectors for transformation of a host
cell, said vector comprising the sequence of one or more of the
polynucleotide sequences of SEQ ID NOs: 124-132, or a fragment
thereof.
[0242] Also disclosed herein are isolated or purified polypeptides
encoded by a polynucleotide sequences wherein the polynucleotide
sequence comprises the sequence of one or more of the
polynucleotide sequences of SEQ ID NOs: 124-132, or a fragment
thereof. Also disclosed herein are isolated or purified
polypeptides encoded by a polynucleotide sequences wherein the
polynucleotide sequence comprises 80%, 85%, 90%, 95%, or 100%
sequence identity to the sequence of one or more of the
polynucleotide sequences of SEQ ID NOs: 124-132, or a fragment
thereof.
EXAMPLES
Example 1
Materials and Methods
[0243] A. platys-infected dogs. Dogs that were naturally infected
with A. platys were identified in Lara, Venezuela, in 2007 by
observation of bacterial inclusions (morulae) in platelets from
blood smears, and cases were confirmed by PCR and sequencing using
primer pairs specific for A. platys 16S rRNA (EP1-EP3 and
EP2-EP3)..sup.29 Naturally infected dogs in Taichung, Taiwan and
the Democratic Republic of Congo were identified and confirmed by
PCR using primer pair EPLAT5-EPLAT..sup.42
[0244] Cloning of p44 Expression Locus from A. platys.
[0245] DNA samples from three dogs from Venezuela and one dog from
Taiwan were used as templates. By aligning the p44/msp2 expression
loci from A. phagocytophilum and A. marginale, several degenerate
primers were designed for conserved regions of the locus (FIG. 1,
Table 1). Using the first and the second primer pairs F1-R1 and
F1-R2, (hemi-) nested touchdown PCR.sup.52 was used to amplify the
tr1 and omp-1X gene sequences from A. platys. In order to avoid
truncating p44 pseudogenes in the A. platys genome, primer F3 was
designed to be upstream of the predicted p44 open reading frame.
p44ES sequences were amplified by nested touchdown PCR using primer
pairs F2-R3 and F3-R4. Amplification was performed as previously
described..sup.68 The amplified DNA fragments were cloned using the
TA cloning kit (Invitrogen, Carlsbad, Calif.) and sequenced with
M13 forward or M13 reverse sequencing primers. All sequencing data
were assembled using the SeqMan program (DNASTAR Inc., Madison,
Wis.). To confirm the assembly the entire locus was amplified using
primers F1 and R5 (Table 1).
[0246] Phylogenetic Analysis.
[0247] The deduced amino acid sequences for Tr1, OMP-1/OMP-1X, and
P44ES from A. marginale, A. phagocytophilum, and A. platys were
aligned using the MegAlign program (DNASTAR Inc.) by the Clustal W
method.
[0248] Protein Structure Analysis Using Bioinformatics Tools.
[0249] SignalP 3.0 server trained on Gram-negative bacteria
(http://www.cbs.dtu.dk/services/SignalP/) was used for signal
peptide sequence analysis. The secondary structures of P44 and
OMP-1X were predicted by PRED-TMBB.sup.4 and hydrophobicity
analysis and the hydrophobic moment profile method, as previously
described..sup.30, 35 The antigenic index and surface probability
were determined using the Protean program (DNASTAR Inc.).
[0250] ELISA Analysis of OMP-1X-Specific Peptide.
[0251] The OMP-1X peptide from A. platys was synthesized at
Biomatik (Wilmington, Del.). The purity of the peptide was greater
than 98%, as assessed by high-performance liquid chromatography.
The wells of a 96-well microtiter plate were coated with 200 ng
peptide/well and the ELISA was performed as previously
described..sup.60 Samples were from three dogs that were
PCR-positive for A. platys (TW 431, TW 270, and TW 210), and three
dogs that were both PCR-negative for A. platys and antigen dot
blot-negative for A. phagocytophilum (E05-290, E10-0062, and
E10-0075). In addition, horse anti-A. phagocytophilum positive sera
(EQ002, EQ006, and E09-0011).sup.65, 71 were used to confirm the
absence of OMP-1X peptide antigen cross-reactivity with anti-A.
phagocytophilum antibodies. The horseradish peroxidase substrate
2,2'-azido-di-(3-ethyl)-benzothiazoline-6-sulfonic acid (Sigma, St.
Louis, Mo.) in 70 mM citrate buffer (pH 4.2) applied, and
absorbance values at 415 and 492 nm were measured in an ELISA plate
reader (Molecular Devices, Sunnyvale, Calif.) as previously
described Results..sup.68 were presented as optical density at 415
nm minus that at 492 nm (OD.sub.415-OD.sub.492), and the cutoff for
a positive reaction was set at greater than the mean
OD.sub.415-OD.sub.492+3 SD for the negative control samples
(OD>0.165). The assay was repeated at least three times.
[0252] Nucleotide Sequence Accession Number.
[0253] The A. platys tr1-omp-1X-p44ES sequences from two naturally
infected dogs from Venezuela were assembled and deposited at
GenBank under accession numbers GQ868750 and GU357491. Additional
p44ES and p44 sequences were deposited at GenBank under accession
numbers GU357492, GU357493, GU357494, GU357495, GU357496, GU357497,
and HQ738571.
Results
[0254] A. platys Tr1-Omp1-p44ES Cluster Sequencing and
Assembly.
[0255] Three degenerate primers and one primer at a highly
conserved region of tr1 upstream region were designed based on
conserved regions of the aligned tr1-omp1X-omp1N-p44 cluster of A.
phagocytophilum and YP.sub.--154239 (hypothetical protein AM1138;
transcriptional regulator)-YP.sub.--154240 (outer membrane protein
1; outer membrane protein 4)-YP.sub.--154241 (msp2 operon
associated gene 3; outer membrane protein 3)-YP.sub.--154243 (msp2
operon associated gene 2)-YP.sub.--154244 (msp2 operon associated
gene 1)-YP.sub.--154245 (msp2) cluster of A. marginale (FIG. 1,
Table 1). The first touchdown PCR was designed to amplify the
entire fragment using primers F1 and R1. The PCR products from the
two dog DNA specimens were then used as templates for hemi-nested
touchdown PCRs using primers F1 and R2 (FIG. 1, Table 1). As a
result, a single band of about 2,100 bp was amplified (fragment A).
The PCR products were TA cloned and sequenced. The sequence results
showed this fragment contained A. platys tr1 and omp-1X. The
downstream region of fragment A was amplified by nested touchdown
PCR, using the PCR products obtained with primers F1 and R1 as
templates, with primer F2 designed based on the fragment A sequence
and primer R3 designed based on p44 (msp2) sequences conserved
between A. phagocytophilum and A. marginale. As a result, a single
band of approximately 1,100 bp was amplified (fragment B). The PCR
products were TA cloned and sequenced. The sequence results showed
that this fragment contained a partial A. platys p44 sequence. To
amplify A. platys full-length p44, primer F3 was designed based on
fragment B sequence and primer R4 was designed based on the
conserved region of valS found downstream of p44 (msp2) in both A.
phagocytophilum and A. marginale. Further touchdown PCR was done
using primers F1 and R4. The PCR products were then used as
templates for hemi-nested touchdown PCRs using primers F3 and R4.
As a result, a single band of about 1,700 bp was amplified
(fragment C). The PCR products were TA cloned and sequenced. The
sequence results showed this fragment contained full length A.
platys p44 sequences. The final assembled sequences 3,957 bp from
dogs 1 and 2, respectively contained the entire A. platys p44ES
locus, and the G+C content was 47.4647.51%.
[0256] The final assembled sequence of 3957 bp from Venezuelan dogs
1 and 2 contained the entire A. platys p44ES locus. The sequence
average coverage of the entire locus was 8.3 fold (5 to 15 fold).
To confirm the assembly was from a complete genomic locus, primer
R5 was designed to be downstream of the predicted p44 open reading
frame and one more touchdown PCR was conducted using primers F1 and
R5. A single band, approximately 3.9 kb was amplified (fragment D),
indicating the fragment containing the entire locus was amplified
from the blood of dog 2 (FIG. 1C). The G+C content was determined
to be 47.46% to 47.51%. The synteny among the entire outer membrane
protein gene clusters of A. platys and the two previously sequenced
Anaplasma species was analyzed using the Artemis Comparison
Tool,.sup.14 The tr1 sequences were conserved among the three
Anaplasma species (FIG. 2). The 5' and 3' regions of p44ES were
conserved between A. platys and A. phagocytophilum, but not between
A. platys and A. marginale (FIG. 2).
[0257] A. platys Tr1 structure. Three similar (97.8% to 99.5%) A.
platys tr1 sequences were obtained from two dogs from Venezuela and
one dog from Taiwan. The predicted molecular mass of A. platys Tr1
was 21.0 to 21.1 kDa with an isoelectric point of 5.50 to 5.80
(Table 2). Tr1 was not predicted to have a signal peptide, thus was
a cytoplasmic protein, as analyzed by SignalP 3.0. Tr1 was
predicted to contain a putative N-terminal helix-turn-helix
DNA-binding domain based on the analysis of the NCBI conserved
domain database, suggesting that it was a transcriptional
regulator. The amino acid sequence identity between A. platys Tr1
and A. phagocytophilum Tr1 (YP.sub.--505749) was 84.8% to 86.4%,
and that between A. platys Tr1 and A. marginale Tr
(YP.sub.--154239) was 73.1% to 74.1%.
[0258] A. platys OMP-1X structure. Three nearly identical (99.1%)
A. platys omp-1X sequences were obtained from two dogs from
Venezuela and one dog from Taiwan. Using SignalP 3.0 server, OMP-1X
was predicted to have a signal peptide with a cleavage site between
positions 23 and 24. The predicted molecular mass of mature A.
platys OMP-1X was 31.9 kDa with an isoelectric point of 7.27 to
7.92 (Table 2). The secondary structure of OMP-1X was then
examined, using PRED-TMBB..sup.4 The discrimination value of the
OMP-1X amino acid sequence was 2.907, which is below the threshold
value of 2.965, making OMP-1X likely to be a .beta.-barrel protein
localized to the outer membrane. Hydrophobicity analysis and the
hydrophobic moment profile program developed for the porin
structure prediction,.sup.35 predicted 14 .beta.-strands in OMP-1X.
The protein sequences most closely related to A. platys OMP-1X were
A. phagocytophilum OMP-1X (YP.sub.--505750; 45.9%-46.3% identity)
and A. marginale OMP-1 (YP.sub.--154240; 39.8% identity). A
phylogenetic analysis showed that OMP-1X homologs in Anaplasma spp.
formed a cluster that was distinct from the cluster of most closely
related OMP-1X homologs in each Ehrlichia spp. (FIG. 3).
[0259] A. platys P44ES Structure.
[0260] Four P44ES sequences (GQ868750, GU357491, GU357492, and
GU357493) were obtained from three dogs from Venezuela. Using
SignalP 3.0 sever P44ES was predicted to have a putative signal
peptide with a cleavage site between positions 21 and 22. The
molecular mass of the mature P44ES protein was predicted to be 41.2
to 41.4 kDa with an isoelectric point of 5.30 to 5.72 (Table 2). By
PRED-TMBB.sup.4 analysis, the discrimination value of the P44 amino
acid sequence was 2.920 which was below the threshold value of
2.965, making P44 likely to be a .beta.-barrel protein localized to
the outer membrane. Hydrophobicity analysis and the hydrophobic
moment profile predicted 16 .beta.-strands in P44. Alignment of
total nine A. platys P44 sequences (the four P44 full-length
proteins from dogs 1, 2, and 3, and the five partial P44 sequences
obtained from dogs 1, 2 and Taiwan) using HVF and HVR primers,
revealed a single central hypervariable region (aa position
193-247) of approximately 55 amino acid residues, and N-terminal
and C-terminal conserved regions of approximately 192 and 159 amino
acid residues, respectively. The conserved amino acids C, C, W, and
A from the P44 hypervariable region of A. phagocytophilum
P44.sup.41 were also detected in the hypervariable region of A.
platys P44. The C terminus of A. platys P44 ends with
phenylalanine, as does the C terminus of A. phagocytophilum
P44..sup.30 The amino acid sequence identity between A. platys
P44ES and A. phagocytophilum P44-18ES (YP.sub.--505752) was 55.0%
to 56.9%, and that between A. platys P44ES and A. marginale Msp2
(YP.sub.--154245) was 41.5% to 42.1%. Phylogenetic analysis placed
full-length A. platys p44s between A. phagocytophilum p44s and A.
marginale msp2s (FIG. 4). The sequence identities of the conserved
N-terminal 192 amino acids and the conserved C-terminal 159 amino
acids of A. platys and A. phagocytophilum P44s were 57.3% and
66.7%, respectively.
[0261] Primer pairs (HVF and HVR, Table 1) designed based on A.
platys p44 conserved region amplified only A. platys DNA, but not
A. phagocytophilum and A. marginale DNA. Alignment of a total of
nine A. platys P44 hypervariable regions and flanking conserved
regions with P44/Msp2 sequences among A. phagocytophilum P44s and
A. marginale Msp2s revealed several A. platys-specific sequences:
TGTAAGSDVDYVSKF (SEQ ID NO: 92; aa position 23-37), TRVEWKAE (SEQ
ID NO: 93; aa position 78-85), AAEIVKFAEAVGTSAK (SEQ ID NO: 94; aa
position 174-189), SWKCTQTG (SEQ ID NO: 95; aa position 207-214),
AAKAEDLS (SEQ ID NO: 96; aa position 248-255) and ATTNKTKEF (SEQ ID
NO: 97; aa position 378-386). These A. platys-specific p44 regions
were utilized as serologic test antigens to distinguish from A.
phagocytophilum or A. marginale infections.
TABLE-US-00001 TABLE 1 Primers used for PCR amplification of A.
platys P44ES cluster Primer Sequence Note F1
5'-ATTATGTATGATTTATCCTAAGTTATCTGAG-3' tr1/tr highly conserved (SEQ
ID NO: 82) upstream region F2 5-GGGATATCGGCGTTGATAGGG-3' (SEQ ID
NO: A. platys omp-1X 83) downstream genomic walking F3
5'-GGTTTGTGTTGCTGGTGATTGGAGG-3' (SEQ ID A. platys p44 upstream NO:
84) genomic walking R1 5'-GCAAACCTAACACCMAAYTCMCCACC-3' (SEQ
p44ES/Msp2 C-terminal ID NO: 85) highly conserved region R2
5'-TATACTAAAAAAGAATTAAGTCAAGAG-3' Conserved intergenetic (SEQ ID
NO: 86) region between omp-1X /omp1 and omp-1N/opag3 R3
5'-ATGGTAGAAASCCCCAGCAAA-3' (SEQ ID NO: p44ES/Msp2 N-terminal 87)
conserved region R4 5'-CACGTNTTTAGTTACTGCCA-3' (SEQ ID NO: 88)
p44ES/Msp2ES from downstream conserved valS gene R5
5'-GTACTAGTCAGCGCCACTAACATCAA-3' (SEQ p44ES/Msp2ES downstream ID
NO: 89) region for complete locus confirmation HVF
5'-GAAGAATACGAAAGCGGCGG-3' (SEQ ID NO: A. platys P44 hypervariable
90) cloning region HVR 5'-TACTTAGGTCTTCCGCTTTCGC-3' (SEQ ID NO: A.
platys P44 hypervariable 91) cloning region
[0262] ELISA Analysis of OMP-1X.
[0263] When the Clustal W method was used to compare A. platys
OMP-1X to its phylogenetically closest OMP-1 homologs-A.
phagocytophilum OMP-1X (YP.sub.--505750), A. marginale OMP1
(YP.sub.--154240), E. canis P30-19 (AAK28680), E. ruminantium
Map1-related protein (YP.sub.--180721), E. ewingii OMP-1-1
(ABO36240), and E. chaffeensis OMP-1M (YP.sub.--507903), we
identified a unique region in the A. platys OMP-1X amino acid
sequence. This sequence, AVQEKKPPEA (SEQ ID NO: 98), is within the
2nd external loop from the N-terminus based on the hydrophobicity
analysis and the hydrophobic moment profile program. The sequence
is predicted to be highly antigenic and surface exposed by Protean
program may aid in differential serodiagnosis (FIG. 14). The A.
platys OMP-1X peptide was synthesized and its reactivity to known
infected dog sera was tested by ELISA. Three A. platys PCR-positive
dog sera reacted with the synthesized OMP-1X peptide antigen. Sera
from A. platys PCR-negative dogs and horse anti-A. phagocytophilum
serum did not react with the OMP-1X peptide antigen (FIG. 15),
suggesting that this antigen can be used for species-specific
serodiagnosis of A. platys.
TABLE-US-00002 TABLE 2 Properties of A. platys p44ES cluster
Upstream Protein Signal Upstream intergenic Gene Size peptide
Molecular intergenic space Length (amino (amino mass.sup.a
Isoelectric space Protein (bp) (bp) acids).sup.a acids).sup.b (Da)
point Protein (bp) Tr1 NA.sup.c 558 185 NA 21010.6- 5.50-5.80.sup.d
Tr1 NA.sup.c 20952.6.sup.d OMP- 306 933 301 23 31885.0-
7.27-7.92.sup.d OMP- 306 1X 31942.1.sup.d 1X P44ES 682 1221 380- 21
41167.3- 5.30-5.72.sup.d P44ES 682 386.sup.d 41359.5.sup.d
.sup.aMature protein. .sup.bPredicted cleavage site. .sup.cNot
applicable. .sup.dRange among strains.
DISCUSSION
[0264] In the present study, the entire 4 kb A. platys p44ES locus,
containing tr1, omp-1X and p44 genes, was sequenced, providing new
insight into the p44 expression locus and the major surface antigen
of A. platys. From each infected dog different p44ES sequences were
detected, showing mixed P44 allele population of A. platys is
present in the dog blood in a given time point similar to A.
phagocytophilum p44 expression in humans and horses.sup.38, 39, 65
or A. marginale msp2 in the blood of cattle..sup.18, 21, 47 In
addition three more different hypervariable regions were detected
in partial A. platys p44 gene sequences, suggesting the
p44-expression locus of A. platys is also the site of active
recombination, and multiple p44 donor sequences also exist in the
A. platys genome.
[0265] The synteny analysis suggested that the major outer membrane
expression locus existed in a common ancestor of the three
Anaplasma species in existence today. Furthermore, the locus
appears to have diverged primarily by duplicating omp-1-like
sequences between tr1 and p44/msp2ES; A. marginale, A.
phagocytophilum, and A. platys have 4, 2, and 1 omp-1-like
sequences, respectively.
[0266] Three species of Anaplasma infect different host cells,
namely neutrophils, erythrocytes, and platelets. The comparative
study of between A. phagocytophilum, A. marginale, and A. platys
P44/Msp2s, and OMP-1 homologs provided a new window of opportunity
to investigate different Anaplasma host cell tropism.
[0267] Tr1, a putative transcription factor, is more highly
expressed in tick cells infected with A. phagocytophilum than in
human leukemic HL-60 cells infected with A. phagocytophilum, which
suggested that Tr1 may regulate genes involved in the bacterial
infection cycle in ticks..sup.44, 64 In contrast, Tr is expressed
similarly in bovine red blood cells and IDE8 tick cell cultures
infected with A. marginale..sup.5
[0268] In A. phagocytophilum tr1, two omp-1s and p44E were
co-expressed..sup.39 In the cattle blood, the aforementioned and
related genes were co-expressed with the exception of the third
msp2-associated genes.sup.48 Omp-1s are major surface antigens of
Ehrlichia species, also has a role in A. platys infection cycle.
OMP-1 homologous proteins are major surface antigens in Ehrlichia
species.sup.23, 46, 57, 62, 66 and OMP-1X functions similarly in
the A. platys infection cycle. A. platys OMP-1X is predicted to
have a .beta.-barrel structure similar to that of E. chaffeensis
P28 and OMP-1F,.sup.37 and is thus a porin.
[0269] The fact that A. phagocytophilum and A. marginale P44/Msp2
transcripts were distinct between mammals and ticks advocates
multiple physiological adaptations between different host
environments..sup.44, 53, 71 Furthermore, A. phagocytophilum p44
gene conversion in mammalian hosts suggested its role in antigenic
variation..sup.8, 19, 38, 65 In cattle, A. marginale MSP2s allows
antigenic variation for persistent infection..sup.6, 11, 47 A.
platys P44, therefore, plays an important role in determining
persistent or cyclical rickettsemia.
[0270] It is not known whether A. platys p44ES undergoes
nonsegmental gene conversion (as in A. phagocytophilum to generate
identical P44s from a large number of donor loci) or segmental gene
conversion (as in A. marginale to generate mosaic Msp2ES from a
small number of donor loci)..sup.39, 47 P44 has a role in the
interaction between A. phagocytophilum and host cells..sup.36, 49,
64 The P44 of A. phagocytophilum is the major surface antigen
useful for serologic diagnosis of human granulocytic anaplasmosis,
and has a role in the interaction between A. phagocytophilum and
host cells. P44s also elicits a porin activity for passive
diffusion of hydrophilic solutes.
[0271] Based on the present study, recombinant or peptide-based
OMP-1X and P44 antigen can be prepared for testing the
applicability of A. platys serodiagnosis. P44 can also serve as a
specific and sensitive target for PCR diagnosis for human
granulocytic anaplasmosis, and thus can be tested for A. platys
infection or exposure.
[0272] A. phagocytophilum is known to infect dogs in regions where
the Ixodes tick is endemic..sup.2, 24, 50, 51 A. platys inclusions
in the platelets of a naturally infected dog cross-reacted with
mouse anti-A. phagocytophilum serum..sup.32 It was important,
therefore, to develop a method for distinguishing A. platys
infection from A. phagocytophilum infection. Since the p44 primer
pair: HVF and HVR described herein, is specific to A. platys, it
was expected to be useful for species-specific PCR diagnosis. P44
of A. phagocytophilum is the major surface antigen used for
serologic diagnosis of human granulocytic anaplasmosis..sup.1, 16,
27, 31, 70 In the present study, several A. platys-specific amino
acid sequences were identified within P44 proteins that can be used
as serologic test antigens to provide differential diagnosis from
other Anaplasma species infection. Additionally, Ehrlichia
OMP-1/P28/P30/MAP families are immunodominant major outer membrane
proteins useful for serodiagnosis..sup.45, 62, 63, 68 The alignment
results showed a distinct fragment (.about.20 amino acids) in A.
platys Omp-1X that was not observed in the closest homologs from
Anaplasma and Ehrlichia spp. Furthermore, this region was identical
in A. platys samples from the geographically separated regions of
Venezuela and Taiwan. This specific OMP-1X peptide antigen did not
cross-react with anti-A. phagocytophilum serum, and therefore can
be suitable for species-specific differential serodiagnosis of A.
platys.
[0273] Since the only available source of A. platys DNA was a small
amount of DNA purified from infected dog blood specimens, touchdown
PCR was employed to amplify the available canine DNA. Incorrect
base pairings resulting from amplification were minimized by using
a high-fidelity Taq polymerase
Example 2
A. platys Expression Locus Analysis
[0274] DNA specimens from three dogs naturally infected with A.
platys at Lara, Venezuela in 2007 were used as the template for the
amplification and sequencing process. A. platys infection of the
dog was confirmed by PCR and sequencing of the 16S rRNA of A.
platys as well as by observation of bacterial inclusions (morulae)
in platelets in the blood smear. By aligning A. phagocytophilum and
A. marginale p44/msp2 expression loci, several degenerate primers
were designed based upon conserved regions (FIG. 1, Table 1). Using
the first primer pair F1 and R1, and the second primer pair F1 and
R2, (hemi-) nested touchdown PCR amplified the A. platys tr1 and
omp-1X sequences. In order to avoid truncated p44 pseudogenes in A.
platys genome, primer F3 for the 5' upstream of the predicted p44
ORF was designed. p44ES sequences were amplified by nested
touchdown PCR with primer pairs F2 and R3, and F3 and R4.
Amplification was performed as previously described. The amplified
DNA fragments were cloned using a TA cloning kit (Invitrogen,
Carlsbad, Calif.) and sequenced with M13 Forward or M13 Reverse
sequencing primers. All sequencing data was assembled using SeqMan
program of DNASTAR software (DNASTAR Inc., Madison, Wis.). The
deduced amino acid sequences of A. marginale, A. phagocytophilum,
and A. platys tr1, Omp-1X and p44ES were aligned using the MegAlign
program of DNASTAR software by the Clustal W method.
[0275] P44 Secondary Structure Prediction.
[0276] The P44 secondary structure was predicted by hydrophobicity
and the hydrophobic moment profile method as previously described.
Antigenic Index and surface probability were examined by Protean
program of DNASTAR.
[0277] All patents, patent applications, and other scientific or
technical writings referred to anywhere herein are incorporated by
reference in their entirety. The invention illustratively described
herein suitably can be practiced in the absence of any element or
elements, limitation or limitations that are not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising", "consisting essentially of", and
"consisting of" can be replaced with either of the other two terms,
while retaining their ordinary meanings. The terms and expressions
which have been employed are used as terms of description and not
of limitation, and there is no intention that in the use of such
terms and expressions of excluding any equivalents of the features
shown and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by embodiments,
optional features, modification and variation of the concepts
herein disclosed can be resorted to by those skilled in the art,
and that such modifications and variations are considered to be
within the scope of this invention as defined by the description
and the appended claims.
[0278] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0279] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
[0280] Nothing herein is to be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention. Further, the dates of publication
provided herein may be different from the actual publication dates,
which can require independent confirmation.
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Sequence CWU 1
1
1321324PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 1Met Lys Lys Arg Ile Arg Val Phe Ala Phe
Ala Val Phe Met Leu Gly 1 5 10 15 Leu Pro Ser Val Ser Phe Ala Ser
Pro Gln Pro Val Asp Phe Ser Tyr 20 25 30 His Glu Gly Ala Ser Gly
Phe Phe Ala Ser Val Gln Tyr Lys Tyr Gly 35 40 45 Ala Pro Tyr Phe
Gly Ser Leu Thr Leu Glu Ser Gly Gly Lys Thr Leu 50 55 60 Asn Leu
Val Ser Ala Val Gln Glu Lys Lys Pro Pro Glu Ala Pro Ala 65 70 75 80
Ala Asp Glu Ala Ala Glu Pro Ala Thr Pro Ala Pro Ser Ser Pro Glu 85
90 95 Gly Phe Gly Ser Ser Thr Asp Asp Phe Gln Gly Arg Tyr Ser Pro
Thr 100 105 110 Tyr Leu Lys Asp Ala Gly Ala Phe Ser Ile Thr Ala Gly
Tyr Thr Thr 115 120 125 Gly Ile Met Arg Phe Glu Ala Glu Ala Met Arg
Ser Arg Phe Gln Val 130 135 140 Asn Gly Ser Lys Trp Asn Pro Val Glu
Asn Ala Tyr Ile Phe Ala Ala 145 150 155 160 Ala Lys Pro Ser Glu Asn
Ile Ser Tyr Pro Ala Gln Ile Leu Glu Ala 165 170 175 Gln Lys Tyr Phe
Val Thr Leu Glu Asn Arg Asp Val Ala Ile Thr Ser 180 185 190 Leu Val
Ala Asn Ala Cys Tyr Asp Met Met Pro Ala Thr Ser Ser Ile 195 200 205
Ala Pro Ser Ala Cys Val Gly Val Gly Val Ser Phe Ala Lys Leu Leu 210
215 220 Gly Val Leu Glu Gln Arg Leu Thr Tyr Gln Phe Lys Gly Gly Leu
Gln 225 230 235 240 Tyr Phe Val Gly Lys Lys Thr Val Ile Phe Leu Ser
Gly Tyr Val Ser 245 250 255 Thr Ile Gly Gly Arg Lys Ile Thr Gln Val
Lys Val Lys His Arg Leu 260 265 270 Ser Thr Pro Gln Thr Ala Ser Val
Gly Ala Glu Gly Ser Gly Ala Gly 275 280 285 Ala Ala Ala Thr Ser Ser
Ala Pro Pro Ala Pro Ile His Leu Leu Tyr 290 295 300 Pro Asp Ala Asn
Leu Ser Leu Ala Tyr Tyr Gly Phe Glu Leu Gly Val 305 310 315 320 Arg
Leu Val Phe 2117PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 2Leu Val Ser Ala Val Gln Glu
Lys Lys Pro Pro Glu Ala Pro Ala Ala 1 5 10 15 Asp Glu Ala Ala Glu
Pro Ala Thr Pro Ala Pro Ser Ser Pro Glu Gly 20 25 30 Phe Gly Ser
Ser Thr Asp Asp Phe Gln Gly Arg Tyr Ser Pro Thr Tyr 35 40 45 Leu
Lys Asp Ala Gly Ala Phe Ser Ile Thr Ala Gly Tyr Thr Thr Gly 50 55
60 Ile Met Arg Phe Glu Ala Glu Ala Met Arg Ser Arg Phe Gln Val Asn
65 70 75 80 Gly Ser Lys Trp Asn Pro Val Glu Asn Ala Tyr Ile Phe Ala
Ala Ala 85 90 95 Lys Pro Ser Glu Asn Ile Ser Tyr Pro Ala Gln Ile
Leu Glu Ala Gln 100 105 110 Lys Tyr Phe Val Thr 115
368PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 3Leu Thr Tyr Gln Phe Lys Gly Gly Leu Gln Tyr
Phe Val Gly Lys Lys 1 5 10 15 Thr Val Ile Phe Leu Ser Gly Tyr Val
Ser Thr Ile Gly Gly Arg Lys 20 25 30 Ile Thr Gln Val Lys Val Lys
His Arg Leu Ser Thr Pro Gln Thr Ala 35 40 45 Ser Val Gly Ala Glu
Gly Ser Gly Ala Gly Ala Ala Ala Thr Ser Ser 50 55 60 Ala Pro Pro
Ala 65 4185PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 4Leu Val Ser Ala Val Gln Glu Lys Lys Pro
Pro Glu Ala Pro Ala Ala 1 5 10 15 Asp Glu Ala Ala Glu Pro Ala Thr
Pro Ala Pro Ser Ser Pro Glu Gly 20 25 30 Phe Gly Ser Ser Thr Asp
Asp Phe Gln Gly Arg Tyr Ser Pro Thr Tyr 35 40 45 Leu Lys Asp Ala
Gly Ala Phe Ser Ile Thr Ala Gly Tyr Thr Thr Gly 50 55 60 Ile Met
Arg Phe Glu Ala Glu Ala Met Arg Ser Arg Phe Gln Val Asn 65 70 75 80
Gly Ser Lys Trp Asn Pro Val Glu Asn Ala Tyr Ile Phe Ala Ala Ala 85
90 95 Lys Pro Ser Glu Asn Ile Ser Tyr Pro Ala Gln Ile Leu Glu Ala
Gln 100 105 110 Lys Tyr Phe Val Thr Leu Thr Tyr Gln Phe Lys Gly Gly
Leu Gln Tyr 115 120 125 Phe Val Gly Lys Lys Thr Val Ile Phe Leu Ser
Gly Tyr Val Ser Thr 130 135 140 Ile Gly Gly Arg Lys Ile Thr Gln Val
Lys Val Lys His Arg Leu Ser 145 150 155 160 Thr Pro Gln Thr Ala Ser
Val Gly Ala Glu Gly Ser Gly Ala Gly Ala 165 170 175 Ala Ala Thr Ser
Ser Ala Pro Pro Ala 180 185 5324PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 5Met Lys Lys Arg
Ile Arg Val Phe Ala Phe Ala Val Phe Met Leu Gly 1 5 10 15 Leu Pro
Ser Val Ser Phe Ala Ser Pro Gln Pro Val Asp Phe Ser Tyr 20 25 30
His Glu Gly Ala Ser Gly Phe Phe Ala Ser Val Gln Tyr Lys Tyr Gly 35
40 45 Ala Pro Tyr Phe Gly Ser Leu Thr Leu Glu Ser Gly Gly Lys Ile
Leu 50 55 60 Asn Leu Val Ser Ala Val Gln Glu Lys Lys Pro Pro Glu
Ala Pro Ala 65 70 75 80 Ala Asp Glu Ala Ala Gly Pro Ala Thr His Ala
Pro Ser Ser Pro Glu 85 90 95 Gly Phe Gly Ser Ser Thr Asp Asp Phe
Gln Gly Arg Tyr Ser Pro Thr 100 105 110 Tyr Leu Lys Asp Ala Gly Ala
Phe Ser Ile Thr Ala Gly Tyr Thr Thr 115 120 125 Gly Ile Met Arg Phe
Glu Ala Glu Ala Met Arg Ser Arg Phe Gln Val 130 135 140 Asn Gly Ser
Lys Trp Asn Pro Val Glu Asn Ala Tyr Ile Phe Ala Ala 145 150 155 160
Ala Lys Pro Ser Glu Asn Ile Ser Tyr Pro Ala Gln Ile Leu Glu Thr 165
170 175 Gln Lys Tyr Phe Val Thr Leu Glu Asn Arg Asp Val Ala Ile Thr
Ser 180 185 190 Leu Val Ala Ser Ala Cys Tyr Asp Met Met Pro Ala Thr
Ser Ser Ile 195 200 205 Ala Pro Ser Ala Cys Val Gly Val Gly Val Ser
Phe Ala Lys Leu Leu 210 215 220 Gly Val Leu Glu Gln Arg Leu Thr Tyr
Gln Phe Lys Gly Gly Leu Gln 225 230 235 240 Tyr Phe Val Gly Lys Lys
Thr Val Ile Phe Leu Ser Gly Tyr Val Ser 245 250 255 Thr Ile Gly Gly
Arg Lys Ile Ser Gln Val Lys Val Lys His Arg Leu 260 265 270 Pro Thr
Pro Gln Thr Ala Ser Val Gly Ala Glu Gly Ser Gly Ser Gly 275 280 285
Ala Ala Ala Thr Ser Ser Ala Pro Pro Ala Leu Ile His Leu Leu Tyr 290
295 300 Pro Asp Ala Asn Leu Ser Leu Ala Tyr Tyr Gly Phe Glu Leu Gly
Val 305 310 315 320 Arg Leu Val Phe 6117PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 6Leu Val Ser Ala Val Gln Glu Lys Lys Pro Pro Glu Ala Pro
Ala Ala 1 5 10 15 Asp Glu Ala Ala Gly Pro Ala Thr His Ala Pro Ser
Ser Pro Glu Gly 20 25 30 Phe Gly Ser Ser Thr Asp Asp Phe Gln Gly
Arg Tyr Ser Pro Thr Tyr 35 40 45 Leu Lys Asp Ala Gly Ala Phe Ser
Ile Thr Ala Gly Tyr Thr Thr Gly 50 55 60 Ile Met Arg Phe Glu Ala
Glu Ala Met Arg Ser Arg Phe Gln Val Asn 65 70 75 80 Gly Ser Lys Trp
Asn Pro Val Glu Asn Ala Tyr Ile Phe Ala Ala Ala 85 90 95 Lys Pro
Ser Glu Asn Ile Ser Tyr Pro Ala Gln Ile Leu Glu Thr Gln 100 105 110
Lys Tyr Phe Val Thr 115 768PRTArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 7Leu Thr Tyr Gln Phe
Lys Gly Gly Leu Gln Tyr Phe Val Gly Lys Lys 1 5 10 15 Thr Val Ile
Phe Leu Ser Gly Tyr Val Ser Thr Ile Gly Gly Arg Lys 20 25 30 Ile
Ser Gln Val Lys Val Lys His Arg Leu Pro Thr Pro Gln Thr Ala 35 40
45 Ser Val Gly Ala Glu Gly Ser Gly Ser Gly Ala Ala Ala Thr Ser Ser
50 55 60 Ala Pro Pro Ala 65 8185PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 8Leu Val Ser Ala
Val Gln Glu Lys Lys Pro Pro Glu Ala Pro Ala Ala 1 5 10 15 Asp Glu
Ala Ala Gly Pro Ala Thr His Ala Pro Ser Ser Pro Glu Gly 20 25 30
Phe Gly Ser Ser Thr Asp Asp Phe Gln Gly Arg Tyr Ser Pro Thr Tyr 35
40 45 Leu Lys Asp Ala Gly Ala Phe Ser Ile Thr Ala Gly Tyr Thr Thr
Gly 50 55 60 Ile Met Arg Phe Glu Ala Glu Ala Met Arg Ser Arg Phe
Gln Val Asn 65 70 75 80 Gly Ser Lys Trp Asn Pro Val Glu Asn Ala Tyr
Ile Phe Ala Ala Ala 85 90 95 Lys Pro Ser Glu Asn Ile Ser Tyr Pro
Ala Gln Ile Leu Glu Thr Gln 100 105 110 Lys Tyr Phe Val Thr Leu Thr
Tyr Gln Phe Lys Gly Gly Leu Gln Tyr 115 120 125 Phe Val Gly Lys Lys
Thr Val Ile Phe Leu Ser Gly Tyr Val Ser Thr 130 135 140 Ile Gly Gly
Arg Lys Ile Ser Gln Val Lys Val Lys His Arg Leu Pro 145 150 155 160
Thr Pro Gln Thr Ala Ser Val Gly Ala Glu Gly Ser Gly Ser Gly Ala 165
170 175 Ala Ala Thr Ser Ser Ala Pro Pro Ala 180 185
923PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 9Gly Gly Lys Thr Leu Asn Ala Val Gln Glu Lys
Lys Pro Pro Glu Ala 1 5 10 15 Pro Ala Ala Asp Glu Ala Ala 20
10324PRTArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 10Met Lys Lys Arg Ile Arg Val Phe Ala Phe Ala
Val Phe Met Leu Gly 1 5 10 15 Leu Pro Ser Val Ser Phe Ala Ser Pro
Gln Pro Val Asp Phe Ser Tyr 20 25 30 His Glu Gly Ala Ser Gly Phe
Phe Ala Ser Val Gln Tyr Lys Tyr Gly 35 40 45 Ala Pro Tyr Phe Gly
Ser Leu Thr Leu Glu Ser Gly Gly Lys Thr Leu 50 55 60 Asn Leu Val
Ser Ala Val Gln Glu Lys Lys Pro Pro Glu Ala Pro Ala 65 70 75 80 Ala
Asp Glu Ala Ala Glu Pro Ala Thr Pro Ala Pro Ser Ser Pro Glu 85 90
95 Gly Phe Gly Ser Ser Thr Asp Asp Phe Gln Gly Arg Tyr Ser Pro Ala
100 105 110 Tyr Leu Lys Asp Ala Gly Ala Phe Ser Ile Thr Ala Gly Tyr
Thr Thr 115 120 125 Gly Ile Met Arg Phe Glu Ala Glu Ala Met Arg Ser
Arg Phe Gln Val 130 135 140 Asn Gly Ser Lys Trp Asn Pro Val Glu Asn
Ala Tyr Ile Phe Ala Ala 145 150 155 160 Ala Lys Pro Ser Glu Asn Ile
Ser Tyr Pro Ala Gln Ile Leu Glu Ala 165 170 175 Gln Lys Tyr Phe Val
Thr Leu Glu Asn Arg Asp Val Ala Ile Thr Ser 180 185 190 Leu Val Ala
Asn Ala Cys Tyr Asp Met Met Pro Ala Thr Ser Ser Ile 195 200 205 Ala
Pro Ser Ala Cys Val Gly Val Gly Val Ser Phe Ala Lys Leu Leu 210 215
220 Gly Val Leu Glu Gln Arg Leu Thr Tyr Gln Phe Lys Gly Gly Ser Gln
225 230 235 240 Tyr Phe Val Gly Lys Lys Thr Val Ile Phe Leu Ser Gly
Tyr Val Ser 245 250 255 Thr Ile Gly Gly Arg Lys Ile Thr Gln Val Lys
Val Lys His Arg Leu 260 265 270 Pro Thr Pro Gln Thr Ala Ser Val Gly
Ala Glu Gly Ser Gly Ala Gly 275 280 285 Ala Ala Ala Thr Ser Ser Ala
Pro Pro Ala Pro Ile His Leu Leu Tyr 290 295 300 Pro Asp Ala Asn Leu
Ser Leu Ala Tyr Tyr Gly Phe Glu Leu Gly Val 305 310 315 320 Arg Leu
Val Phe 11975DNAArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 11atgaagaaga gaatccgggt
tttcgcgttt gcagtgttca tgcttgggtt gccctccgta 60tcatttgctt ctccacaacc
cgtggatttt tcgtaccacg agggtgcatc cggctttttt 120gccagcgttc
agtataagta cggtgcaccc tatttcggaa gccttacact ggaaagcggc
180gggaagacgc tgaatcttgt gagcgcggtg caggaaaaga aacccccgga
agcccccgca 240gctgacgaag ctgctgaacc tgccacccct gctccctcaa
gccctgaagg gtttggttca 300tctaccgatg attttcaagg taggtactcg
ccaacctatt taaaggacgc aggagccttt 360tcaataacag cgggttatac
caccggtatt atgagatttg aagcagaggc tatgcgttcg 420cgtttccagg
taaatggcag taaatggaat cctgtagaga acgcttatat atttgccgct
480gctaaaccca gtgagaatat atcgtaccct gctcagatac tggaggcaca
aaagtacttc 540gtcaccttgg aaaacaggga tgttgccatc acttcgctgg
tggcaaatgc ctgctacgac 600atgatgccag caacctctag catcgcacct
agtgcgtgcg tgggtgtagg ggttagcttt 660gccaaactgt taggtgttct
ggaacaaagg ctaacctatc aattcaaagg tggattgcaa 720tattttgtcg
gcaaaaagac cgtcatcttc ctatctggat acgtatccac aataggtgga
780aggaaaatca ctcaggtaaa agtgaaacat cgtttgtcca cgcctcagac
cgcgtcagtt 840ggtgcagaag gcagtggagc aggtgctgct gcaacatctt
ctgctccccc agcccccatt 900cacctgttgt acccagatgc aaatttatcg
ctagcgtact acgggtttga gcttggggtg 960cgtcttgttt tctaa
97512975DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 12atgaagaaga gaatccgggt tttcgcgttt
gcagtgttca tgcttgggtt gccctccgta 60tcatttgctt ctccacaacc cgtggatttt
tcgtaccacg agggtgcatc cggctttttt 120gccagcgttc agtataagta
cggtgcaccc tatttcggaa gccttacact ggaaagcggc 180gggaagattc
tgaatcttgt gagcgcggtg caggaaaaga aacccccgga agcccccgca
240gctgacgaag ctgctggacc tgccacccat gctccctcaa gccctgaagg
gtttggttca 300tctaccgatg attttcaagg taggtactcg ccaacctatt
taaaggacgc aggagccttt 360tcaataacag cgggttatac caccggtatt
atgagatttg aagcagaggc tatgcgttcg 420cgtttccagg taaatggcag
taaatggaat cctgtagaga acgcttatat atttgccgct 480gctaaaccca
gtgagaatat atcgtaccct gctcagatac tggagacaca aaagtacttc
540gtcaccttgg aaaacaggga tgttgccatc acttcgctgg tggcaagtgc
ctgctacgac 600atgatgccag caacctctag catcgcacct agtgcgtgcg
tgggtgtagg ggttagcttt 660gccaaactgt taggtgttct ggaacaaagg
ctaacctatc aattcaaagg tggattgcaa 720tattttgtcg gcaaaaagac
cgtcatcttc ctatctggat acgtatccac aataggtgga 780aggaaaatct
ctcaggtaaa agtgaaacat cgtttgccca cgcctcagac cgcgtcagtt
840ggtgcagaag gcagtggatc aggtgctgct gcaacatctt ctgctccccc
agccctcatc 900cacctgttgt acccagatgc aaatttatcg ctagcgtact
acgggtttga gcttggggtg 960cgtcttgttt tctaa 97513975DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 13atgaagaaga gaatccgggt tttcgcgttt gcagtgttca tgcttgggtt
gccctccgta 60tcatttgctt ctccacaacc cgtggatttt tcgtaccacg agggtgcatc
cggctttttt 120gccagcgttc agtataagta cggtgcaccc tatttcggaa
gccttacact ggaaagcggc 180gggaagacgc tgaatcttgt gagcgcggtg
caggaaaaga aacccccgga agcccccgca 240gctgacgaag ctgctgaacc
tgccacccct gctccctcaa gccctgaagg gtttggttca 300tctaccgatg
attttcaagg taggtactcg ccagcctatt taaaggacgc aggagccttt
360tcaataacag cgggttatac caccggtatt atgagatttg aagcagaggc
tatgcgttcg 420cgtttccagg taaatggcag taaatggaat cctgtagaga
acgcttatat atttgccgct 480gctaaaccca gtgagaatat atcgtaccct
gctcagatac tggaggcaca aaagtacttc 540gtcaccttgg aaaacaggga
tgttgccatc acttcgctgg tggcaaatgc ctgctacgac 600atgatgccag
caacctctag catcgcacct agtgcgtgcg tgggtgtagg
ggttagcttt 660gccaaactgt taggtgttct ggaacaaagg ctaacctatc
aattcaaagg tggatcgcaa 720tattttgtcg gcaaaaagac cgtcatcttc
ctatctggat acgtatccac aataggtgga 780aggaaaatca ctcaggtaaa
agtgaaacat cgtttgccca cgcctcagac cgcgtcagtt 840ggtgcagaag
gcagtggagc aggtgctgct gcaacatctt ctgctccccc agcccccatt
900cacctgttgt acccagatgc aaatttatcg ctagcgtact acgggtttga
gcttggggtg 960cgtcttgttt tctaa 97514360DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 14ctgaatcttg tgagcgcggt gcaggaaaag aaacccccgg aagcccccgc
agctgacgaa 60gctgctgaac ctgccacccc tgctccctca agccctgaag ggtttggttc
atctaccgat 120gattttcaag gtaggtactc gccaacctat ttaaaggacg
caggagcctt ttcaataaca 180gcgggttata ccaccggtat tatgagattt
gaagcagagg ctatgcgttc gcgtttccag 240gtaaatggca gtaaatggaa
tcctgtagag aacgcttata tatttgccgc tgctaaaccc 300agtgagaata
tatcgtaccc tgctcagata ctggaggcac aaaagtactt cgtcaccttg
36015204DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 15ctaacctatc aattcaaagg tggattgcaa
tattttgtcg gcaaaaagac cgtcatcttc 60ctatctggat acgtatccac aataggtgga
aggaaaatca ctcaggtaaa agtgaaacat 120cgtttgtcca cgcctcagac
cgcgtcagtt ggtgcagaag gcagtggagc aggtgctgct 180gcaacatctt
ctgctccccc agcc 20416564DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 16ctgaatcttg
tgagcgcggt gcaggaaaag aaacccccgg aagcccccgc agctgacgaa 60gctgctgaac
ctgccacccc tgctccctca agccctgaag ggtttggttc atctaccgat
120gattttcaag gtaggtactc gccaacctat ttaaaggacg caggagcctt
ttcaataaca 180gcgggttata ccaccggtat tatgagattt gaagcagagg
ctatgcgttc gcgtttccag 240gtaaatggca gtaaatggaa tcctgtagag
aacgcttata tatttgccgc tgctaaaccc 300agtgagaata tatcgtaccc
tgctcagata ctggaggcac aaaagtactt cgtcaccttg 360ctaacctatc
aattcaaagg tggattgcaa tattttgtcg gcaaaaagac cgtcatcttc
420ctatctggat acgtatccac aataggtgga aggaaaatca ctcaggtaaa
agtgaaacat 480cgtttgtcca cgcctcagac cgcgtcagtt ggtgcagaag
gcagtggagc aggtgctgct 540gcaacatctt ctgctccccc agcc
5641751DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 17gcggtgcagg aaaagaaacc cccggaagcc
cccgcagctg acgaagctgc t 511819DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 18ctgaatcttg
tgagcgcgg 191923DNAArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 19caaggtgacg aagtactttt gtg
232027DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 20ctaacctatc aattcaaagg tggattg
2721406PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 21Met Lys Glu Arg Lys Leu Ala Leu Ser
Gly Ala Val Ala Met Thr Val 1 5 10 15 Leu Val Ser Thr Ala Gly Thr
Gly Thr Ala Ala Gly Ser Asp Val Asp 20 25 30 Tyr Val Ser Lys Phe
Gly Glu Gly Ser Phe Tyr Val Gly Leu Asn Tyr 35 40 45 Ser Pro Ala
Phe Ser Lys Ile Asn Gly Phe Glu Ile Arg Glu Ser Thr 50 55 60 Gly
Glu Thr Ala Ala Val Tyr Pro Tyr Met Lys Asp Gly Thr Arg Val 65 70
75 80 Glu Trp Lys Ala Glu Lys Phe Asp Trp Asn Thr Pro Asp Pro Arg
Ile 85 90 95 Lys Phe Lys Asn Asn Pro Ile Val Ala Leu Glu Gly Ser
Val Gly Tyr 100 105 110 Ser Ile Gly Val Ala Arg Val Glu Leu Glu Ile
Gly Tyr Glu Gln Phe 115 120 125 Lys Thr Lys Gly Ile Arg Asp Thr Gly
Ser Lys Glu Glu Glu Ala Asp 130 135 140 Ala Val Tyr Leu Leu Ala Lys
Lys Leu Pro His Thr Leu Val Ser Asp 145 150 155 160 Gln Ser Asp Lys
Phe Leu Glu Glu Leu Lys Asn Thr Lys Ala Ala Glu 165 170 175 Ile Val
Lys Phe Ala Glu Ala Val Gly Thr Ser Ala Lys Asp Ile Asp 180 185 190
Gly Lys Val Cys Lys Lys Gly Gly Ser Gly Asn Ala Ala Gly Ser Trp 195
200 205 Lys Cys Thr Gln Thr Gly Ser Asn Gly Val Ser Thr Ala Glu Phe
Ser 210 215 220 Lys Ile Phe Thr Lys Ala Asp Val Asn Thr Asp Asn Lys
Gly Lys Ala 225 230 235 240 Trp Pro Asn Gly Asn Asn Asp Ala Ala Lys
Ala Glu Asp Leu Ser Ile 245 250 255 Ala Leu Asn Arg Glu Leu Thr Ser
Ala Glu Lys Asn Lys Val Ala Gly 260 265 270 Leu Leu Thr Arg Thr Ile
Ser Gly Gly Glu Val Val Glu Ile Arg Ala 275 280 285 Val Ser Thr Thr
Ser Val Met Leu Asn Gly Cys Tyr Asp Leu Gln Ser 290 295 300 Glu Gly
Phe Ser Ile Val Pro Tyr Ala Cys Leu Gly Val Gly Ala Asn 305 310 315
320 Phe Val Gly Ile Val Asp Gly His Val Thr Pro Lys Leu Ala Tyr Lys
325 330 335 Val Lys Ala Gly Leu Ser Tyr Glu Leu Ser Pro Glu Ile Ser
Met Phe 340 345 350 Ala Gly Gly Phe Tyr His Arg Val Leu Gly Glu Gly
Glu Tyr Asp Asp 355 360 365 Leu Pro Val Gln Arg Leu Val Asp Asp Ala
Thr Thr Asn Lys Thr Lys 370 375 380 Glu Phe Ala Lys Ala Ser Phe Lys
Met Ala Tyr Thr Gly Ala Glu Ile 385 390 395 400 Gly Val Arg Ser Ala
Phe 405 22406PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 22Met Lys Glu Arg Lys Leu Ala
Leu Ser Gly Ala Val Ala Met Thr Val 1 5 10 15 Leu Val Ser Thr Ala
Gly Thr Gly Thr Ala Ala Gly Ser Asp Val Asp 20 25 30 Tyr Val Ser
Lys Phe Gly Glu Gly Ser Phe Tyr Val Gly Leu Asn Tyr 35 40 45 Ser
Pro Ala Phe Ser Lys Ile Asn Gly Phe Glu Ile Arg Glu Ser Thr 50 55
60 Gly Glu Thr Ala Ala Val Tyr Pro Tyr Met Lys Asp Gly Thr Arg Val
65 70 75 80 Glu Trp Lys Ala Glu Lys Phe Asp Trp Asn Thr Pro Asp Pro
Arg Ile 85 90 95 Lys Phe Lys Asn Asn Pro Ile Val Ala Leu Glu Gly
Ser Val Gly Tyr 100 105 110 Ser Ile Gly Val Ala Arg Val Glu Leu Glu
Ile Gly Tyr Glu Gln Phe 115 120 125 Lys Thr Lys Gly Ile Arg Asp Thr
Gly Ser Lys Glu Glu Glu Ala Asp 130 135 140 Ala Val Tyr Leu Leu Ala
Lys Lys Leu Pro His Thr Leu Val Ser Asp 145 150 155 160 Gln Ser Asp
Lys Phe Leu Glu Glu Leu Lys Asn Thr Lys Ala Ala Glu 165 170 175 Ile
Val Lys Phe Ala Glu Ala Val Gly Thr Ser Ala Lys Asp Ile Asp 180 185
190 Gly Lys Val Cys Lys Lys Gly Gly Ser Gly Asn Ala Ala Gly Ser Trp
195 200 205 Lys Cys Thr Gln Thr Gly Ser Asn Gly Val Ser Thr Ala Glu
Phe Ser 210 215 220 Lys Ile Phe Thr Lys Ala Asp Val Asn Thr Asp Asn
Lys Gly Lys Ala 225 230 235 240 Arg Pro Asn Gly Asn Asn Asp Ala Ala
Lys Ala Glu Asp Leu Ser Ile 245 250 255 Ala Leu Asn Arg Glu Leu Thr
Ser Ala Glu Lys Asn Lys Val Ala Gly 260 265 270 Leu Leu Thr Arg Thr
Ile Ser Gly Gly Glu Val Val Glu Ile Arg Ala 275 280 285 Val Ser Thr
Thr Ser Val Met Leu Asn Gly Cys Tyr Asp Leu Gln Ser 290 295 300 Glu
Gly Phe Ser Ile Val Pro Tyr Ala Cys Leu Gly Val Gly Ala Asn 305 310
315 320 Phe Val Gly Ile Val Asp Gly His Val Thr Pro Lys Leu Ala Tyr
Lys 325 330 335 Val Lys Ala Gly Leu Ser Tyr Glu Leu Ser Pro Glu Ile
Ser Met Phe 340 345 350 Ala Gly Gly Phe Tyr His Arg Val Leu Gly Glu
Gly Glu Tyr Asp Asp 355 360 365 Leu Pro Val Gln Arg Leu Val Asp Asp
Ala Thr Thr Asn Lys Thr Lys 370 375 380 Glu Phe Ala Lys Ala Ser Phe
Lys Met Ala Tyr Thr Gly Ala Glu Ile 385 390 395 400 Gly Val Arg Phe
Ala Phe 405 23400PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 23Met Lys Glu Arg Lys Leu Ala
Leu Ser Gly Ala Val Ala Met Thr Val 1 5 10 15 Leu Val Ser Thr Ala
Gly Thr Gly Thr Ala Ala Gly Ser Asp Val Asp 20 25 30 Tyr Val Ser
Lys Phe Gly Glu Gly Ser Phe Tyr Val Gly Leu Asn Tyr 35 40 45 Ser
Pro Ala Phe Ser Lys Ile Asn Gly Phe Glu Ile Arg Glu Ser Thr 50 55
60 Gly Glu Thr Ala Ala Val Tyr Pro Tyr Met Lys Asp Gly Thr Arg Val
65 70 75 80 Glu Trp Lys Ala Glu Lys Phe Asp Trp Asn Thr Pro Asp Pro
Arg Ile 85 90 95 Lys Phe Lys Asn Asn Pro Ile Val Ala Leu Glu Gly
Ser Val Gly Tyr 100 105 110 Ser Ile Gly Val Ala Arg Val Glu Leu Glu
Ile Gly Tyr Glu Gln Phe 115 120 125 Lys Thr Lys Gly Ile Arg Asp Thr
Gly Ser Lys Glu Glu Glu Ala Asp 130 135 140 Ala Val Tyr Leu Leu Ala
Lys Lys Leu Pro His Thr Leu Val Ser Asp 145 150 155 160 Gln Ser Asp
Lys Phe Leu Glu Glu Leu Lys Asn Thr Lys Ala Ala Glu 165 170 175 Ile
Val Lys Phe Ala Glu Ala Val Gly Thr Ser Ala Lys Asp Ile Asp 180 185
190 Lys Lys Val Cys Lys Lys Thr Glu Asn Thr Glu Asp Ser Trp Lys Cys
195 200 205 Thr Gln Thr Gly Asn Asp Gly Ser Asp Lys Glu Phe Ser Lys
Ile Phe 210 215 220 Thr Lys Lys Asn Val Asp Thr Ser Gly Lys Ala Trp
Pro Asn Gly Ser 225 230 235 240 Asp Ala Ala Lys Ala Glu Asp Leu Ser
Thr Ala Leu Asn Arg Glu Leu 245 250 255 Thr Ser Ala Glu Lys Asn Lys
Val Ala Gly Leu Leu Thr Arg Thr Ile 260 265 270 Ser Gly Gly Glu Val
Val Glu Ile Arg Ala Val Ser Thr Thr Ser Val 275 280 285 Met Leu Asn
Gly Cys Tyr Asp Leu Gln Ser Glu Gly Phe Ser Ile Val 290 295 300 Pro
Tyr Ala Cys Leu Gly Val Gly Ala Asn Phe Val Gly Ile Val Asp 305 310
315 320 Gly His Val Thr Ser Lys Leu Ala Tyr Lys Val Lys Ala Gly Leu
Ser 325 330 335 Tyr Glu Leu Ser Pro Glu Ile Ser Met Phe Ala Gly Gly
Phe Tyr His 340 345 350 Arg Val Leu Gly Glu Gly Glu Tyr Asp Asp Leu
Pro Val Gln Arg Leu 355 360 365 Val Asp Asp Ala Thr Thr Asn Lys Thr
Lys Glu Phe Ala Lys Ala Ser 370 375 380 Phe Lys Met Ala Tyr Thr Gly
Ala Glu Ile Gly Val Arg Phe Ala Phe 385 390 395 400
24400PRTArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 24Met Lys Glu Arg Lys Leu Ala Leu Ser Gly Ala
Val Ala Met Thr Val 1 5 10 15 Leu Val Ser Thr Ala Gly Thr Gly Thr
Ala Ala Gly Ser Asp Val Asp 20 25 30 Tyr Val Ser Lys Phe Gly Glu
Gly Ser Phe Tyr Val Gly Leu Asn Tyr 35 40 45 Ser Pro Ala Phe Ser
Lys Ile Asn Gly Phe Glu Ile Arg Glu Ser Thr 50 55 60 Gly Glu Thr
Ala Ala Val Tyr Pro Tyr Met Lys Asp Gly Thr Arg Val 65 70 75 80 Glu
Trp Lys Ala Glu Lys Phe Asp Trp Asn Thr Pro Asp Pro Arg Ile 85 90
95 Lys Phe Lys Asn Asn Pro Ile Val Ala Leu Glu Gly Ser Val Gly Tyr
100 105 110 Ser Ile Gly Val Ala Arg Val Glu Leu Glu Ile Gly Tyr Glu
Gln Phe 115 120 125 Lys Thr Lys Gly Ile Arg Asp Thr Gly Ser Lys Glu
Glu Glu Ala Asp 130 135 140 Ala Val Tyr Leu Leu Ala Lys Lys Leu Pro
His Thr Leu Val Ser Asp 145 150 155 160 Gln Ser Asp Lys Phe Leu Glu
Glu Leu Lys Asn Thr Lys Ala Ala Glu 165 170 175 Ile Val Lys Phe Ala
Glu Ala Val Gly Thr Ser Ala Lys Asp Ile Asp 180 185 190 Lys Lys Val
Cys Lys Lys Thr Glu Asn Thr Glu Asp Ser Trp Lys Cys 195 200 205 Thr
Gln Thr Gly Asn Asp Gly Gly Asp Lys Glu Phe Ser Lys Ile Phe 210 215
220 Thr Lys Lys Asn Val Asp Thr Ser Gly Lys Ala Trp Pro Asn Gly Ser
225 230 235 240 Asp Ala Ala Lys Ala Glu Asp Leu Ser Thr Ala Leu Asn
Arg Glu Leu 245 250 255 Thr Ser Ala Glu Lys Asn Lys Val Ala Gly Leu
Leu Thr Arg Thr Ile 260 265 270 Ser Gly Gly Glu Val Val Glu Ile Arg
Ala Val Ser Thr Thr Ser Val 275 280 285 Met Leu Asn Gly Cys Tyr Asp
Leu Gln Ser Glu Gly Phe Ser Ile Val 290 295 300 Pro Tyr Ala Cys Leu
Gly Val Gly Ala Asn Phe Val Gly Ile Val Asp 305 310 315 320 Gly His
Val Thr Pro Lys Leu Ala Tyr Lys Val Lys Ala Gly Leu Ser 325 330 335
Tyr Glu Leu Ser Pro Glu Ile Ser Met Phe Ala Gly Gly Phe Tyr His 340
345 350 Arg Val Leu Gly Glu Gly Glu Tyr Asp Asp Leu Pro Val Leu Arg
Leu 355 360 365 Val Asp Asp Ala Thr Thr Asn Lys Thr Lys Glu Phe Ala
Lys Ala Ser 370 375 380 Phe Lys Met Ala Tyr Thr Gly Ala Glu Ile Gly
Val Arg Phe Ala Phe 385 390 395 400 25280PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 25Met Lys Glu Arg Lys Leu Ala Leu Ser Gly Ala Val Ala Met
Thr Val 1 5 10 15 Leu Val Ser Thr Ala Gly Thr Gly Thr Ala Ala Gly
Ala Asp Val Asp 20 25 30 Tyr Val Ser Lys Phe Gly Glu Gly Ser Phe
Tyr Val Gly Leu Asn Tyr 35 40 45 Ser Pro Ala Phe Ser Lys Ile Asn
Gly Phe Glu Ile Arg Glu Ser Thr 50 55 60 Gly Glu Thr Ala Ala Val
Tyr Pro Tyr Met Lys Asp Gly Thr Arg Val 65 70 75 80 Glu Trp Lys Ala
Glu Lys Phe Asp Trp Asn Thr Pro Asp Pro Arg Ile 85 90 95 Lys Phe
Lys Asn Asn Pro Ile Val Ala Leu Glu Gly Ser Val Gly Tyr 100 105 110
Ser Ile Gly Ile Ala Arg Val Glu Leu Glu Ile Gly Tyr Glu Gln Phe 115
120 125 Lys Thr Lys Gly Ile Arg Asp Thr Gly Ser Lys Glu Glu Glu Ala
Asp 130 135 140 Ala Val Tyr Leu Leu Ala Lys Lys Leu Pro His Thr Leu
Val Ser Asp 145 150 155 160 Gln Ser Asp Lys Phe Leu Glu Glu Leu Lys
Asn Thr Lys Ala Ala Glu 165 170 175 Ile Val Lys Phe Ala Glu Ala Val
Gly Thr Ser Ala Lys Asp Ile Asp 180 185 190 Gly Lys Val Cys Lys Lys
Thr Gly Asn Glu Ala Asp Ser Trp Lys Cys 195 200 205 Thr Gln Thr Gly
Asn Gly Ser Gly Asn Ala Thr Glu Phe Ser Lys Ile 210 215 220 Phe Thr
Lys Lys Asn Val Asp Ala Glu Gly Lys Gly Lys Ala Trp Pro 225 230 235
240 Asn Gly His Thr Asp Ser Ala Ala Lys Ala Glu Asp Leu Ser Thr Ala
245 250 255 Leu Asn Arg Glu Leu Thr Ser Ala Glu Lys Asn Lys Val Ala
Gly Leu
260 265 270 Leu Thr Arg Thr Ile Ser Gly Gly 275 280
2686PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 26Lys Asn Thr Lys Ala Ala Glu Ile Val Lys Phe
Ala Glu Ala Val Gly 1 5 10 15 Thr Ser Ala Lys Asp Ile Asp Gly Lys
Val Cys Lys Lys Gly Gly Ser 20 25 30 Gly Asn Ala Ala Gly Ser Trp
Lys Cys Thr Gln Thr Gly Ser Asn Gly 35 40 45 Val Ser Thr Ala Glu
Phe Ser Lys Ile Phe Thr Lys Ala Asp Val Asn 50 55 60 Thr Asp Asn
Lys Gly Lys Ala Trp Pro Asn Gly Asn Asn Asp Ala Ala 65 70 75 80 Lys
Ala Glu Asp Leu Ser 85 2784PRTArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 27Lys Asn Thr Lys
Ala Ala Glu Ile Val Lys Phe Ala Glu Ala Val Gly 1 5 10 15 Thr Ser
Ala Lys Asp Ile Asp Lys Lys Val Cys Lys Lys Thr Asp Asn 20 25 30
Thr Glu Gly Ser Trp Lys Cys Thr Gln Thr Gly Asn Asp Gly Gly Asp 35
40 45 Lys Glu Phe Ser Lys Thr Phe Thr Lys Thr Gly Val Asn Glu Ala
Thr 50 55 60 Lys Gly Lys Ala Trp Pro Asn Gly His Thr Asp Ser Ala
Ala Lys Ala 65 70 75 80 Glu Asp Leu Ser 2884PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 28Lys Asn Thr Lys Ala Ala Glu Ile Val Lys Phe Ala Glu Ala
Val Gly 1 5 10 15 Thr Ser Ala Lys Asn Ile Asp Lys Lys Val Cys Lys
Lys Thr Gly Asn 20 25 30 Asp Ala Asn Ser Trp Lys Cys Leu Gln Thr
Gly Arg Asp Asp Ser Thr 35 40 45 Ser Gly Lys Lys Phe Ser Glu Ile
Phe Thr Lys Ala Asp Val Asn Thr 50 55 60 Asp Asn Lys Gly Lys Ala
Trp Pro Asn Gly Asn Glu Ala Ala Lys Ala 65 70 75 80 Glu Asp Leu Ser
2986PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 29Lys Asn Thr Lys Ala Ala Glu Ile Val Lys Phe
Ala Glu Ala Val Gly 1 5 10 15 Thr Ser Ala Lys Asn Ile Asp Lys Lys
Val Cys Lys Lys Thr Gly Asn 20 25 30 Asp Ala Asn Ser Trp Lys Cys
Glu Gln Thr Gly Ser Gly Ala Glu Thr 35 40 45 Ser Ala Lys Ala Phe
Ser Glu Ile Phe Thr Lys Ala Gly Val Asn Glu 50 55 60 Ala Thr Lys
Gly Lys Ala Trp Pro Asn Gly His Thr Gly Gly Ala Ala 65 70 75 80 Lys
Ala Glu Asp Leu Ser 85 301221DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 30atgaaggaaa
gaaaacttgc gctaagtgga gcggtggcga tgacagtttt ggtgtcgact 60gctggtaccg
ggactgcggc agggtcggac gtggactatg taagtaagtt cggtgagggc
120agcttctacg taggtctaaa ctatagtccg gcgtttagta agataaatgg
gtttgagata 180agagagagta ccggggaaac tgcggcagta tatccgtaca
tgaaagatgg aactagagtg 240gagtggaaag ctgagaagtt cgactggaac
acaccagatc cgaggattaa gtttaaaaac 300aatcctatcg tggcgttaga
aggaagtgtg ggctacagta tcggggtagc gagagtagaa 360ctggagatcg
gctatgaaca gttcaagacg aaaggaataa gagatacggg aagtaaggaa
420gaagaagctg atgccgtgta cctgttggct aagaagctac cgcataccct
ggtgagtgac 480cagagcgata aattcctgga ggagctgaag aatacgaaag
cggcggagat cgttaaattt 540gctgaggctg ttggcacatc ggcaaaggat
attgatggaa aggtttgtaa gaagggcggc 600agcggcaatg ccgcgggcag
ctggaagtgt acgcagactg gcagcaacgg cgtcagcacc 660gcagagttca
gtaaaatatt tacgaaggca gacgtaaata ctgacaacaa aggcaaagca
720tggcctaacg ggaacaacga cgctgcgaaa gcggaagacc taagtattgc
gttgaataga 780gaactaacca gcgccgaaaa gaacaaggta gctggcctac
taaccaggac tatatccggt 840ggtgaggtag tggagatccg tgcggtgtcg
acaacgtcag taatgttgaa tggttgttat 900gatctgcaga gtgaagggtt
tagtatagta ccttatgcat gtcttggtgt aggtgctaac 960ttcgttggca
ttgttgacgg acacgtcact cctaaactgg cttacaaggt caaggctggt
1020ttgagttatg agttgtcgcc ggaaatctca atgttcgctg gtgggttcta
tcatcgggtg 1080ctgggtgaag gtgagtacga tgatctgcca gtgcagaggc
ttgtagacga tgcgactacg 1140aacaagacta aagagttcgc taaagcgtcg
ttcaagatgg cgtacactgg tgctgaaatc 1200ggtgttaggt ctgcgttcta a
1221311221DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 31atgaaggaaa gaaaacttgc gctaagtgga
gcggtggcga tgacagtttt ggtgtcgact 60gctggtaccg ggactgcggc agggtcggac
gtggactatg taagtaagtt cggtgagggc 120agcttctacg taggtctaaa
ctatagtccg gcgtttagta agataaatgg gtttgagata 180agagagagta
ccggggaaac tgcggcagta tatccgtaca tgaaagatgg aactagagtg
240gagtggaaag ctgagaagtt cgactggaac acaccagatc cgaggattaa
gtttaaaaac 300aatcctatcg tggcgttaga aggaagtgtg ggctacagta
tcggggtagc gagagtagaa 360ctggagatcg gctatgaaca gttcaagacg
aaaggaataa gagatacggg aagtaaggaa 420gaagaagctg atgccgtgta
cctgttggct aagaagctac cgcataccct ggtgagtgac 480cagagcgata
aattcctgga ggagctgaag aatacgaaag cggcggagat cgttaaattt
540gctgaggctg ttggcacatc ggcaaaggat attgatggaa aggtttgtaa
gaagggcggc 600agcggcaatg ccgcgggcag ctggaagtgt acgcagactg
gcagcaacgg cgtcagcacc 660gcagagttca gtaaaatatt tacgaaggca
gacgtaaata ctgacaacaa aggcaaagca 720cggcctaacg ggaacaacga
cgctgcgaaa gcggaagacc taagtattgc gttgaataga 780gaactaacca
gcgccgaaaa gaacaaggta gctggcctac taaccaggac tatatccggt
840ggtgaggtag tggagatccg tgcggtgtcg acaacgtcag taatgttgaa
tggttgttat 900gatctgcaga gtgaagggtt tagtatagta ccttatgcat
gtcttggtgt aggtgctaac 960ttcgttggca ttgttgacgg acacgtcact
cctaaactgg cttacaaggt caaggctggt 1020ttgagttatg aattgtcgcc
ggaaatctca atgttcgctg gtgggttcta tcatcgggtg 1080ctgggtgaag
gtgagtacga tgatctgcca gtgcagaggc ttgtagacga tgcgactacg
1140aacaagacta aagagttcgc taaagcgtcg ttcaagatgg cgtacactgg
tgctgaaatc 1200ggtgttaggt ttgcgttcta a 1221321203DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 32atgaaggaaa gaaaacttgc gctaagtgga gcggtggcga tgacagtttt
ggtgtcgact 60gctggtaccg ggactgcggc agggtcggac gtggactatg taagtaagtt
cggtgagggc 120agcttctacg taggtctaaa ctatagtccg gcgtttagta
agataaatgg gtttgagata 180agagagagta ccggggaaac tgcggcagta
tatccgtaca tgaaagatgg aactagagtg 240gagtggaaag ctgagaagtt
cgactggaac acaccagatc cgaggattaa gtttaaaaac 300aatcctatcg
tggcgttaga aggaagtgtg ggctacagta tcggggtagc gagagtagaa
360ctggagatcg gctatgaaca gttcaagacg aaaggaataa gagatacggg
aagtaaggaa 420gaagaagctg atgccgtgta cctgttggct aagaagctac
cgcataccct ggtgagtgac 480cagagcgata aattcctgga ggagctgaag
aatacgaaag cggcggagat cgttaagttt 540gctgaggctg tcggtacatc
ggcaaaggat attgataaga aggtttgtaa gaagactgaa 600aatacagaag
acagttggaa gtgtacgcag actggcaacg acggcagcga caaggagttc
660agtaaaatat ttacgaagaa aaacgtagat actagcggca aagcatggcc
taacggaagc 720gacgccgcga aagcggaaga cctaagtact gcgctgaata
gagaactaac cagcgccgaa 780aagaacaagg tagctggcct actaaccagg
actatatccg gtggtgaggt agtggagatc 840cgtgcggtgt cgacaacgtc
agtaatgttg aatggttgtt atgatctgca gagtgaaggg 900tttagtatag
taccttatgc atgtcttggt gtaggtgcta acttcgttgg cattgttgac
960ggacacgtca cttctaaact ggcttacaag gtcaaggctg gtttgagtta
tgagttgtcg 1020ccggaaatct caatgttcgc tggtgggttc tatcatcggg
tgctgggtga aggtgagtac 1080gatgatctgc cagtgcagag gcttgtagac
gatgcgacta cgaacaagac taaagagttc 1140gctaaagcgt cgttcaagat
ggcgtacact ggtgctgaaa tcggtgttag gtttgcgttc 1200taa
1203331203DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 33atgaaggaaa gaaaacttgc gctaagtgga
gcggtggcga tgacagtttt ggtgtcgact 60gctggtaccg ggactgcggc agggtcggac
gtggactatg taagtaagtt cggtgagggc 120agcttctacg taggtctaaa
ctatagtccg gcgtttagta agataaatgg gtttgagata 180agagagagta
ccggggaaac tgcggcagta tatccgtaca tgaaagatgg aactagagtg
240gagtggaaag ctgagaagtt cgactggaac acaccagatc cgaggattaa
gtttaaaaac 300aatcctatcg tggcgttaga aggaagtgtg ggctacagta
tcggggtagc gagagtagaa 360ctggagatcg gctatgaaca gttcaagacg
aaaggaataa gagatacggg aagtaaggaa 420gaagaagctg atgccgtgta
cctattggct aagaagctac cgcataccct ggtgagtgac 480cagagcgata
aattcctgga ggagctgaag aatacgaaag cggcggagat cgttaagttt
540gctgaggctg ttggtacatc ggcaaaggat attgataaga aggtttgtaa
gaagactgaa 600aatacagaag acagttggaa gtgtacgcag actggcaacg
acggcggcga caaggagttc 660agtaaaatat ttacgaagaa aaacgtagat
actagcggca aagcatggcc taacggaagc 720gacgccgcga aagcggaaga
cctaagtact gcgctgaata gagaactaac cagcgccgaa 780aagaacaagg
tagctggcct actaaccagg actatatccg gtggtgaggt agtggagatc
840cgtgcggtgt cgacaacgtc agtaatgttg aatggttgtt atgatctgca
gagtgaaggg 900tttagtatag taccttatgc atgtcttggt gtaggtgcta
acttcgttgg cattgttgac 960ggacacgtca ctcctaaact ggcttacaag
gtcaaggctg gtttgagtta tgagttgtcg 1020ccggaaatct caatgttcgc
tggtgggttc tatcatcggg tgctgggtga aggtgagtac 1080gatgatctgc
cagtgctgag gcttgtagac gatgcgacta cgaacaagac taaagagttc
1140gctaaagcgt cgttcaagat ggcgtacact ggtgctgaaa tcggtgttag
gtttgcgttc 1200taa 120334260DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 34gaagaatacg
aaagcggcgg agatcgttaa atttgctgag gctgttggca catcggcaaa 60ggatattgat
ggaaaggttt gtaagaaggg cggcagcggc aatgccgcgg gcagctggaa
120gtgtacgcag actggcagca acggcgtcag caccgcagag ttcagtaaaa
tatttacgaa 180ggcagacgta aatactgaca acaaaggcaa agcatggcct
aacgggaaca acgacgctgc 240gaaagcggaa gacctaagta
26035254DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 35gaagaatacg aaagcggcgg agatcgttaa
atttgctgag gctgttggta catcggcaaa 60ggatattgat aagaaggttt gtaagaagac
tgacaataca gaaggcagtt ggaagtgtac 120gcagactggc aacgacggcg
gcgacaagga gttcagtaaa acatttacga agacgggcgt 180gaatgaggcc
accaagggca aagcatggcc taacgggcac accgacagcg ccgcgaaagc
240ggaagaccta agta 25436254DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 36gaagaatacg
aaagcggcgg agatcgttaa atttgctgag gctgttggca catcggcaaa 60gaatattgat
aagaaggttt gtaagaagac tggaaatgac gcgaacagtt ggaagtgctt
120gcagactggc agagacgaca gcacgtcggg gaaaaagttc agtgaaatat
ttacgaaggc 180agacgtaaat actgacaaca aaggcaaagc atggcctaac
gggaacgaag ccgcgaaagc 240ggaagaccta agta 25437260DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 37gaagaatacg aaagcggcgg agatcgttaa atttgctgag gctgttggca
catcggcaaa 60gaatattgat aagaaggttt gtaagaagac tggaaatgac gcgaacagtt
ggaagtgcga 120gcagactggc agcggcgccg agacaagcgc caaggcgttc
agtgaaatat ttacgaaggc 180gggcgtgaat gaggccacca agggcaaagc
atggcctaac gggcacaccg gcggcgccgc 240gaaagcggaa gacctaagta
26038842DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 38atgaaggaaa gaaaacttgc gctaagtgga
gcggtggcga tgacagtttt ggtgtcgact 60gctggtaccg ggactgcggc aggggcggac
gtggactatg taagtaagtt cggtgagggc 120agcttctacg taggtctaaa
ctatagtccg gcgtttagta agataaatgg gtttgagata 180agagagagta
ccggggaaac tgcggcagta tatccgtaca tgaaagatgg aactagagtg
240gagtggaaag ctgagaagtt cgactggaac acaccagatc cgaggattaa
gtttaaaaac 300aatcctatcg tggcgttgga aggaagtgtg ggctacagta
tcgggatagc gagagtagaa 360ctagagatcg gctatgaaca gttcaagacg
aaaggaataa gagatacggg aagtaaggaa 420gaagaagctg atgccgtgta
cctgttggct aagaagctac cgcataccct ggtgagtgac 480cagagcgata
aattcctgga ggagctgaag aatacgaaag cggcggagat cgttaagttt
540gctgaggctg ttggtacatc ggcaaaggat attgatggaa aggtttgtaa
gaagactgga 600aatgaggcgg acagttggaa gtgtacgcag actggcaacg
gcagcggcaa cgccacagag 660tttagtaaaa tatttacgaa gaaaaacgta
gatgctgagg gcaaaggcaa agcatggcct 720aacgggcaca ccgacagcgc
cgcgaaagcg gaagacctaa gtactgcgtt gaatagagaa 780ctaaccagcg
ccgaaaagaa caaggtagct ggcctactaa ccaggactat atccggtggc 840ga
8423918PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 39Gly Thr Gly Thr Ala Ala Gly Ser Asp
Val Asp Tyr Val Ser Lys Phe 1 5 10 15 Gly Glu 409PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 40Gly Thr Arg Val Glu Trp Lys Ala Glu 1 5
4140PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 41Lys Lys Leu Pro His Thr Leu Val Ser Asp Gln
Ser Asp Lys Phe Leu 1 5 10 15 Glu Glu Leu Lys Asn Thr Lys Ala Ala
Glu Ile Val Lys Phe Ala Glu 20 25 30 Ala Val Gly Thr Ser Ala Lys
Asp 35 40 428PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 42Ser Trp Lys Cys Thr Gln Thr
Gly 1 5 4322PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 43Ala Ala Lys Ala Glu Asp Leu
Ser Ile Ala Leu Asn Arg Glu Leu Thr 1 5 10 15 Ser Ala Glu Lys Asn
Lys 20 449PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 44Ala Thr Thr Asn Lys Thr Lys Glu Phe 1
5 45106PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 45Gly Thr Gly Thr Ala Ala Gly Ser Asp
Val Asp Tyr Val Ser Lys Phe 1 5 10 15 Gly Glu Gly Thr Arg Val Glu
Trp Lys Ala Glu Lys Lys Leu Pro His 20 25 30 Thr Leu Val Ser Asp
Gln Ser Asp Lys Phe Leu Glu Glu Leu Lys Asn 35 40 45 Thr Lys Ala
Ala Glu Ile Val Lys Phe Ala Glu Ala Val Gly Thr Ser 50 55 60 Ala
Lys Asp Ser Trp Lys Cys Thr Gln Thr Gly Ala Ala Lys Ala Glu 65 70
75 80 Asp Leu Ser Ile Ala Leu Asn Arg Glu Leu Thr Ser Ala Glu Lys
Asn 85 90 95 Lys Ala Thr Thr Asn Lys Thr Lys Glu Phe 100 105
4654DNAArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 46ggtaccggga ctgcggcagg gtcggacgtg gactatgtaa
gtaagttcgg tgag 544727DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 47ggaactagag
tggagtggaa agctgag 2748120DNAArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 48aagaagctac
cgcataccct ggtgagtgac cagagcgata aattcctgga ggagctgaag 60aatacgaaag
cggcggagat cgttaaattt gctgaggctg ttggcacatc ggcaaaggat
1204924DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 49agctggaagt gtacgcagac tggc
245066DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 50gctgcgaaag cggaagacct aagtattgcg ttgaatagag
aactaaccag cgccgaaaag 60aacaag 665127DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 51gcgactacga acaagactaa agagttc 275217DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 52ggtaccggga ctgcggc 175327DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 53ctcaccgaac ttacttacat agtccac 275423DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 54ggaactagag tggagtggaa agc 235516DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 55ctcagctttc cactcc 165621DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 56aagaagctac cgcataccct g 215721DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 57atcctttgcc gatgtaccaa c 215822DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 58agctggaagt gtacgcagac tg 225920DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 59gccagtctgc gtacacttcc 206018DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 60gctgcgaaag cggaagac 186119DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 61cttgttcttt tcggcgctg
196224DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 62gcgactacga acaagactaa agag
246326DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 63gaactcttta gtcttgttcg tagtcg
266419DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 64gcggtgcagg aaaagaaac 196518DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 65agcagcttcg tcagctgc 186624DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 66aacatatgaa tcttgtgagc gcgg 246725DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 67ggggatccgg ctgggggagc agaag 256831DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 68aagctagcca aggtgacgaa gtacttttgt g 316935DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 69aagctagcct aacctatcaa ttcaaaggtg gattg
357024DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 70ggccatgggt accgggactg cggc
247124DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 71ttcatatgct cagctttcca ctcc
247235DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 72ccctgcagct caccgaactt acttacatag tccac
357331DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 73ggctgcaggg aactagagtg gagtggaaag c
317429DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 74aaggatccaa gaagctaccg cataccctg
297528DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 75ccgtcgacgc cagtctgcgt acacttcc
287629DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 76aagaattcat cctttgccga tgtaccaac
297730DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 77ttgaattcag ctggaagtgt acgcagactg
307826DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 78aaaagcttgc tgcgaaagcg gaagac
267934DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 79ccctcgagga actctttagt cttgttcgta gtcg
348027DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 80ttggatccct tgttcttttc ggcgctg
278132DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 81aaggatccgc gactacgaac aagactaaag ag
328231DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 82attatgtatg atttatccta agttatctga g
318321DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 83gggatatcgg cgttgatagg g 218425DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 84ggtttgtgtt gctggtgatt ggagg 258526DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 85gcaaacctaa caccmaaytc mccacc 268627DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 86tatactaaaa aagaattaag tcaagag 278721DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 87atggtagaaa sccccagcaa a 218820DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 88cacgtnttta gttactgcca 208926DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 89gtactagtca gcgccactaa catcaa 269020DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 90gaagaatacg aaagcggcgg 209122DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 91tacttaggtc ttccgctttc gc 229215PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 92Thr Gly Thr Ala Ala Gly Ser Asp Val Asp Tyr Val Ser Lys
Phe 1 5 10 15 938PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 93Thr Arg Val Glu Trp Lys Ala
Glu 1 5 9416PRTArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 94Ala Ala Glu Ile Val Lys Phe
Ala Glu Ala Val Gly Thr Ser Ala Lys 1 5 10 15 958PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 95Ser Trp Lys Cys Thr Gln Thr Gly 1 5 968PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 96Ala Ala Lys Ala Glu Asp Leu Ser 1 5 979PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 97Ala Thr Thr Asn Lys Thr Lys Glu Phe 1 5
9810PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 98Ala Val Gln Glu Lys Lys Pro Pro Glu Ala 1 5
10 9917DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 99ggctggggga gcagaag
1710018PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 100Gly Thr Gly Thr Ala Ala Gly Ser Asp
Val Asp Tyr Val Ser Lys Phe 1 5 10 15 Gly Glu 10118PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 101Gly Thr Gly Thr Ala Ala Gly Ser Asp Val Asp Tyr Val
Ser Lys Phe 1 5 10 15 Gly Glu 10218PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 102Gly Thr Gly Thr Ala Ala Gly Ser Asp Val Asp Tyr Val
Ser Lys Phe 1 5 10 15 Gly Glu 10318PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 103Gly Thr Gly Thr Ala Ala Gly Ser Asp Val Asp Tyr Val
Ser Lys Phe 1 5 10 15 Gly Glu 1049PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 104Gly Thr Arg
Val Glu Trp Lys Ala Glu 1 5 1059PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 105Gly Thr Arg
Val Glu Trp Lys Ala Glu 1 5 1069PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 106Gly Thr Arg
Val Glu Trp Lys Ala Glu 1 5 1079PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 107Gly Thr Arg
Val Glu Trp Lys Ala Glu 1 5 10840PRTArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 108Lys Lys Leu
Pro His Thr Leu Val Ser Asp Gln Ser Asp Lys Phe Leu 1 5 10 15 Glu
Glu Leu Lys Asn Thr Lys Ala Ala Glu Ile Val Lys Phe Ala Glu 20 25
30 Ala Val Gly Thr Ser Ala Lys Asp 35 40 10941PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 109Ala Lys Lys Leu Pro His Thr Leu Val Ser Asp Gln Ser
Asp Lys Phe 1 5 10 15 Leu Glu Glu Leu Lys Asn Thr Lys Ala Ala Glu
Ile Val Lys Phe Ala 20 25 30 Glu Ala Val Gly Thr Ser Ala Lys Asp 35
40 11040PRTArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 110Lys Lys Leu Pro His Thr Leu Val Ser
Asp Gln Ser Asp Lys Phe Leu 1 5 10 15 Glu Glu Leu Lys Asn Thr Lys
Ala Ala Glu Ile Val Lys Phe Ala Glu 20 25 30 Ala Val Gly Thr Ser
Ala Lys Asp 35 40 11141PRTArtificial SequenceDescription of
Artificial Sequence note = synthetic construct 111Ala Lys Lys Leu
Pro His Thr Leu Val Ser Asp Gln Ser Asp Lys Phe 1 5 10 15 Leu Glu
Glu Leu Lys Asn Thr Lys Ala Ala Glu Ile Val Lys Phe Ala 20 25 30
Glu Ala Val Gly Thr Ser Ala Lys Asp 35 40 1128PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 112Ser Trp Lys Cys Thr Gln Thr Gly 1 5 1138PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 113Ser Trp Lys Cys Thr Gln Thr Gly 1 5 1148PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 114Ser Trp Lys Cys Thr Gln Thr Gly 1 5 1158PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 115Ser Trp Lys Cys Thr Gln Thr Gly 1 5 11622PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 116Ala Ala Lys Ala Glu Asp Leu Ser Ile Ala Leu Asn Arg
Glu Leu Thr 1 5 10 15 Ser Ala Glu Lys Asn Lys 20 11722PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 117Ala Ala Lys Ala Glu Asp Leu Ser Ile Ala Leu Asn Arg
Glu Leu Thr 1 5 10 15 Ser Ala Glu Lys Asn Lys 20 11822PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 118Ala Ala Lys Ala Glu Asp Leu Ser Thr Ala Leu Asn Arg
Glu Leu Thr 1 5 10 15 Ser Ala Glu Lys Asn Lys 20 11921PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 119Ala Lys Ala Glu Asp Leu Ser Thr Ala Leu Asn Arg Glu
Leu Thr Ser 1 5 10 15 Ala Glu Lys Asn Lys 20 1209PRTArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 120Ala Thr Thr Asn Lys Thr Lys Glu Phe 1 5
1219PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 121Ala Thr Thr Asn Lys Thr Lys Glu Phe 1 5
1229PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 122Ala Thr Thr Asn Lys Thr Lys Glu Phe 1 5
1239PRTArtificial SequenceDescription of Artificial Sequence note =
synthetic construct 123Ala Thr Thr Asn Lys Thr Lys Glu Phe 1 5
12411DNAArtificial SequenceDescription of Artificial Sequence note
= synthetic construct 124ggtggtgagg t 1112515DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 125gttgttatga tctgc 1512611DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 126ccttatgcat g 1112714DNAArtificial SequenceDescription
of Artificial Sequence note = synthetic construct 127cctcctaact
tcct 1412814DNAArtificial SequenceDescription of Artificial
Sequence note = synthetic construct 128ttgttatgat ctgc
1412914DNAArtificial SequenceDescription of Artificial Sequence
note = synthetic construct 129gtaccttatg catg 1413014DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 130cctcctaact tcct 1413117DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 131aaggctggtt tgagtta 1713214DNAArtificial
SequenceDescription of Artificial Sequence note = synthetic
construct 132ccctcttacc tctg 14
* * * * *
References