Note: Descriptions are shown in the official language in which they were submitted.
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HOMOLOGOUS 28-KILODALTON IMMUNODOMINANT PROTEIN
GENES OF EHRLICHIA CAN.IS AND USES THEREOF
S
BACKGROUND OF THE INVENTION
Fi~ld of y~e_. TnvPnti~n
The present invention relates generally to the field o f
molecular biology. More specifically, the present invention relates t o
molecular cloning and characterization of homologous 28-kDa
protein genes in Ehrlichia canis and a multigene locus encoding the
28-kDa homologous proteins of Ehrlichia cams and uses thereof.
Description ~f the Rel~teri Ar
Canine ehrlichiosis, also known as canine tropical
pancytopenia, is a tick-borne rickettsial disease of dogs first described
in Africa in 1935 and the United States in 1963 (Donatien and
Lestoquard, 1935; Ewing, 1963). The disease became better
recognized after an epizootic outbreak occurred in United S fates
military dogs during the Vietnam War (Walker et al., 1970)
The etiologic agent of canine ehrlichiosis is Ehrlichia canis,
a small, gram-negative, obligate intracellular bacterium which exhibits
tropism for mononuclear phagocytes (Nyindo er al., 197 / ) and is
transmitted by the brown dog tick, Rhipiceplaalus sangecineccs (Groves
et al., /975). The progression of canine ehrlichiosis occurs in three
phases, acute, subclinical and chronic. The acute phase is
characterized by fever, anorexia, depression, lymphadenopathy and
mild thrombocytopenia (Troy and Forrester, 1990). Dogs typically
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recover from the acute phase, but become persistently infected
carriers of the organism without clinical signs of disease for months
or even years (Harrus et al., 1998). A chronic phase develops i n
some cases that is characterized by thrombocytopenia,
hyperglobulinemia, anorexia, emaciation, and hemorrhage,
particularly epistaxis, followed by death (Troy and Forrester, 1990).
Molecular taxonomic analysis based on the 16S rRNA gene
has determined that E. canis and E. chaffeens is, the etiologic agent o f
human monocytic ehrlichiosis (HME), are closely related (Anderson a t
al., 1991; Anderson et al., 1992; Dawson et al.; I991; Chen et al.,
1994). Considerable cross reactivity of the 64, 47, 40, 30, 29 and 2 3 -
kDa antigens between E. canis and E. chaffeensis has been reported
(Chen et al., 1994; Chen et al., 1997; Rikihisa et al., 1994; Rikihisa a t
al., 1992). Analysis of immunoreactive antigens with human and
canine convalescent phase sera by immunobIot has resulted in tha
identification of numerous immunodominant proteins of E. cahis,
including a 30-kDa protein (Chen et al., 1997). In addition, a 30-kDa
protein of E. canis has been described as a major immunodominant
antigen recognized early in the immune response that is antigenically
distinct from the 30-kDa protein of E. chaffeensis {Rikihisa et al.,
1992; Rikihisa et al., 1994). Other immunodominant proteins of E
canis with molecular masses ranging from 20 to 30-kDa have also
been identified {Brouqui et al., 1992; Nyindo et al., 1991; Chen et al.,
1994; Chen et al., 1997).
Recently, cloning and sequencing of a multigene family
(omp-1 ) encoding proteins of 23 to 28-kDa have been described for E
chaffeensis (Ohashi et al., 1998). The 28-kDa immunodominant outer
membrane protein gene (p28) of E. chaffeensis, homologous to the
Cowdria racmiraantium map-1 gene, was cloned. Mice immunized with
recombinant P28 were protected against challenge infection with the
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homologous strain according to PCR analysis of periperal blood 5 days
after challenge (Ohashi et al., 1998). Molecular cloning of two
similar, but nonidentical, tandernly arranged 28-kDa genes of E. canis
homologous to E. chaffeensis omp-I gene family and C. rumanintium
rnap- I gene has also been reported (Reddy et al., 1998).
The prior art is deficient in the lack of cloning and
characterization of new homologous 28-kDa immunoreactive protein
genes of Ehrlichia canis and a single multigene locus containing the
homologous 28-kDa protein genes. Further, The prior art is deficient
in the lack of recombinant proteins of such immunoreactive genes of
Ehrlichia canis. The present invention fulfills this long-standing need
and desire in the art.
SUMMARY OF THE INVENTION
The present invention describes the molecular cloning,
sequencing, characterization, and expression of homologous mature
28-kDa immunoreactive protein genes of Ehrlichia canis (designated
Eca28-l, ECa28SA3 and ECa28SA2), and the identification of a single
locus (5.592-kb) containing five 28-kDa protein genes of Ehrlichia
cams (ECa28SAl, ECa28SA2, ECa28SA3, Eca28-I and ECa28-2).
Comparison with E. chaffeensis and among E. canis 28-kDa protein
genes revealed that ECa28-1 shares the most amino acid homology
with the E. chaffeensi,r ornp-I multigene family and is highly conserved
among E. cams isolates. The five 28-kDa proteins were predicted t o
have signal peptides resulting in mature proteins, and had amino acid
homology ranging from Si to 72%. Analysis of intergenic regions
revealed hypothetical promoter regions for each gene, suggesting that
these genes may be independently and differentially expressed.
3 0 Intergenic noncoding regions ranged in size from 299 to 355-bp, a n d
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were 48 to 71 % homologous.
In one embodiment of the present invention, there are
provided DNA sequences encoding a 30-kDa immunoreactive protein
of Ehrlichia canis. Preferably, the protein has an amino acid sequence
selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and
SEQ ID No. 6, and the gene has a nucleic acid sequence selected from
the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5
and is a member of a polymorphic multiple gene family. Generally,
the protein has an N-terminal signal sequence which is cleaved after
i0 post-translational process resulting in the production of a mature 28-
kDa protein. Still preferably, the DNAs encoding 28-kDa proteins are
contained in a single multigene locus, which has the size of 5.592 k b
and encodes all five homologous 28-kDa proteins of Ehrlichia canis.
In another embodiment of the present invention, there is
provided an expression vector comprising a gene encoding a 28-kDa
immunoreaetive protein of Ehrlichia canis and capable of expressing
the gene when the vector is introduced into a cell.
In still another embodiment of the present invention, there
is provided a recombinant protein comprising an amino acid s a q a a n c a
selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 a n d
SEQ ID No. 6. Preferably, the amino acid sequence is encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
No. 1, SEQ ID No. 3 and SEQ ID No. 5. Preferably, the recombinant
protein comprises four variable regions which are surface exposed,
hydrophilic and antigenic. The recombinant protein may be useful as
an antigen.
In yet another embodiment of the present invention, there
is provided a method of producing the recombinant protein,
comprising the steps of obtaining a vector that comprises a n
expression region comprising a sequence encoding the amino acid
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sequence selected from the group consisting of SEQ ID No. 2. SEQ ID
Na. 4 and SEQ ID No. 6 operatively linked to a promoter; transfecting
the vector into a cell; and culturing the cell under conditions effective
for expression of the expression region.
The invention may also be described in certain
embodiments as a method of inhibiting Ehrlichia canis infection in a
subject comprising the steps of: identifying a subject suspected o f
being exposed to or infected with Ehrlichia canis; and administering a
composition comprising a 28-kDa antigen of Ehrlichia canis in a n
amount effective to inhibit an Ehrlichia canis infection. The inhibition
may occur through any means such as, i.e. the stimulation of the
subject's humoral or cellular immune responses, or by other means
such as inhibiting the normal function of the 28-kDa antigen, or even
competing with the antigen for interaction with some agent in th a
subject's body.
Other and further aspects, features, and advantages of the
present invention will be apparent from the following description of
the presently preferred embodiments of the invention given for the
purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which will
become clear, are attained and can be understood in detail, m o r a
particular descriptions of the invention briefly summarized above may
be had by reference to certain embodiments thereof which are
illustrated in the appended drawings. These drawings form a part o f
the specification. It is to be noted, however, that the appended
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drawings illustrate preferred embodiments of the invention and
therefore are not to be considered limiting in their scope.
Figure i shows nucleic acid sequence (SEQ ID No. 1 ) a n d
deduced amino acid sequence (SEQ ID No. 2) of ECa28-I gene
including adjacent 5' and 3' non-coding sequences. The ATG start
colon and TAA termination are shown in bold, and the 23 amino acid
leader signal sequence is underlined.
Figure 2 shows SDS-PAGE of expressed 50-kDa
recombinant ECa28-1-thioredoxin fusion protein (Lane l, arrow) and
16-kDa thioredoxin control (Lane 2, arrow), and corresponding
immunoblot of recombinant ECa28-1-thioredoxin fusion protein
recognized by covalescent-phase E. canis canine antiserum (Lane 3 ) .
Thiroredoxin control was not detected by E.canis antiserum ( n o t
shown).
Figure 3 shows alignment of ECa28-1 protein (SEQ ID NO.
2), and ECa28SA2 (partial sequence, SEQ ID NO. 7) and ECa28SA 1 (SEQ
ID NO. 8), E. chaffeensis P28 (SEQ ID NO. 9}, E. chaffeensis OMP-1
family (SEQ ID NOs: 10-14) and C. ruminantium MAP-1 (SEQ ID NO.
15) amino acid sequences. The ECa28-1 amino acid sequence is
presented as the consensus sequence. Amino acids not shown are
identical to ECa28-1 and are represented by a dot. Divergent amino
acids are shown with the corresponding one letter abbreviation. Gaps
introduced for maximal alignment of the amino acid sequences are
denoted with a dash. Variable regions are underlined and denoted
(VR1, VR2, VR3, and VR4). The arrows indicate the predicted signal
peptidase cleavage site for the signal peptide.
Figure 4 shows phylogenetic relatedness of E. canis ECa28-
1 with the ECa28SA2 (partial sequence) and ECa28SA1, 6 members of
the E.chaffeensis omp-I multiple gene family, and C. rumanintium
map-I from deduced amino acid sequences uxilizing unbalanced tree
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construction. The length of each pair of branches represents the
distance between the amino acid sequence of the pairs. The scale
measures the distance between sequences.
Figure 5 shows Southern blot analysis of E. canis genomic
DNA completely digested with six individual restriction enzymes a n d
hybridized with a ECa28-1 DIG-labeled probe (Lanes 2-7); DIG-labeled
molecular weight markers {Lanes. 1 and 8).
Figure 6 shows comparison of predicted protein
characteristics of ECa28-1 (Jake strain) and E. chaffeensis P28
(Arkansas strain). Surface probability predicts the surface residues
by using a window of hexapeptide. A surface residue is any residue
with a >2.0 nmz of water accessible surface area. A hexapeptide with
a value higher than 1 was considered as surface region. The antigenic
index predicts potential antigenic determinants. The regions with a
value above zero are potential antigenic determinants. T-cell motif
locates the potential T-cell antigenic determinants by using a motif o f
5 amino acids with residue 1-glycine or polar, residue 2-hydrophobic,
residue 3-hydrophobic, residue 4-hydrophobic or proline, and residue
5-polar or glycine. The scale indicates amino acid positions.
Figure 7 shows nucleic acid sequences and deduced
amino acid sequences of the E. canis 28-kDa protein genes ECa28SA2
(nucleotide 1-849: SEQ ID No. 3; amino acid sequence: SEQ ID No. 4 )
and ECa28SA3 (nucleotide 1195-2031: SEQ ID No. S; amino acid
sequence: SEQ ID No. 6) including intergenic noncoding sequences
2 5 (NC2, nucleotide 850-1194: SEQ ID No. 31 ). The ATG start codon a n d
termination condons are shown in bold.
Figure 8 shows schematic of the five E. canis 28-kDa
protein gene locus (5.592-Kb) indicating genomic orientation a n d
intergenic noncoding regions (28NC1-4). The 28-kDa protein genes
shown in Locus 1 and 2 (shaded} have been described (McBride et al.,
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1999; Reddy et al., 1998: Ohashi et al., 1998 ). The complete s a q a a n c a
of ECaSA2 and a new 28-kDa protein gene designated (ECa28SA3
unshaded) was sequenced. The noncoding intergenic regions (28NC2-
3) between ECaSA2. ECa28SA3 and ECa28-I were completed joining the
previously unlinked loci 1 and 2.
Figure 9 shows phylogenetic relatedness of the five E
canis 28-kDa protein gene members based on amino acid sequences
utilizing unbalanced tree construction. The length of each pair of
branches represents the distance between amino acid pairs. The scale
measures the distance beteween sequences.
Figure 14 shows alignment of E. canis 28-kDa protein
gene intergenic noncoding nucleic acid sequences (SEQ ID Nos. 30-
33). Nucleic acids not shown, denoted with a dot (.), are identical t o
noncoding region 1 (28NC 1 ). Divergence is shown with t h a
corresponding one letter abbreviation. Gaps introduced for maximal
alignment of the amino acid sequences are denoted with a dash (-).
Putative transcriptional promoter regions (-10 and -35) and ribosomal
binding site (RBS) are boxed.
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes cloning, sequencing and
expression of homologous genes encoding a 30-kilodalton (kDa)
protein of Ehrlichia canis. A comparative molecular analysis of
homologous genes among seven E. cams isolates and the E. chaffeensis
omp-1 multigene family was also performed. Two new 28-kDa protein
genes are identified, ECa28-1 and ECa28SA3. ECa28-1 has an 834-by
open reading frame encoding a protein of 278 amino acids (SEQ ID
No. 2) with a predicted molecular mass of 30.5-kDa. An N-terminal
signal sequence was identified suggesting that the protein is post-
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translationally modified to a mature protein of 27.7-kDa. ECa28SA3
has an 840-by open reading frame encoding a 280 amino acid protein
(SEQ ID No. 6).
Using PCR to amplify 28-kDa protein genes of E. canis, a
previously unsequenced region of Eca28SA2 was completed. Sequence
analysis of ECa28SA2 revealed an 849-by open reading frame encoding
a 283 amino acid protein (SEQ ID Na. 4): PCR amplification using
primers specific for 28-kDa protein gene intergenic noncoding regions
linked two previously separate Ioci, identifying a single locus (5.592
kb) containing alI five 28-kDa protein genes. The five 28-kDa
proteins were predicted to have signal peptides resulting in mature
proteins, and had amino acid homology ranging from 51 to 72%.
Analysis of intergenic regions revealed hypothetical promoter regions
for each gene, suggesting that these genes may be independently a n d
differentially expressed. Intergenic noncoding regions (28NC1-4)
ranged in size from 299 to 355-bp, and were 48 to 71 % homologous.
The present invention is directed to two new homologous
28-kDa protein genes in Elzrlichia canis, Eca28-1 and ECa28SA3, and a
complete sequence of previously partially sequenced ECa28SA2. Also
disclosed is a multigene locus encoding all five homologous 28-kDa
outer membrane proteins of Ehrlichia canis.
In one embodiment of the present invention, there are
provided DNA sequences encoding a 30-kDa immunoreactive protein
of Ehrlichia canis. Preferably, the protein has an amino acid s a qu a nc a
selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and
SEQ ID No. 6, and the gene has a nucleic acid sequence selected from
the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5
and is a member of a polymorphic multiple gene family. More
preferably, the protein has an N-terminal signal sequence which is
cleaved after post-translational process resulting in the production o f
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a mature 28-kDa protein. Still preferably, the DNAs encoding 28-kDa
proteins are contained in a single multigene Iocus, which has the size
of 5.592 kb and encodes alI five homologous 28-kDa proteins o f
Ehrlichia canis.
S In another embodiment of the present invention, there i s
provided an expression vector comprising a gene encoding a 28-kDa
immunoreactive protein of Ehrlichia cams and capable of expressing
the gene when the vector is introduced into a cell.
In still another embodiment of the present invention, there
is provided a recombinant protein comprising an amino acid sequence
selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and
SEQ iD No. 6. Preferably, the amino acid sequence is encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
No. I , SEQ iD No. 3 and SEQ ID No. 5. Preferably, the r a c o m b i n a n t
I S protein comprises four variable regions which are surface exposed,
hydrophilic and antigenic. Still preferably, the recombinant protein is
an antigen.
In yet another embodiment of the present invention, there
is provided a method of producing the recombinant protein,
comprising the steps of obtaining a vector that comprises a n
expression region comprising a sequence encoding the amino acid
sequence selected from the group consisting of SEQ ID No. 2, SEQ ID
No. 4 and SEQ ID No. 6 operatively linked to a promoter; transfecting
the vector into a cell; and culturing the cell under conditions effective
for expression of the expression region.
The invention may also be described in certain
embodiments as a method of inhibiting Ehrlichia canis infection in a
subject comprising the steps of: identifying a subject suspected o f
being exposed to or infected with Ehrlichia cams; and administering a
34 composition comprising a 28-kDa antigen of Ehrlichia cams in a n
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amount effective to inhibit nn Ehrlichiu cunis infection. The inhibition
may occur through any means such as, i.e. the stimulation of the
subject's humoral or cellular immune responses, or by other means
such as inhibiting the normal function of the 28-kDa antigen, or even
competing with the antigen for interaction with some agent in t h a
subject's body.
In accordance with the present invention there may b a
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g., Maniatis,
Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual ( 19 8 2 ) ;
"DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed.
1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid
Hybridization" [B.D. Homes & S.J. Higgins eds. (1985)]; "Transcription
and Translation" [B.D. Homes & S.J. Higgins eds. (1984)]; "Animal Cell
Culture" [R.I. Freshney, ed. ( 1986)]; "Immobilized Cells And Enzymes"
[IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning"
( 1984).
Therefore, if appearing herein, the following terms shall
have the definitions set out below.
A "replicon" is. any genetic element (e:g., plasmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo; i.e., capable of replication under its own control.
A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment.
A "DNA molecule" refers to the polymeric form o f
deoxyribonucleatides (adenine, guanine, thymine, or cytosine) in its
either single stranded . form, or a double-stranded helix. This term
refers only to the primary and secondary structure of the molecule,
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and does not limit it to any particular tertiary forms. Thus, this term
includes double-stranded DNA found, inter olio, in linear DNA
molecules (e.g., restriction fragments), viruses, plasmids, and
chromosomes. In discussing the structure herein according to tha
normal convention of giving only the sequence in the 5' to 3' direction
along the nontranscribed strand of DNA (i.e., the strand having a
sequence homologous to the mRNA).
A DNA "coding sequence" is a double-stranded DNA
sequence which is transcribed and translated into a polypeptide i n
vivo when placed under the control of appropriate regulatory
sequences. The boundaries of the coding sequence are determined b y
a start codon at the 5' (amino) terminus and a translation stop codon
at the 3' (carboxyl) terminus. A coding sequence can include, but is
not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to the
coding sequence.
Transcriptional and translational control sequences are
DNA regulatory sequences, such as promoters, enhancers,
polyadenylatian signals, terminators, and the like, that provide for the
expression of a coding sequence in a host cell.
A "promoter sequence" is a DNA regulatory region capable
of binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3 '
terminus by the transcription initiation site and extends upstream ( 5'
direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
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transcription initiationsite, as well protein binding domains
as
(consensus sequences)responsible for the binding of RNA
polymerase. Eukaryoticpromoters often, but not always, contain
"TATA" boxes and "CAT" boxes. Prokaryoticpromoters contain Shine-
S Dalgarno seq uences addition to the -10 and -3S consensus
in
sequences.
An "expression control sequence" is a DNA sequence that
controls and regulates Ehe transcription and translation of another
DNA sequence. A coding sequence is "under the control" o f
transcriptional and translational control sequences in a cell when RNA
polymerase transcribes the coding sequence into mRNA, which is then
translated into the protein encoded by the coding sequence.
A "signal sequence" can be included near the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
1 S polypeptide, that communicates to the host cell to direct th a
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell before th a
protein leaves the cell. Signal sequences can be found associated with
a variety of proteins native to prokaryotes and eukaryotes.
The term "oligonucleotide", as used herein in referring to the
probe of the present invention, is defined as a molecule comprised o f
two or more ribonucleotides, preferably more than three. its exact
size will depend upon many factors which, in turn, depend upon t h a
ultimate function and use of the oligonucleotide.
The term "primer" as used herein refers to a n
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of acting
as a point of initiation of synthesis when placed under conditions i n
which synthesis of a primer extension product, which is
complementary to a nucleic acid strand, is induced, i.e., in the
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presence of nucleotides and an inducing agent such as a DNA
polymerase and at a suitable temperature and pH. The primer may b a
either single-stranded or double-stranded and must be sufficiently
long to prime the synthesis of the desired extension product in the
presence of the inducing agent. The exact length of the primer will
depend upon many factors, including temperature, source of primer
and use the method. For example, for diagnostic applications,
depending on the complexity of the target sequence, t h a
oligonucleotide primer typically contains 15-25 or more nucleotides,
although it may contain fewer nucleotides.
The primers herein are selected to be "substantially"
complementary to different strands of a particular target DNA
sequence. This means that the primers must be sufficiently
complementary to hybridize with their respective strands. Therefore,
i5 the primer sequence need not reflect the exact sequence of tha
template. For example, a non-complementary nucleotide fragment
may be attached to the 5' end of the primer, with the remainder of the
primer sequence being complementary to the strand. Alternatively,
non-complementary bases or longer sequences can be interspersed
into the primer, provided that the primer sequence has sufficient
complementarity with the sequence or hybridize therewith and
thereby form the template for the synthesis of the extension product.
A cell has been "transformed" by exogenous o r
heteroIogous DNA when such DNA has been introduced inside the cell.
The transforming DNA may or may not be integrated {covalently
linked) into the genome of the cell. In prokaryotes, yeast, a n d
mammalian cells for example, the transforming DNA may b a
maintained on an episornal element such as a plasmid. With respect
to eukaryotic cells, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that
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it is inherited by daughter cells through chromosome replication.
This stability is demonstrated by the ability of the eukaryotic cell t o
establish cell lines or clones comprised of a population of daughter
cells containing the transforming DNA. A "clone" is a population o f
cells derived from a single cell or ancestor by mitosis. A "cell line" is
a clone of a primary cell that is capable of stable growth in vitro for
many generations.
Two DNA sequences are "substantially homologous" when
at least about 75% {preferably at least about 80%, and most
preferably at Ieast about 90% or 95%) of the nucleotides match over
the defined length of the DNA sequences. Sequences that are
substantially homologous can be identified by comparing th a
sequences using standard software available in sequence data banks,
or in a Southern hybridization experiment under, for example,
stringent conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the art. See,
e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic
Acid Hybridization, supra.
A "heterolagous' region of the DNA construct is a n
identifiable segment of DNA within a larger DNA molecule that is n o t
found in association with the larger molecule in nature. Thus, when
the heterologous region encodes a mammalian gene, the gene will
usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genorne of the source organism. In another
2S example, coding sequence is a construct where the coding sequence
itself is not found in nature {e.g., a cDNA where the genomic coding
sequence contains introns, or synthetic sequences having c o d o n s
different than the native gene). Allelic variations or naturally
occurring mutational events do not give rise to a heterologaus region
3 0 of DNA as defined herein.
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The labels most commonly employed for these studies are
radioactive elements, enzymes, chemicals which fluoresce when
exposed to untraviolet Iight, and others. A number of fluorescent
materials are known and can be utilized as labels. These include, for
example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue
and Lucifer Yellow. A particular detecting material is anti-rabbit
antibody prepared in goats and conjugated with fluorescein through
an isothiocyanate.
Proteins can also be labeled with a radioactive element o r
with an enzyme. The radioactive label can be detected by any of the
currently available counting procedures. The preferred isotope may
be selected from 3H, 1~C, 32P, 3sS, ~~Cl, S~Cr, S~Co, SgCo, 59Fe, 9aY, I25I,
131I, and ~86Re.
Enzyme labels are likewise useful, and can be detected b y
any of the presently utilized colorimetric, spectrophotometric,
fluorospectrophotornetric, amperometric or gasometric techniques.
The enzyme is conjugated to the selected particle by reaction with
bridging molecules such as carbodiimides, diisocyanates,
glutaraldehyde and the like. Many enzymes which can be used i n
these procedures are known and can be utilized. The preferred are
peroxidase, (3-glucuronidase, ~i-D-glucosidase, ~3-D-galactosidase,
urease, glucose oxidase plus peroxidase and alkaline phosphatase.
U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are referred t o
by way of example for their disclosure of alternate labeling material
and methods.
As used herein, the term "host" is meant to include n o t
only prokaryotes but also eukaryotes such as yeast, plant and animal
cells. A recombinant DNA molecule or gene which encodes a 28-kDa
immunoreactive protein of Ehrlichia cahis of the present invention can
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be used to transform a host using any of the techniques commonly
known to those of ordinary skill in the art. Especially preferred is the
use of a vector containing coding sequences for a gene encoding a 28-
kDa immunoreactive protein of Ehrlichia canis of the present
invention for purposes of prokaryote transformation.
Prokaryotic hosts may include E. coli, S. tymphimurium,
Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include
yeasts such as Pichia pastoris, mammalian cells and insect cells.
In general, expression vectors containing promoter
sequences which facilitate the efficient transcription of the inserted
DNA fragment are used in connection with the host. The expression
vector typically contains an origin of replication, promoter(s),
terminator(s), as well as specific genes which are capable of providing
phenotypic selection in transformed cells. The transformed hosts can
I 5 be fermented and cultured according to means known in the art t o
achieve optimal cell growth.
The invention includes a substantially pure DNA encoding
a 28-kDa immunoreactive protein of Ehrlichia canis, a strand of which
DNA will hybridize at high stringency to a probe containing a
sequence of at least 15 consecutive nucleotides of SEQ ID Na. 1 or SEQ
ID No. 3 or SEQ ID No. 5. The protein encoded by the DNA of this
invention may share at least 80% sequence identity (preferably 85 %,
more preferably 90%, and most preferably 95%) with the amino acids
listed in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6. M o r a
preferably, the DNA includes the coding sequence of the nucleotides
of SEQ ID No. i or SEQ ID No. 3 or SEQ ID No. 5, or a degenerate
variant of such a sequence.
The probe to which the DNA of the invention hybridizes
preferably consists of a sequence of at least 20 consecutive
nucleotides, more preferably 40 nucleotides, even more preferably 5 0
I7
CA 02352466 2001-05-28
WO 00/32745 PCT/IJS99/28075
nucleotides, and most preferably 100 nucleotides or more (up t o
100%) of the coding sequence of the nucleotides listed in SEQ ID No. 1
or SEQ ID No. 3 or SEQ ID No. 5 or the complement thereof. Such a
probe is useful for detecting expression of the 28-kDa
immunoreactive protein of Ehrlichia canis in a human cell by a
method including the steps of (a) contacting mRNA obtained from the
cell with the labeled hybridization probe; and {b) detecting
hybridization of the probe with the mRNA.
This invention also includes a substantially pure DNA
containing a sequence of at least 15 consecutive nucleotides
(preferably 20, more preferably 30, even more preferably 50, a n d
most preferably all) of the region from the nucleotides listed in SEQ ID
No 1 or SEQ ID No. 3 or SEQ ID No. 5.
By "high stringency" is meant DNA hybridization and wash
conditions characterized by high temperature and low salt
concentration, e.g., wash conditions of 65°C at a salt concentration o
f
approximately 0.1 x SSC, or the functional equivalent thereof. For
example, high stringency conditions may include hybridization a t
about 42°C in the presence of about 50% formamide; a first wash a t
about 65°C with about 2 x SSC containing 1% SDS; followed by a
second wash at about 65°C with about 0.1 x SSC.
By "substantially pure DNA" is meant DNA that is not p a r t
of a milieu in which the DNA naturally occurs, by virtue of separation
{partial or total purification) of some or all of the molecules of that
milieu, or by virtue of alteration of sequences that flank the claimed
DNA. The term therefore includes, for example, a recombinant DNA
which is incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote o r
eukaryote; or which exists as a separate molecule (e.g., a cDNA or a
genomic or cDNA fragment produced by polymerase chain reaction
18
CA 02352466 2001-05-28
WO 00132745 PCT/US99/2$075
(PCR) or restriction endonuclease digestion) independent of other
sequences. It also includes a recombinant DNA which is part of a
hybrid gene encoding additional polypeptide sequence, e.g., a fusion
protein. Also included is a recombinant DNA which includes a portion
of the nucleotides listed in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No.
5 which encodes an alternative splice variant of a gene encoding a 28-
kDa immunoreactive protein of Ehrlichia canis.
The DNA may have at least about 70% sequence identity t o
the coding sequence of the nucleotides listed in SEQ ID No. 1 or SEQ ID
l 0 No. 3 or SEQ ID No. 5, preferably at least 75% (e.g. at least 80%); a n d
most preferably at least 90%. The identity between two sequences is a
direct function of the number of matching or identical positions.
When a subunit position in both of the two sequences is occupied b y
the same monomeric subunit, e.g., if a given position is occupied by
an adenine in each of two DNA molecules, then they are identical a t
that position. For example, if 7 positions in a sequence
10 nucleotides in length are identical to the corresponding positions
in a second 10-nucleotide sequence, then the two sequences have 70%
sequence identity. The length of comparison sequences will generally
be at least 50 nucleotides, preferably at least 60 nucleotides, more
preferably at least 75 nucleotides, and most preferably
100 nucleotides. Sequence identity is typically measured using
sequence analysis software (e.g., Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, WI 53705).
The present invention comprises a vector comprising a
DNA sequence coding for a which encodes a gene encoding a 28-kDa
immunoreactive protein of Ehrlichia canis and said vector is capable
of replication in a host which comprises, in operable linkage: a) a n
origin of replication; b) a promoter; and c) a DNA sequence coding
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WO 00/32745 PCT/US99/28075
for said protein. Preferably, the vector of the present invention
contains a portion of the DNA sequence shown in SEQ ID No. I or SEQ
ID No. 3 or SEQ ID No. 5.
A "vector" may be defined as a repiicable nucleic acid
construct, e.g., a plasmid or viral nucleic acid. Vectors may be a s ed
to amplify and/or express nucleic acid encoding a 28-kDa
immunoreactive protein of Ehrlichia canis. An expression vector is a
replicable construct in which a nucleic acid sequence encoding a
polypeptide is operably linked to suitable control sequences capable
of effecting expression of the polypeptide in a cell. The need for s a c h
control sequences will vary depending upon the cell selected and th a
transformation method chosen. Generally, control sequences include
a transcriptional promoter and/or enhances, suitable mRNA ribosomal
binding sites, and sequences which control the termination of
I 5 transcription and translation. Methods which are well known to th o s a
skilled in the art can be used to construct expression vectors
containing appropriate transeriptional and translational control
signals. See for example, the techniques described in Sambrook et al.,
1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring
Harbor Press, N.Y. A gene and its transcription control sequences are
defined as being "operably linked" if the transcription control
sequences effectively control the transcription of the gene. Vectors o f
the invention include, but are not limited to, plasmid vectors and viral
vectors. Preferred viral vectors of the invention are those derived
from retroviruses, adenovirus, adeno-associated virus, SV40 virus, o r
herpes viruses.
By a "substantially pure protein" is meant a protein which
has been separated from at least some of those components which
naturally accompany it. Typically, the protein is substantially pure
3 0 when it is at least 60%, by weight, free from the proteins and o th a r
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
naturally-occurring organic molecules with which it is naturally
associated in vivo. Preferably, the purity of the preparation is at least
75%, more preferably at least 90%, and most preferably at least 99%,
by weight. A substantially pure 28-kDa immunoreactive protein o f
S Ehrlichia cams may be obtained, for example, by extraction from a
natural source; by expression of a recombinant nucleic acid encoding
a 28-kDa immunoreactive protein of Ehrlichia canis; or by chemically
synthesizing the protein. Purity can be measured by any appropriate
method, e.g., column chromatography such as immunoaffinity
chromatography using an antibody specific for a 28-kDa
immunoreactive protein of Ehrlichia canis, polyacrylamide gel
electrophoresis, or HPLC analysis. A protein is substantially free o f
naturally associated components when it is separated from at least
some of those contaminants which accompany it in its natural state.
Thus, a protein which is chemically synthesized or produced in a
cellular system different from the cell from which it naturally
originates will be, by definition, substantially free from its naturally
associated components. Accordingly, substantially pure proteins
include eukaryotic proteins synthesized in E. coli, .other prokaryotes,
or any other organism in which they do not naturally occur.
In addition to substantially full-length proteins, the
invention also includes fragments {e.g., antigenic fragments} of the
28-kDa immunoreactive protein of Ehrlichia cams (SEQ ID No. 2 o r
SEQ ID No. 4 or SEQ ID No. 6). As used herein, "fragment," as applied
to a polypeptide, will ordinarily be at Ieast 10 residues, more typically
at least 20 residues, and preferably at least 30 (e.g., 50) residues in
length, but less than the entire, intact sequence. Fragments of the 2 8 -
kDa immunoreactive protein of Ehrlichia canis can be generated by
methods known to those skilled in the art, e.g., by enzymatic digestion
of naturally occurring or recombinant 28-kDa immunoreactive
21
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
protein of Ehrlichia canis, by recombinant DNA techniques using a n
expression vector that encodes a defined fragment of 28-kDa
immunoreactive protein of Ehrlichia canis, or by chemical synthesis.
The ability of a candidate fragment to exhibit a characteristic of 28-
kDa immunoreactive protein of Ehrlichia canis {e.g., binding to a n
antibody specific for 28-kDa immunoreactive protein of Ehrlichia
canis) can be assessed by methods described herein. Purified 28-kDa
immunoreactive protein of Ehrlichia cams or antigenic fragments of
28-kDa immunoreactive protein of Ehrlichia canis can be used t o
generate new antibodies or to test existing antibodies (e.g., as positive
controls in a diagnostic assay) by employing standard protocols
known to those skilled in the art. Included in this invention are
polyclonal antisera generated by using 28-kDa immunoreactive
protein of Ehrlichia canis or a fragment of 28-kDa immunoreactive
protein of Ehrlichia canis as the immunogen in, e.g., rabbits. Standard
protocols for monoclonal and polyclonal antibody production known
to those skilled in this art are employed. The monoclonal antibodies
generated by this procedure can be screened for the ability to identify
recombinant Ehrlichia canis cDNA clones, and to distinguish them
from known cDNA clones.
Further included in this invention are fragments of the 2 8 -
kDa immunoreactive protein of Ehrlichia canis which are encoded at
least in part by portions of SEQ ID No. 1 or SEQ ID No. 3 or SEQ m No.
5, e.g., products of alternative mRNA splicing or alternative protein
processing events, or in which a section of the sequence has been
deleted. The fragment, or the intact 28-kDa immunoreactive protein
of Ehrlichia canis, may be covalently linked to another polypeptide,
e.g. which acts as a label, a ligand or a means to increase antigenicity.
The phrase "pharmaceutically acceptable" refers t o
3 0 molecular entities and compositions that do not produce an allergic
22
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
or similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as a n
active ingredient is well understood in the art. Typically, s a c h
compositions are prepared as injectables, either as liquid solutions o r
suspensions; solid forms suitable for solution in, or suspension in,
liquid prior to injection can also be prepared. The preparation can
also be emulsified.
A protein may be formulated into a composition in a
neutral or salt forma Pharmaceutically acceptable salts, include the
acid addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as, f o r
example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine;
trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a
manner compatible with the dosage formulation and in such am o a n t
as is therapeutically effective. The formulations are easily
administered in a variety of dosage forms such as injectable solutions.
For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and the
liquid diluent first rendered isotonic with sufficient saline or glucose.
2S These particular aqueous solutions are especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal
administration. In this connection, sterile aqueous media which can
be employed will be known to those of skill in the art in light of the
present disclosure. For example, one dosage could be dissolved in 1
mL of isotonic NaCl solution and either added to 1000mL of
23
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th Edition,
pages 1035-1038 and 1570-1580). Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any event,
determine the appropriate dose for the individual subject.
As is well known in the art, a given polypeptide may vary
in its immunogenicity. It is often necessary therefore to couple the
immunogen (e.g., a polypeptide of the present invention) with a
carrier. Exemplary and preferred carriers are keyhole limpet
hemocyanin (KLH) and human serum albumin. Other carriers may
include a variety of lymphokines and adjuvants such as IL2, IL4, IL8
and others.
Means for conjugating a polypeptide to a carrier protein
are well known in the art and include glutaraldehyde, m -
maleimidobenzoyl-N-hydroxysuccinimide ester, carbo-diimide and
bis-biazotized benzidine, It is also understood that the peptide may
be conjugated to a protein by genetic engineering techniques that axe
well known in the art.
As is also well known in the art, immunogenicity to a
particular immunogen can be enhanced by the use of non-specific
stimulators of the immune response known as adjuvants. Exemplary
and preferred adjuvants include complete BCG, Detox, {RIBI,
Immunochem Research Inc.) ISCOMS and aluminum hydroxide
adjuvant {Superphos, Biosector).
As used herein the term "complement" is used to define
the strand of nucleic acid which will hybridize to the first nucleic acid
sequence to form a double stranded molecule under stringent
conditions. Stringent conditions are those that allow hybridization
between two nucleic acid sequences with a high degree of homology,
24
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
but precludes hybridization of random sequences. For example,
hybridization at low temperature and/or high ionic strength is t a r m a d
low stringency and hybridization at high temperature andlor low ionic
strength is termed high stringency. The temperature and ionic
strength of a desired stringency are understood to be applicable t o
particular probe lengths, to the length and base content of the
sequences and to the presence of formamide in the hybridization
mixture.
As used herein, the term "engineered" or "recombinant"
cell is intended to refer to a cell into which a recombinant gene, s a c h
as a gene encoding an Ehrlichia chaffeensis antigen has been
introduced. Therefore, engineered cells are distinguishable from
naturally occurring cells which do not contain a recombinantly
introduced gene. Engineered cells are thus cells having a gene o r
genes introduced through the hand of man. Recombinantly
introduced genes will either be in the form of a cDNA gene, a copy o f
a genomic gene, or will include genes positioned adjacent to a
promoter not naturally associated with the particular introduced
gene. In addition, the recombinant gene may be integrated into the
host genome, or it may be contained in a vector, or in a bacterial
genome transfected into the host cell.
The following examples are given for the purpose o f
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
Ehrlichiae and Purificati~~
Ehrlichia canis (Florida strain and isolates Dernon, DJ,
Jake, and Fuzzy) were provided by Dr. Edward Breitschwerdt, (College
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28U75
of Veterinary Medicine, North Carolina State University, Raleigh, NC).
E. canis (Louisiana strain) was provided by Dr. Richard E. Corstvet
(School of Veterinary Medicine, Louisiana State University, Baton
Rouge, LA) and E. canis (Oklahoma strain) was provided by Dr.
S - Jacqueline Dawson {Centers for Disease Control and Prevention,
Atlanta, GA). Propagation of ehrlichiae was performed in DH82 cells
with DMEM supplemented with 10% bovine calf serum and 2 mM L-
glutaminc at 37°C. The intracellular growth in DH82 cells was
monitored by presence of E cams morulae using general cytologic
staining methods. CeIIs were harvested when 100% of the cells were
infected with ehrlichiae and were then pelleted in a centrifuge a t
17,000 x g for 20 min. Cell pellets were disrupted with a Braun-Sonic
2000 sonicator twice at 40W for 30 sec on ice. Ehrlichiae were
purified as described previously (Weiss et al., 1975). The lysate was
1S loaded onto discontinuous gradients of 42%-36%-30% renografin, and
centrifuged at 80,000 x g for 1 hr. Heavy and light bands containing
ehrlichiae were collected and washed with sucrose-phosphate-
glutamate buffer (SPG, 218 mM sucrose, 3.8 mM KHZP04, 7.2 m M
KZHP04, 4.9 mM glutamate, pH 7.0) and pelleted by centrifugation.
Nzcleic Acid Pr~arati~
Ehrlichia cams genomic DNA was prepared b y
2S resuspending the renografin-purified ehrlichiae in 600 p,l of i0 mM
Tris-HCl buffer (pH ?.S) with 1% sodium dodecyl sulfate (SDS, w/v)
and i00 ng/ml of proteinase K as described previously (McBride et al.,
1996). This mixture was incubated for 1 hr at S6° C, and the nucleic
acids were extracted twice with a mixture o f
phenol/chloroformlisoamyl alcohol {24:24:1). DNA was pelleted by
26
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
absolute ethanol precipitation, washed once with 70% ethanol, dried
and resuspended in l OmM Tris (pH 7.S). Plasmid DNA was purified b y
using High Pure Plasmid Isolation Kit (Boehringer Mannheim,
Indianapolis, IN), and PCR products were purified using a QIAquick PCR
S Purification Kit {Qiagen, Santa Clarita, CA).
PC'_R Amnlifica inn of the F cr~nis 2A-kIW nrn in CTPn,~e
IO Regions of the E, canis ECa28-I gene selected for PCR
amplification were chosen based on homology observed (>90%) in the
consensus sequence generated from Jotun-Hein aligorithm alignment
of E. chaffeensis p28 and Cowdria ruminantium map-I genes. Forward
primer 793 (S-GCAGGAGCTGTTGGTTACTC-3') (SEQ ID NO. 16) a n d
1 S reverse primer 1330 (S'-CCTTCCTCCAAGTTCTATGCC-3') (SEQ ID NO.
17) corresponded to nucleotides 313-332 and 823-843 of C.
ruminantium MAP-1 and 307-326 and 834-814 of E. chaffeensis P28.
E. canis (a North Carolina isolate, Jake) DNA was amplified with
primers 793 and 1330 with a thermal cycling profile of 9S°C for 2
20 min, and 30 cycles of 9S°C for 30 sec, 62° C for 1 min,
72°C for 2 min
followed by a 72°C extension for IO min and 4°C hold. PCR
products
were analyzed on 1% agarose gels. This amplified PCR product was
sequenced directly with primers 793 and 1330.
Primers specific for ECa28SA2 gene designated 46f (5'
2S ATATACTTCCTACCTAATGTCTCA-3', SEQ ID No. i 8) and primer 13 3 0
(SEQ ID No. 17) were used to amplify the targeted region. The
amplified product was gel purified and cloned into a TA cloning vector
{Invitrogen, Santa Clarita, CA). The clone was sequenced
bidirectionally with primers: M13 reverse from the vector, 46f,
30 ECa28SA2 {S'-AGTGCAGAGTCTTCGGTTTC-3', SEQ ID No. 19), ECa5.3
27
CA 02352466 2001-05-28
WO 00132745 PCTILJS99128075
(5'-GTTACTTGCGGAGGACAT-3', SEQ ID No. 20). DNA was amplified
with a thermal cycling profile of 95°C for 2 min, and 30 cycles of
95°C
for 30 sec, 48°C for 1 min, 72°C for 1 min followed by a
72°C
extension for 10 min and 4°C hold.
~~g Llnkn~wn 5' ,~,~ Regions of ~k]'~e F.Ca2f3- I C'rene
The full length sequence of ECa28-1 was determined using
a Universal GenorneWalker Kit (CLONTECH, Palo Alto, CA) according t o
the protocol supplied by the manufacturer. Genomic E. canis (Jake
isolate) DNA was digested completely with five restriction enzymes
(DraI, EcoRV, PvuII, ScaI, StuI) which produce blunt-ended DNA. An
adapter (APl) supplied in the kit was ligated to each end of E. cams
DNA. The genomic libraries were used as templates to find the
unknown DNA sequence of the ECa28-1 gene by PCR using a primer
complementary to a known portion of the ECa28-1 sequence and a
primer specific for the adapter AP1. Primers specific for ECa28-1 a s a d
for genome walking were designed from the known DNA s a q a a n c a
derived from PCR amplification of ECa28-1 with primers 793 {SEQ ID
NO. 16) and 1330 (SEQ. ID NO. 17). Primers 394 (5'-
GCATTTCCACAGGATCATAGGTAA-3'; nucleotides 687-7I0, SEQ ID NO.
2 I ) arid 394C (5'-TTACCTATGATCCTGT GGAAATGC-3; n a c 1 a o ti d a s
710-687, SEQ ID NO. 22) were used in conjunction with supplied
primer APl to amplify the unknown 5' and 3' regions of the ECa28-1
gene by PCR. A PCR product corresponding to the 5' region of the
ECa28-1 gene amplified with primers 3940 and AP1 {2000-bp) w,as
sequenced unidirectionally with primer 793C (5'-GAGTA
ACCAACAGCTCCTGC-3', SEQ ID No. 23). A PCR product corresponding
28
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99128075
to the 3' region of the ECa28-1 gene amplified with primers 394 a n d
APl (580-bp) was sequenced bidirectionally with the same primers.
Noncoding regions on the 5' and 3' regions adjacent to the open
reading frame were sequenced, and primers EC280M-F (5'-
TCTACTTTGCACTTCC ACTATTGT-3', SEQ ID NO. 24) and EC280M-R ( 5 ' -
ATTCTITTGCCACTATTT TTCTTT-3', SEQ ID NO. 25) complementary t o
these regions were designed in order to amplify the entire ECa28-1
gene.
E~ADZELE.~
y .~ncing ~f E.. rani.c isolates
DNA was sequenced with an ABI Prism 377 DNA Sequencer
{Perkin- Elmer Applied Biosystems, Foster City, CA). The entire Eca28
1 genes of seven E. canis isolates (four from North Carolina, and o n a
each from Oklahoma, Florida, and Louisiana) were amplified by PCR
with primers EC280M-F (SEQ ID No. 24) and EC28OM-R (SEQ ID No.
25) with a thermal cycling profile of 95°C fox 5 minutes, and 30 cycles
of 95°C for 30 seconds, 62°C for 1 minutes, and 72°C for
2 minutes
and a 72°C extension for 10 minutes. The resulting PCR products were
bidirectionally sequenced with the same primers.
f"'lons~lg and .xnregsion c~ F.. rani.c F. .a28-1
The entire E. canis ECa28-1 gene was PCR-amplified with
primers-EC280M-F and EC280M-R and cloned into pCR2.l-TOPO TA
cloning vector to obtain the desired set of restriction enzyme cleavage
sites (Invitrogen, Carlsbad, CA). The insert was excised from pCR2.1-
29
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
TOPO with BstX 1 and ligated into pcDNA 3.1 eukaryotic expression
vector {Invitrogen, Carlsbad, CA} designated pcDNA3.l/EC28 for
subsequent studies. The pcDNA3.l/EC28 plasmid was amplified, a n d
the gene. was excised with a KpnI-XbaI double digestion and
directionally ligated into pThioHis prokaryotic expression vector
(Invitrogen, Carlsbad, CA). The clone (designated pThioHis/EC28)
produced a recombinant thioredoxin fusion protein in Escherichia coli
BL21. The recombinant fusion protein was crudely purified in th a
insoluble phase by centrifugation. The control thioredoxin fusion
protein was purified from soluble cell lysates under native conditions
using nickel-NTA spin columns (Qiagen, Santa Clarita, CA).
W~~ rn Im~n~h1 of ,u.al;rsi ss
Recombinant E. canis ECa28-1 fusion protein was subjected
to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4-15% Tris-
HCl gradient gels (Bio-Rad, Hercules, CA) and transferred to pure
nitrocellulose (Schleicher & Schuell, Keene, NH} using a semi-dry
transfer cell (Bio-Rad, Hercules, CA). The membrane was incubated
with convalescent phase antisera from an E. canis-infected dog diluted
1:5000 for 1 hour, washed, and then incubated with an anti-canine IgG
(H & L) alkaline phosphatase-conjugated affinity-purified secondary
antibody at 1:1000 for I hour (Kirkegaard & Perry Laboratories,
Gaithersburg, MD). Bound antibody was visualized with 5-bromo-4-
chloro-3-indolyl phosphatelnitroblue tetrazolium (BCIP/NBT)
substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
30
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
~nuthern ~IQt Analysis
To determine if multiple genes homologous to the ECa28-I
gene were present in the E. canis genome, a genomic Southern blot
analysis was performed using a standard procedure (Sambrook et al.
1989). E. canis genomic DNA digested completely with each of th a
restriction enzymes BanII, EcoRV, HaeII, KpnI and SpeI, which do n o t
cut within the ECa28-1 gene, and AseI which digests ECa28-I a t
nucleotides 34, 43 and 656. The probe was produced by ~ PGR
amplification with primers EC284M-F and EC284M-R and digoxigenin
{DIG)-labeled deoxynucleotide triphosphates {dNTPs) (Boehringer
Mannheim, Indianapolis, IN) and digested with AseI. The digested
probe (566-bp} was separated by agarose gel electrophoresis, gel-
purified and then used for hybridization. The completely digested
genomic E. canis DNA was electrophoresed and transferred to a nylon
membrane (Boehringer Mannheim, Indianapolis, IN} and hybridized a t
40°C for 16 hr with the ECa28-1 gene DIG-labeled probe in DIG Easy
Hyb buffer according to the manufacturer's protocol (Boehringer
Mannheim, Indianapolis, IN). Bound probe was detected with a anti-
DIG alkaline phosphatase-conjugated antibody and a luminescent
substrate (Boehringer Mannheim, Indianapolis, IN) and exposed t o
BioMax scientific imaging film (Eastman Kodak, Rochester, NY).
2 5 EXA P~dILF _9
~n ~ .n . . Anal~~.c_L an C'.nmnarasinn
E. chaffeensis p28 and C. rumi~cantium map-1 DNA
sequences were obtained from the National Center of Biotechnology
information (NCBI) (World Wide Web site at URL:
31
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
http://www.ncbi.nlm.nih.gov/Entrez). Nucleotide and deduced amino
acid sequences, and protein and phylogenetic analyses were
performed with LASI~GINE software (DNASTAR, Inc., Madison, WI).
Analysis of post-translational processing was performed by the
method of McGeoch and von Heijne for signal sequence recognition
using the PSORT program (McGeoch, 1985; von Heijne, 1986) (World
Wide Web site at URL: PRIVATE HREF "http:l/www.imcb.osaka-
u.ac.jp/nakai/form.htm", MACROBUTTON HtmlResAnchor
http:/lwww.imcb.osaka-u.ac.jp/nakai/form.htm).
GenBank accession numbers for nucleic acid and amino
acid sequences of the E. canis ECa28-1 genes described in this study
are: Jake, AF082744; Louisiana, AF082745; Oklahoma, AF082746;
Demon, AF082747; DJ, AF082748; Fuzzy, AF082749; Florida,
AF082750.
Sequence analysis of ECa28-1 from seven different strains
of E. canis was performed with primers designed to amplify the entire
gene. Analysis revealed the sequence of this gene was conserved
among the isolates from North Carolina (four), Louisiana, Florida a n d
Oklahoma.
PC'R Amnlifica tOll~SlOnin~~llPnrttl~ and Fxnre~~i~n ~f FC'n~R 1
Alignment of nucleic acid sequences from E. chaffeensis
p28 and Cowdria ruminantium map-1 using the Jotun-Hein aligorithm
produced a consensus sequence with regions of high homology
(>90%). These homologous regions (nucleotides 313-332 and 823-
843 of C. ruminantium map-l; 307-326 and 814-834 of E. chaffeensis
p28) were targeted as primer annealing sites for PCR amplification.
PCR amplification of the E. canis ECa28-I and E. chaffeensis p28 gene
32
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was accomplished with primers 793 and 1330, resulting in a 518-by
PCR product. The nucleic acid sequence of the E. canis PCR product
was obtained by sequencing the product directly with primers 793 and
1330. Analysis of the sequence revealed an open reading frame
S encoding a protein of 170 amino acids, and alignment of the 518-by
sequence obtained from PCR amplification of E. canis with the DNA
sequence of E. chaffeensis p28 gene revealed a similarity greater than
70°l0, indicating that the genes were homologous. Adapter PCR with
primers 394 and 793C was performed to determine the 5' and 3 '
segments of the sequence of the entire gene. Primer 394 p r o d a c a d
four PCR products (3-kb, 2-kb, 1-kb, and 0.8-kb), and the 0.8-by
product was sequenced bidirectionally using primers 394 and AP1.
The deduced sequence overlapped with the 3' end of the 518-by
product, extending the open reading frame 12-by to a termination
codon. An additional 625-by of non-coding sequence at the 3' end o f
the ECa28-1 gene was also sequenced. Primer 394C was used t o
amplify the 5' end of the ECa28-1 gene with supplied primer AP1.
Amplification with these primers resulted in three PCR products (3.3,
3-kb, and 2-kb). The 2-kb fragment was sequenced unidirectionally
with primer 793C. The sequence provided the putative start codon o f
the ECa28-1 gene and completed the 834-by open reading frame
encoding a protein of 278 amino acids. An additional 144-by o f
readable sequence in the 5' noncoding region of the ECa28-I gene was
generated. Primers EC280M-F and EC280M-R were designed from
complementary non-coding regions adjacent to the ECa28-1 gene.
The PCR product amplified with these primers was
sequenced directly with the same primers. The complete DNA
sequence (SE(~ ID NO. 1) for the E. canis ECa28-I gene is shown in
Figure 1. The ECa28-1 PCR fragment amplified with these primers
contained the entire open reading frame and 17 additional amino
33
CA 02352466 2001-05-28
WO 00/32745 PCT1US99/28075
acids from the S' non-coding primer region. The gene was
directionally subcloned into pThioHis expression vector, and E. coli
(BL2I) were transformed with this construct. The expressed ECa28-1-
thioredoxin fusion protein was insoluble. The expressed protein h ad
S an additional 114 amino acids associated with the thioredoxin, S
amino acids for the enterokmase recognition site, and 32 amino acids
from the multiple cloning site and S' non-coding primer region at the
N-terminus. Convalescent-phase antiserum from an E. canis infected
dog recognized the expressed recombinant fusion protein, but did n o t
l0 react with the thioredoxin control (Figure 2).
~eq~~t~~~ce H~mologT
Z 5 The nucleic acid sequence of ECa28-1 (834-bp) and the E
chaffeensis omp-I family of genes including signal sequences (ECa28-
1, omp-lA, B, G, D, E, and F) were aligned using the Clustal method t o
examine homology between these genes (alignment not shown).
Nucleic acid homology was equally conserved (68.9%) between ECa28-
20 l, and E. chaffeensis p28 and omp-1F. Other putative outer
membrane protein genes in the E. chaffeensis omp-1 family, omp-1 D
(68.2%), omp-lE (66.7%), omp-1C (64.1%), Cowdria ruminantium
map-I (61.8%), E. canis 28-kDa protein 1 gene (60%) and 28-kDa
protein 2 gene (partial) {S9.S%) were also homologous to ECa28-1. E
25 chaffeensis omp-IB had the least nucleic acid homology {45:1%) with
E.Ca28-1.
Alignment of the predicted amino acid sequences o f
ECa28-1 (SEQ ID NO. 2) and E: chaffeensis P28 revealed amino acid
substitutions resulting in four variable regions (VR). Substitutions o r
30 deletions in the amino acid sequence and the locations of variable
34
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WO 00/32745 PCT/US99I28075
regions of ECa28-1 and the E. chaffeensis OMP-1 family were identified
(Figure 3}. Amino acid comparison including the signal peptide
revealed that ECa28-1 shared the most homology with OMP-1F (68%)
of the E. chaffeensis OMP-1 family, followed by E. chaffeensis P28
(65.5%), OMP-lE (65.1%), OMP-1D {62.9%), OMP-1C (62.9%),
Cowdria runainantium MAP-1 (59.4%), E. canis 28-kDa protein 1
(55.6%) and 28-kDa protein 2 (partial) (53.6%), and OMP-1B
(43.2%). The phylogenetic relationships based on amino acid
sequences show that ECa28-1 and C. ruminantium MAP-1,
chaffeensis OMP-1 proteins, and E. cams 28-kDa proteins 1 and 2
(partial) are related (Figure 4).
Predicted ~nrfa . . Prnhahilit?~and TmmunnrPartivitv
Analysis of E. canis ECa28-1 using hydropathy amd
hydrophilicity profiles predicted surface-exposed regions on ECa28-1
(Figure 6). Eight major surface-exposed regions consisting of 3 to 9
amino acids were identified on ECa28-1 and were similar to the profile
of surface-exposed regions on E. chaffeensis P28 {Figure 6}. Five o f
the larger surface-exposed regions on ECa28-1 were located in the N-
terminal region of the protein. Surface-exposed hydrophilic regions
were found in all four of the variable regions of ECa28-1. Ten T-cell
motifs were predicted in the ECa28-1 using the Rothbard-Taylor
2S aligorithm (Rothbard and Taylor, 1988), and high antigenicity of the
ECa28-1 was predicted by the Jameson-Wolf antigenicity aligorithm
(Figure 6) (Jameson and Wolf, 1988). Similarities in antigenicity and
T-cell motifs were observed between ECa28-1 and E. chaffeensis P28.
3S
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WO 00/32745 PCT/U599/28075
Detection of Homoloøonc Cienomis C'~cnie-5 cf ~FC'a2R 1 C'T n
Genomic Southern blot analysis of E. canis DNA completely
digested independently with restriction enzymes BanII, EcoRV, HaeII,
Kpni, SpeI, which do not have restriction endonuclease sites in the
ECa28-1 gene, and AseI, which has internal restriction endonuclease
sites at nucleotides 34, 43 and 656, revealed the presence of at least
three homologous ECa28-1 gene copies (Figure 5). Although ECa28-1
has internal Ase I internal restriction sites, the DIG-labeled probe used
in the hybridization experiment targeted a region of the gene within a
single DNA fragment generated by the AseI digestion of the gene.
Digestion with AseI produced 3 bands (approximately 566-bp, 850 -
bp, and 3-kb) that hybridized with the ECa28-1 DNA probe indicating
the presence of multiple genes homologous to ECa28-1 in the genome.
Digestion with EcoRV and SpeI produced two bands that hybridized
with the ECa28-1 gene probe.
Identification of 2R-kT~a Prnt in (''TPn I~~
Specific primers designated ECaSA3-2 (5'-CTAGGATTA
GGTTATAGTATAAGTT-3', SEQ ID No. 26) corresponding to regions
within ECa28SA3 and primer 793C (SEQ ID No. 23) which anneals to a
region with ECa28-1 were used to amplify the intergenic region
between gene SA3 and ECa28-1. The 800-by product was sequenced
with the same primers. DNA was amplified with a thermal cycling
profile of 95°C for 2 min, and 30 cycles of 95°C fox 30 sec,
50°C for 1
min, 72°C for 1 min followed by a 72°C extension for 10 min and
4°C
hold.
36
CA 02352466 2001-05-28
WO 00l3274S f'CT/US99/28075
P_C'.R mnlific~tion cf 2f~-kDa Pro ein C; .n .s and Tdentificatinn ~f h a
Mtlti.pje yen . .ocLs
In order to specifically amplify possible unknown genes
downstream of ECa28SA2, primer 4bf specific for ECa28SA2, a n d
primer 1330 which targets a conserved region on the 3' end of ECa28-
1 gene were used for amplification. A 2-kb PCR product was amplified
with these primers that contained 2 open reading frames. The first
open reading frame contained the known region of gene, ECaSA2, and
a previously unsequenced 3' portion of the gene. Downstream from
ECaSA2 an additional non identical, but homologous 28-kDa protein
gene was found, and designated ECa28SA3. The two known loci were
joined by amplification with primer SA3-2 specific for the 3' end o f
ECa28SA3 gene was used in conjunction with a reverse primer 793C,
which anneals at 5' end of ECa28-1. An 800-by PCR product was
amplified which contained the 3' end of Eca28SA3, the intergenic
region between ECa28SA3 and ECa28-I (28NC3) and the 5' end o f
Eca28-1, joining the previously separate loci (Figure 8). The 849-by
open reading frame of ECa28SA2 encodes a 283 amino acid protein,
and ECa28SA3 has an 840-by open reading frame encoding a 2 8 0
amino acid protein. The intergenic noncoding region between
ECa28SA3 and ECa28-1 was 345-by in length (Figures 7 and 8)
2 5 EXAMP_L L
jyLCleic and Amino Ar:~ Homolouv
The nucleic and amino acid sequences of all five E. canis
28-kDa protein genes were aligned using the Clustal method t o
examine the homology between these genes. The nucleic acid
37
CA 02352466 2001-05-28
WO 00/32745 PCT/US99128075
homology ranged from 58 to 75% and a similar amino acid homology
of ranging from 67 to 72% was observed between the E. canis 28-kDa
protein gene members (Figure 9).
EXA,D~ELE..1Z
Transcriptional Promo Pr ~gi~nc
The intergenic regions between the 28-kDa protein genes
were analyzed for promoter sequences by comparison with consensus
Escherichia coli promoter regions and a promoter from E. chaffeensis
(Yu et al., 1997; McClure, 1985).
Putative promoter sequences including RBS, -10 and - 3 5
regions were identified in 4 intergenic sequences corresponding t o
genes ECa28SA2, ECa28SA3, ECa28-l, and ECa28-2 (Figure 10). The
upstream noncoding region of ECa28SAl is not known and was n o t
analyzed.
N-Terminal Signal ~ean_encP
The amino acid sequence analysis revealed that entire
E.canis ECa28-1 has a deduced molecular mass of 30.5-kDa and the
entire ECa28SA3 has a deduced molecular mass of 30.7-kDa. Both
proteins have a predicted N-terminal signal peptide of 23 amino acids
(MNCKKILITTALMSLMYYAPSIS, SEQ ID No. 27), which is similar to that
predicted for E. chaffeensis P28 (IVINYKI~.,TTSALISLISSLPGV SFS, SEQ ID
NO. 28), and the OMP-1 protein family (Yu et al., 1998; Ohashi et al.,
1998b). A preferred cleavage site for signal peptidases (SIS; Ser-X-
Ser) (Oliver, 1985) is found at amino acids 21, 22, and 23 of ECa28-1.
An additional putative cleavage site at amino acid position 2 5
38
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
(MNCKKILITTALISLMYSIPSISSFS, SEQ ID NO. 29) identical to the
predicted cleavage site of E. chaffeensis P28 (SFS) was also present,
and would result in a mature ECa28-1 with a predicted molecular m a s s
of 27.7-kDa. Signal cleavage site of the previously reported partial
sequence of ECa28SA2 is predicted at amino acid 30. However, signal
sequence analysis predicted that ECa28SA1 had an uncleavable signal
sequence.
SLmmarv
Proteins of similar molecular mass have been identified
and cloned from multiple rickettsial agents including E. canis, E
chaffeensis, and C. ru»ainantium (Reddy et al., 1998; Jongejan et al.,
1993; Ohashi et al., 1998}. A single locus in Ehrdichia chaffeensis with
6 homologous p28 genes, and 2 loci in E. canis, each containing some
homologous 28-kDa protein genes have been previously described.
The present invention demonstrated the cloning,
expression and characterization of genes encoding a mature 28-kDa
protein of E. canis that are homologous to the omp-1 multiple gene
family of E. chaffeensis and the C. ruminantium map-I gene. Two new
28-kDa protein genes were identidfied, Eca28-1 and ECa28SA3.
Another E.canis 28-kDa protein gene, ECa28SA2, partially sequenced
previously (Reddy et al., 1998}, was sequenced completely in the
present invention. Also disclosed is the identification and
characterization of a single locus in E.canis containing ail five E.canis
28-kDa protein genes.
The E.canis 28-kDa protein are homologous to
E.chaffeensis OMP-i family and the MAP-1 protein of C. rumanintium.
The most homologous E. cards 28-kDa proteins (ECa28SA3, ECa28-1
and ECa28-2) are sequentially arranged in the locus. Homology o f
these proteins ranged from 67.5% to 72.3%. Divergence among these
28-kDa proteins was 27.3% to 38.6%. E. canis 28-kDa proteins
39
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
ECa28SA1 and ECa28SA2 were the least homologous with homology
ranging from 50.9% to 59.4% and divergence of 53.3 to 69.9%.
Differences between the genes lies primarily in the four hypervariable
regions and suggests that these regions are surface exposed a n d
subject to selective pressure by the immune system. Conservation o f
ECa28-I among seven E. canis isolates has been reported (McBride a t
al., 1999), suggesting that E.canis may be clonal in North America.
Conversely, significant diversity of p28 among E. chaffeensis isolates
has been reported (Yu et al., 1998).
All of the E. canis 28-kDa proteins appear to be post
translationally processed from a 30-kD protein to a mature 28-kD
protein. Recently, a signal sequence was identified on E. chaffeensis
P28 {Yu et al., 1998), and N-terminal amino acid sequencing has
verified that the protein is post-translationally processed resulting i n
cleavage of the signal sequence to produce a mature protein (Ohashi
et al., 1998). The leader sequences of OMP-1F and OMP-lE have also
been proposed as leader signal peptides (Ohashi et ad., 1998). Signal
sequences identified on E. chaffeensis OMP-1F, OMP-lE and P28 are
homologous to the leader sequence of E. canis 28-kDa protein.
Promoter sequences for the p28 genes have not been determined
experimentally, but putative promoter regions were identified b y
comparison with consensus sequences of the RBS, -10 and -35
promoter regions of E. coli and other ehrlichiae (Yu et al., 1997;
McClure, 1985). Such promoter sequences would allow each gene t o
potentially be transcribed and translated, suggesting that these genes
may be differentially expressed in the host. Persistence of infection in
dogs may be related to differential expression of p28 genes resulting
in antigenic changes in vivo, thus allowing the organism to evade tha
immune response.
CA 02352466 2001-05-28
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The E. canis 28-kda protein genes were found to exhibit
nucleic acid and amino acid sequence homology with the E
chaffeensis omp-1 gene family and C. ruminantiunz nzap-1 gene.
Previous studies have identified a 30-kDa protein of E canis that
reacts with convalescent phase antisera against E chaffeensis, but was
believed to be antigenically distinct (Rikihisa et al., 1994}. Findings
based on comparison of amino acid substitutions in four variable
regions of E. canis 28-kDa proteins support this possibility. Together
these findings also suggest that the amino acids responsible for th a
antigenic differences between E. canis and E, chaffeensis P28 are
located in these variable regions and are readily accessible to the
immune system. It was reported that immunoreactive peptides were
located in the variable regions of the 28-kDa proteins of C.
ruminantium, E. chaffeensis and E. canis (Reddy et al., 1998). Analysis
of E. canis and E. chaffeensis P28 revealed that all of the variable
regions have predicted surface-exposed amino acids. A study in dogs
demonstrated lack of cross protection between E. canis and E
chaffeensis (Dawson and Ewing, 1992). This observation may b a
related to antigenic differences in the variable regions of P28 as well
as in other immunologically important antigens of these ehrlichial
species. Another study found that convalescent phase human antisera
from E. chaffeensis-infected patients recognized 29/28-kDa proteins}
of E. chaffeensis and also reacted with homologous proteins of E. canis
(Chen et al., 1997). Homologous and crossreactive epitopes on the E
cams 28-kDa protein and E chaffeensis P28 appear to be recognized
by the immune system.
E. canis 28-kDa proteins may be important
immunoprotective antigens. Several reports have demonstrated that
the 30-kDa antigen of E canis exhibits strong immunoreactivity
(Rikihisa et al., 1994; Rikihisa et al., 1992). Antibodies in
41
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
convalescent phase antisera from humans and dogs have consistently
reacted with proteins in this size range from E. chaffeensis and E
canis, suggesting that they may be important immunoprotective
antigens (Rikihisa et al., 1994; Chen et al., 1994; Chen et al., I997). In
addition, antibodies to 30, 24 and 21-kDa proteins developed early i n
the immune response to E. canis (Rikihisa et al., 1994; Rikihisa et al.,
1992), suggesting that these proteins may be especially important in
the immune responses in the acute stage of disease. Recently, a family
of homologous genes encoding outer membrane proteins with
I 0 molecular masses of 28-kDa have been identified in E. chaffeensis, a n d
mice immunized with recombinant E. chaffeensis P28 appeared t o
have developed immunity against homologous challenge (Ohashi a t
al., 1998). The P28 of E. chaffeensis has been demonstrated to b a
present in the outer membrane, and immunoelectron microscopy has
i 5 localized the P28 on the surface on the organism, and thus suggesting
that it may serve as an adhesin {Ohashi et al., 1998). It is likely that
the 28-kDa proteins of E. canis identified in this study have the s am a
location and possibly serve a similar function.
Comparison of ECa28-1 from different strains of E. canis
20 revealed that the gene is apparently completely conserved. Studies
involving E. chaffeensis have demonstrated immunologic and
molecular evidence of diversity in the ECa28-1. Patients infected with
E. chaffeensis have variable immunoreactivity to the 29/28-kDa
proteins, suggesting that there is antigenic diversity (Chen et al.,
25 1997). Recently molecular evidence has been generated to support
antigenic diversity in the p28 gene from E. chaffeensis {Yu et al.,
1998). A comparison of five E. chaffeensis isolates revealed that two
isolates (Sapulpa and St. Vincent) were 100% identical, but three
others (Arkansas, Sax, 9IHE17) were divergent by as much as 13.4%
30 at the amino acid Level. The conservation of ECa28-I suggests that E
42
CA 02352466 2001-05-28
WO 00132745 PCTIUS99/28075
canis strains found in the United States may be genetically identical,
and thus E. canis 28-kDa protein is an attractive vaccine candidate f o r
canine ehrlichiosis in the United States. Further analysis of E. canis
isolates outside the United States may provide information regarding
the origin and evolution of E. canis. Conservation of the 28-kDa
protein makes it an important potential candidate for reliable
serodiagnosis of canine ehrlichiosis.
The role of multiple homologous genes is not known a t
this point; however, persistence of E.canis infections in dogs could
conceivably be related to antigenic variation due to variable
expression of homologous 28-kDa protein genes, thus enabling E
canis to evade immune surveillance. Variation of n2sp-3 genes in A.
marginale is partially responsible for variation in the MSP-3 protein,
resulting in persistent infections (Alleman et al., 1997). Studies t o
examine 28-kDa protein gene expression by E. canis in acutely and
chronically infected dogs would provide insight into the role of the
28-kDa protein gene family in persistence of infection.
The following references were cited herein.
Alleman A.R., et al., (1997) Infect Irnmun 65: i56-163.
Anderson B.E., et al., (1991) J Clin Microbiol 29: 2838-2842.
Anderson B.E., et al., ( 1992) Int J Syst Bacteriol 42: 299-302.
Brouqui P., et al., ( 1992) J Clin Microbiol 30: 1062-1066.
Chen S.M., et al., ( 1997) Clin Diag Lab Immunol 4: 731-735.
Chen S.M., et al., (1994} Am J Trop Med Hyg 50: 52-58.
Dawson J.E., et aL, (1992) Am J Vet Res 53: 1322-1327.
Dawson J.E., et al., (1991) J Infect Dis 1b3: 564-567.
Donatien, et al., (1935) Bull Soc Pathol Exot 28: 418-9.
Swing, (1963) J Am Vet Med Assoc 143: 503-6.
Groves M.G., et al., ( 1975) Am J Vet Res 36: 937-940.
Harrus S., et al., (1998) J Clisa Microbiol 36: 73-76.
43
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
Jameson B.A., et al., ( 1988) CABIOS 4: 181-186.
Jongejan F., et al., (1993) Rev Elev Med Vet Pays Trop 46: 145-152.
McBride J.W., et al., {I996) J Vet Diag Invest 8: 441-447.
McBride; et al.,. ( 1999) Clin Diagn Lab Immunol.; (In press).
McCIure, (1985} Ann Rev Biochem 54: I71-204.
McGeoch D.J. (1985) Virus Res 3: 271-286.
Nyindo M., et al., {1991) Am J Vet Res 52: 1225-1230.
Nyindo, et al., (1971) Am J Vet Res 32: 1651-58.
Ohashi, et al., (1998) Infect Immun 66: 132-9.
Ohashi, et al., (1998} J Clin Microb 36: 2671-80
Reddy, et al., ( 1998) Biochem Biophys Res Comm 247: 636-43.
Rikihisa, et al., (1994) J Clin Microbiol32: 2107-12.
Rothbard J.B., et al., (1988) The EMBO J7: 93-100.
Sambrook J., et al., ( 1989) In Molecular Cloning: A Laboratory
I5 Manual. Cold Spring Harbor: Cold Spring Harbor Press.
Troy G.C., et al., (1990} Canine ehrlichiosis. In Infectious diseases o f
the dog and cat . Green C.E. (ed). Philidelphia: W.B. Sauders Co.
von Heijne, ( 1986) Nucl Acids Res I4: 4683-90.
Walker, et al., ( 1970) J Am Vet Med Assoc 157: 43-55.
Weiss E., et al., (1975) Appl Microbiol30: 456-463.
Yu, et al., (1997) Gene 184: 149-154.
Yu, et al.; ( 1998) J. Clin. Microbiol. (In press).
Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which tha
invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was individually indicated to be incorporated b y
reference.
One skilled in the art will readily appreciate that tha
present invention is well adapted to carry out the objects and obtain
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CA 02352466 2001-05-28
WO 00/32745 PCT/1JS99128075
the ends , and advantages mentioned, as well as those inherent therein.
The present examples along with the methods, procedures,
treatments, molecules, and specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the art
which are encompassed within the spirit of the invention as defined b y
the scope of the claims.
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
SEQUENCE LISTING
<110> Walker, David H.
McBride, Jere W.
Yu, Xue-Jie
<120> Homologous 28-Kilodalton Immunodominant Protein
Genes of Ehrlichia can.is and Uses Thereof
<130> D6152PCT
<141> 1999-11-30
<150> 09/261,358
<151> 1999-03-03
<160> 33
<210> 1
<211> 1607
<212> DNA
<213> Ehrlichia cams
<220>
<223> nucleic acid sequence of ECa28-1
<400> 1
attttattta ttaccaatct tatataatat attaaatttc tcttacaaaa 50
atctctaatg ttttatacct aatatatata ttctggcttg tatctacttt 100
gcacttccac tattgttaat ttattttcac tattttaggt gtaatatgaa 150
ttgcaaaaaa attcttataa caactgcatt aatatcatta atgtactcta 200
ttccaagcat atctttttct gatactatac aagatggtaa catgggtggt 250
aacttctata ttagtggaaa gtatgtacca agtgtctcac attttggtag 300
cttctcagct aaagaagaaa gcaaatcaac tgttggagtt tttggattaa 350
aacatgattg ggatggaagt ccaatactta agaataaaca cgctgacttt 400
actgttccaa actattcgtt cagatacgag aacaatccat ttctagggtt 450
tgcaggagct atcggttact caatgggtgg cccaagaata gaattcgaaa 500
tatcttatga agcattcgac gtaaaaagtc ctaatatcaa ttatcaaaat 550
gacgcgcaca ggtactgcgc tctatctcat cacacatcgg cagccatgga 600
agctgataaa tttgtcttct taaaaaacga agggttaatt gacatatcac 650
ttgcaataaa tgcatgttat gatataataa atgacaaagt acctgtttct 700
ccttatatat gcgcaggtat tggtactgat ttgatttcta tgtttgaagc 750
tacaagtcct aaaatttcct accaaggaaa actgggcatt agttactcta 800
ttaatccgga aacctctgtt ttcatcggtg ggcatttcca caggatcata 850
ggtaatgagt ttagagatat tcctgcaata gtacctagta actcaactac 900
aataagtgga ccacaatttg caacagtaac actaaatgtg tgtcactttg 950
SEQ 1/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
gtttagaact tggaggaaga tttaacttct aattttattg ttgccacata 1000
ttaaaaatga tctaaacttg tttttawtat tgctacatac aaaaaaagaa 1050
aaatagtggc aaaagaatgt agcaataaga gggggggggg ggaccaaatt 1100
tatcttctat gcttcccaag ttttttcycg ctatttatga cttaaacaac 1150
agaaggtaat atcctcacgg aaaacttatc ttcaaatatt ttatttatta 1200
ccaatcttat ataatatatt aaatttctct tacaaaaatc actagtattt 1250
tataccaaaa tatatattct gacttgcttt tcttctgcac ttctactatt 1300
tttaatttat ttgtcactat taggttataa taawatgaat tgcmaaagat 1350
ttttcatagc aagtgcattg atatcactaa tgtctttctt acctagcgta 1400
tctttttctg aatcaataca tgaagataat ataaatggta acttttacat 1450
tagtgcaaag tatatgccaa gtgcctcaca ctttggcgta ttttcagtta 1500
aagaagagaa aaacacaaca actggagttt tcggattaaa acaagattgg 1550
gacggagcaa cactaaagga tgcaagcwgc agccacacaw tagacccaag 1600
tacaatg 1607
<210> 2
<211> 278
<212> PRT
<213> Ehriichia canis
<220>
<223> amino acid sequence of ECa28-1 protein
<400> 2
Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Ile Ser Leu
10 15
Met Tyr Ser Ile Pro Ser Ile Ser Phe Ser Asp Thr Ile Gln Asp
20 25 30
Gly Asn Met Gly Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Val Pro
35 40 45
Ser Val Ser His Phe Gly Ser Phe Ser Ala Lys Glu Glu Ser Lys
50 55 60
Ser Thr Val Gly Val Phe Gly Leu Lys His Asp Trp Asp Gly Ser
65 70 75
Pro Ile Leu Lys Asn Lys His Ala Asp Phe Thr Val Pro Asn Tyr
80 85 90
Ser Phe Arg Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105
Ile Gly Tyr Ser Met Gly Gly Pro Arg Ile Glu Phe Glu Ile Ser
110 115 120
SEQ 2/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US9912$07S
Tyr Glu Ala Phe Asp Val Lys Ser Pro Asn Ile Asn Tyr Gln Asn
125 1.30 135
Asp Ala His Arg Tyr Cys Ala Leu Ser His His Thr Ser Ala Ala
140 145 150
Met Glu Ala Asp Lys Phe Val Phe Leu Lys Asn Glu Gly Leu Ile
155 160 165
Asp Ile Ser Leu Ala Ile Asn Ala Cys Tyr Asp Ile Ile Asn Asp
170 175 180
Lys Val Pro Val Ser Pro Tyr Ile Cys Ala Gly Ile Gly Thr Asp
185 190 195
Leu Ile Ser Met Phe Glu Ala Thr Ser Pro Lys I1e Ser Tyr G1n
200 205 210
Gly Lys Leu G1y Ile Ser Tyr Ser Ile Asn Pro Glu Thr Ser Val
215 220 225
Phe Ile Gly Gly His Phe His Arg Ile I1e Gly Asn Glu Phe Arg
230 235 240
Asp Tle Pro Ala Ile Val Pro Ser Asn Ser Thr Thr Ile Ser Gly
245 250 255
Pro Gln Phe Ala Thr Val Thr Leu Asn Val Cys His Phe Gly Leu
260 265 270
Glu Leu Gly Gly Arg Phe Asn Phe
275
<210> 3
<211> 849
<212> DNA
<213> Ehrlichia can.is
<220>
<221> mat_peptide
<223> nucleic acid sequence of ECa28SA2
<400> 3
atgaattgta aaaaagtttt cacaataagt gcattgatat catccatata 50
cttcctacct aatgtctcat actctaaccc agtatatggt aacagtatgt 100
atggtaattt ttacatatca ggaaagtaca tgccaagtgt tcctcatttt 150
ggaatttttt cagctgaaga agagaaaaaa aagacaactg tagtatatgg 200
cttaaaagaa aactgggcag gagatgcaat atctagtcaa agtccagatg 250
ataattttac cattcgaaat tactcattca agtatgcaag caacaagttt 300
SEQ 3/24
CA 02352466 2001-05-28
WO OEl/32745 PCT/US99/28U75
ttagggtttg cagtagctat tggttactcg ataggcagtc caagaataga 350
agttgagatg tcttatgaag catttgatgt gaaaaatcca ggtgataatt 400
acaaaaacgg tgcttacagg tattgtgctt tatctcatca agatgatgcg 450
gatgatgaca tgactagtgc aactgacaaa tttgtatatt taattaatga 500
aggattactt aacatatcat ttatgacaaa catatgttat gaaacagcaa 550
gcaaaaatat acctctctct ccttacatat gtgcaggtat tggtactgat 600
ttaattcaca tgtttgaaac tacacatcct aaaatttctt atcaaggaaa 650
gctagggttg gcctacttcg taagtgcaga gtcttcggtt tcttttggta 700
tatattttca taaaattata aataataagt ttaaaaatgt tccagccatg 750
gtacctatta actcagacga gatagtagga ccacagtttg caacagtaac 800
attaaatgta tgctactttg gattagaact tggatgtagg ttcaacttc 849
<210> 4
<211> 283
<212> PRT
<213> Ehrlichia canis
<220>
<223> amino acid sequence of ECa28SA2 protein
<400> 4
Met Asn Cys Lys Lys Val Phe Thr Ile Ser Ala Leu Ile Sex Ser
10 15
Ile Tyr Phe Leu Pro Asn Val Ser Tyr Ser Asn Pro Val Tyr Gly
20 25 30
Asn Ser Met Tyr Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45
Ser Val Pro His Phe Gly Ile Phe Ser Ala Glu Glu Glu Lys Lys
50 55 60
Lys Thr Thr Val Val Tyr Gly Leu Lys Glu Asn Trp Ala Gly Asp
65 70 75
Ala Ile Ser Ser Gln Ser Pro Asp Asp Asn Phe Thr Ile Arg Asn
80 85 90
Tyr Ser Phe Lys Tyr Ala Ser Asn Lys Phe Leu Gly Phe Ala Val
95 100 105
Ala Ile Gly Tyr Ser Ile Gly Ser Pro Arg Ile Glu Val Glu Met
110 115 120
Ser Tyr Glu A1a Phe Asp Val Lys Asn Pro Gly Asp Asn Tyr Lys
125 130 135
SEQ 4124
CA 02352466 2001-05-28
WO 00132745 PCTIUS99/28075
Asn Gly Ala Tyr Arg Tyr Cys Ala Leu Ser His Gln Asp Asp Ala
140 145 150
Asp Asp Asp Met Thr Ser Ala Thr Asp Lys Phe Val Tyr Leu Ile
155 160 165
Asn Glu Gly Leu Leu Asn Ile Ser Phe Met Thr Asn Ile Cys Tyr
170 175 180
Glu Thr A1a Ser Lys Asn Ile Pro Leu Ser Pro Tyr Tle Cys Ala
185 190 195
Gly I1e Gly Thr Asp Leu Ile His Met Phe Glu Thr Thr His Pro
zoo 205 210
Lys Ile Ser Tyr Gln Gly Lys Leu Gly Leu Ala Tyr Phe Val Ser
215 220 225
A1a Glu Ser Ser Val Ser Phe Gly Ile Tyr Phe His Lys Ile I1e
230 235 240
Asn Asn Lys Phe Lys Asn Val Pro Ala Met Va1 Pro Ile Asn Ser
245 250 255
Asp Glu Ile Val Gly Pro Gln Phe Ala Thr Val Thr Leu Asn Val
260 265 270
Cys Tyr Phe Gly Leu Glu Leu Giy Cys Arg Phe Asn Phe
275 280
<210> 5
<211> 840
<212> DNA
<213> Ehrlichia canis
<220>
<221> mat_peptide
<223> nucleic acid sequence of ECa28SA3
<400> 5
atgaattgca aaaaaattct tataacaact gcattaatgt cattaatgta 50
ctatgctcca agcatatctt tttctgatac tatacaagac gataacactg 100
gtagcttcta catcagtgga aaatatgtac caagtgtttc acattttggt 150
gttttctcag ctaaagaaga aagaaactca actgttggag tttttggatt 200
aaaacatgat tggaatggag gtacaatatc taactcttct ccagaaaata 250
tattcacagt tcaaaattat tcgtttaaat acgaaaacaa cccattctta 300
gggtttgcag gagctattgg ttattcaatg ggtggcccaa gaatagaact 350
tgaagttctg tacgagacat tcgatgtgaa aaatcagaac aataattata 400
SEQ 5/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99128075
agaacggcgc acacagatac tgtgctttat ctcatcatag ttcagcaaca 450
agcatgtcct ccgcaagtaa caaatttgtt ttcttaaaaa atgaagggtt 500
aattgactta tcatttatga taaatgcatg ctatgacata ataattgaag 550
gaatgccttt ttcaccttat atttgtgcag gtgttggtac tgatgttgtt 600
tccatgtttg aagctataaa tcctaaaatt tcttaccaag gaaaactagg 650
attaggttat agtataagtt cagaagcctc tgtttttatc ggtggacact 700
ttcacagagt cataggtaat gaatttagag acatccctgc tatggttcct 750
agtggatcaa atcttccaga aaaccaattt gcaatagtaa cactaaatgt 800
gtgtcacttt ggcatagaac ttggaggaag atttaacttc 840
<210> 6
<211> 280
<212> PRT
<213> Ehrlichia caxsis
<220>
<223> amino acid sequence of ECa28SA3 protein
<400> 6
Met Asn Cys Lys Lys Ile Leu I1e Thr Thr Ala Leu Met Ser Leu
10 15
Met Tyr Tyr Ala Pro Ser Ile Ser Phe Ser Asp Thr Ile Gln Asp
20 25 30
Asp Asn Thr Gly Ser Phe Tyr Ile Ser G1y Lys Tyr Val Pro Ser
35 40 45
Val Ser His Phe Gly Val Phe Ser Ala Lys G1u Glu Arg Asn Ser
50 55 60
Thr Val Gly Va1 Phe Gly Leu Lys His Asp Trp Asn Gly Gly Thr
65 70 75
Ile Ser Asn Ser Ser Pro Glu Asn Ile Phe Thr VaI GLn Asn Tyr
80 85 90
Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105
Ile Gly Tyr Ser Met Gly Gly Pro Arg Tle Glu Leu Glu Val Leu
110 115 120
Tyr Glu Thr Phe Asp Val Lys Asn Gln Asn Asn Asn Tyr Lys Asn
125 130 135
Gly Ala His Arg Tyr Cys Ala Leu Ser His His Ser Ser Ala Thr
140 145 150
SEQ 6124
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
Ser Met Ser Ser Ala Ser Asn Lys Phe Val Phe Leu Lys Asn Glu
155 160 165
Gly Leu Ile Asp Leu Ser Phe Met Ile Asn Ala Cys Tyr Asp Tle
170 175 180
Ile Ile Glu Gly Met Pro Phe Ser Pro Tyr Ile Cys Ala Gly Val
185 190 195
Gly Thr Asp Val Val Ser Met Phe Glu Ala Ile Asn Pro Lys Ile
200 205 210
Ser Tyr Gln Gly Lys Leu Gly Leu Gly Tyr Ser Ile Ser Ser Glu
215 220 225
Ala Ser Val Phe Ile Gly Gly His Phe His Arg Val Ile Gly Asn
230 235 240
Glu Phe Arg Asp Ile Pro Ala Met Val Pro Ser Gly Ser Asn Leu
245 250 255
Pro Glu Asn Gln Phe Ala Ile Val Thr Leu Asn Val Cys His Phe
260 265 270
Gly Ile Glu Leu Gly G1y Arg Phe Asn Phe
275 280
<210> 7
<211> 133
<212> PRT
<213> Ehrlichia canis
<220>
<223> partial amino acid sequence of ECa28SA2 protein
<400> 7
Met Asn Cys Lys Lys Val Phe Thr Ile Ser Ala Leu Ile Ser Ser
10 15
Ile Tyr Phe Leu Pro Asn Val Ser Tyr Ser Asn Pro Val Tyr Gly
20 25 30
Asn Ser Met Tyr Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45
Ser Val Pro His Phe Gly Ile Phe Ser Ala Glu G1u Glu Lys Lys
50 55 60
Lys Thr Thr Val Val Tyr Gly Leu Lys G1u Asn Trp Ala Gly Asp
65 70 75
Ala Ile Ser Ser Gln Ser Pro Asp Asp Asn Phe Thr Ile Arg Asn
80 85 90
SEQ 7124
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
Tyr Ser Phe Lys Tyr Ala Ser Asn Lys Phe Leu Gly Phe Ala Val
95 100 105
Ala Ile Gly Tyr Ser Ile Gly Ser Pro Arg Ile Glu Val Glu Met
110 115 120
Ser Tyr G1u Ala Phe Asp Val Lys Asn Gln Gly Asn Asn
125 130
<210> 8
<211> 287
<212> PRT
<213> Ehr.~icliia cams
<220>
<223> amino acid sequence of ECa28SA1 protien
<400> 8
Met Lys Tyr Lys Lys Thr Phe Thr Val Thr Ala Leu Val Leu Leu
10 15
Thr Ser Phe Thr His Phe Ile Pro Phe Tyr Ser Pro Ala Arg Ala
20 25 30
Ser Thr Ile His Asn Phe Tyr Ile Ser G1y Lys Tyr Met Pro Thr
35 40 45
Ala Ser His Phe Gly Ile Phe Ser Ala Lys Glu Glu Gln Ser Phe
50 55 60
Thr Lys Val Leu Val Gly Leu Asp Gln Arg Leu Ser His Asn Ile
65 70 75
Ile Asn Asn Asn Asp Thr Ala Lys Ser Leu Lys Val Gln Asn Tyr
80 85 90
Ser Phe Lys Tyr Lys Asn Asn,Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105
Ile Gly Tyr Ser Ile Gly Asn Ser Arg Ile Glu Leu Glu Val Ser
110 115 120
His Giu Ile Phe Asp Thr Lys Asn Pro Gly Asn Asn Tyr Leu Asn
125 130 135
Asp Ser His Lys Tyr Cys Ala Leu Ser His Gly Ser His Ile Cys
140 145 150
Ser Asp Gly Asn Ser Gly Asp Trp Tyr Thr Ala Lys Thr Asp Lys
155 160 165
Phe Val Leu Leu Lys Asn Glu Gly Leu Leu Asp Val Ser Phe Met
170 175 180
SEQ 8/24
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
Leu Asn Ala Cys Tyr Asp Ile Thr Thr Glu Lys Met Pro Phe Ser
185 190 195
Pro Tyr Ile Cys Ala Gly Ile Gly Thr Asp Leu Ile Ser Met Phe
200 205 210
Glu Thr Thr Gln Asn Lys Ile Ser Tyr Gln Gly Lys Leu Gly Leu
215 220 225
Asn Tyr Thr Ile Asn Ser Arg Val Ser Val Phe Ala Gly Gly His
230 235 240
Phe His Lys Val Ile G1y Asn Glu Phe Lys G1y Ile Pro Thr Leu
245 250 255
Leu Pro Asp Gly Ser Asn Ile Lys Va1 Gln Gln Ser Ala Thr Val
260 265 270
Thr Leu Asp Val Cys His Phe Gly Leu Glu Ile Gly Ser Arg Phe
275 280 285
Phe Phe
<210> 9
<211> 281
<212> PRT
<213> Ehrlichia chaffeensis
<220>
<223> amino acid sequence of E. chaffeensis P28
<400> 9
Met Asn Tyr Lys Lys Val Phe Ile Thr Ser Ala Leu Ile Ser Leu
10 15
Ile Ser Ser Leu Pro Gly Val Ser Phe Ser Asp Pro Ala Gly Ser
20 25 30
Gly Ile Asn Gly Asn Phe Tyr Ile Ser G1y Lys Tyr Met Pro Ser
35 40 45
Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Arg Asn Thr
50 55 60
Thr Val Gly Va1 Phe G1y Leu Lys Gln Asn Trp Asp G1y Ser Ala
65 70 75
Ile Ser Asn Ser Ser Pro Asn Asp Val Phe Thr Val Ser Asn Tyr
80 85 90
Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105
SEQ 9124
CA 02352466 2001-05-28
WO 00132745 PCTIUS99l28075
Ile Gly Tyr Ser Met Asp Gly Pro Arg Ile Glu Leu Glu Val Ser
110 115 120
Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys Asn
125 130 135
Glu Ala His Arg Tyr Cys Ala Leu Ser His Asn Ser Ala Ala Asp
140 145 150
Met Ser Ser Ala Ser Asn Asn Phe Val Phe Leu Lys Asn Glu Gly
155 160 165
Leu Leu Asp Ile Ser Phe Met Leu Asn Ala Cys Tyr Asp Val Val
170 175 180
Gly Glu Gly I1e Pro Phe Ser Pro Tyr Ile Cys Ala Gly Ile Gly
185 190 195
Thr Asp Leu Val Ser Met Phe Glu Ala Thr Asn Pro Lys Ile Ser
200 205 210
Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Ser I1e Sex Pro Glu Ala
215 220 225
Ser Val Phe Ile Gly Gly His Phe His Lys Val Ile Gly Asn Glu
230 235 240
Phe Arg Asp Ile Pro Thr Ile Ile Pro Thr Gly Ser Thr Leu Ala
245 250 255
Gly Lys Gly Asn Tyr Pro Ala Ile Val Ile Leu Asp Val Cys His
260 265 270
Phe Gly Tle Glu Leu Gly Gly Arg Phe A1a Phe
275 280
<210> 10
<221> 283
<212> PRT
<213> Ehrlichia chaffeensis
<220>
<223> amino acid sequence of E. chaffeensis OMP-1B
<400> 10
Met Asn Tyr Lys Lys Ile Phe Val Ser Ser Ala Leu Ile Ser Leu
10 15
Met Ser Ile Leu Pro Tyr Gln Ser Phe Ala Asp Pro Val Thr Ser
20 25 30
SEQ 10/24
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99I28075
Asn Asp Thr Gly Ile Asn Asp Ser Arg Glu Gly Phe Tyr Ile Ser
35 40 45
Val Lys Tyr Asn Pro Ser Ile Ser His Phe Arg Lys Phe Ser Ala
50 55 60
Glu Glu Ala Pro I1e Asn Gly Asn Thr Ser I1e Thr Lys Lys Val
65 70 75
Phe Gly Leu Lys Lys Asp Gly Asp I1e Ala Gln Ser Ala Asn Phe
so s5 90
Asn Arg Thr Asp Pro Ala Leu Glu Phe Gln Asn Asn Leu Ile Ser
95 100 105
Gly Phe Ser Gly Ser Ile Gly Tyr Ala Met Asp Gly Pro Arg Ile
110 115 120
Glu Leu Glu Ala Ala Tyr Gln Lys Phe Asp Ala Lys Asn Pro Asp
125 130 135
Asn Asn Asp Thr Asn Ser Gly Asp Tyr Tyr Lys Tyr Phe Gly Leu
140 145 150
Ser Arg Glu Asp Ala Ile Ala Asp Lys Lys Tyr Val Val Leu Lys
155 160 165
Asn Glu Gly Ile Thr Phe Met Ser Leu Met Va1 Asn Thr Cys Tyr
170 175 180
Asp Ile Thr Ala Glu Gly Val Pro Phe Ile Pro Tyr Ala Cys Ala
185 190 195
Gly Val G1y Ala Asp Leu Ile Asn Val Phe Lys Asp Phe Asn Leu
200 205 210
Lys Phe Ser Tyr Gln Gly Lys Ile Gly Ile Ser Tyr Pro Tle Thr
215 220 225
Pro Glu Val Ser Ala Phe Ile,Gly Gly Tyr Tyr His Gly Val Ile
230 235 240
Gly Asn Asn Phe Asn Lys Ile Pro Val Ile Thr Pro Val Val Leu
245 250 255
Glu Gly Ala Pro Gln Thr Thr Ser Ala Leu Val Thr Ile Asp Thr
260 265 270
Gly Tyr Phe Gly Gly Glu Val Gly Val Arg Phe Thr Phe
275 280
<210> 11
<211> 280
SEQ 11/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
<212> PRT
<213> .Ehrlichia chaffeensis
<220>
<223> amino acid sequence of E. chaffeensis OMP-1C
<400> 11
Met Asn Cys Lys Lys Phe Phe Ile Thr Thr Ala Leu Ala Leu Pro
10 15
Met Ser Phe Leu Pro G1y Ile Leu Leu Ser Glu Pro Val Gln Asp
20 25 30
Asp Ser Val Ser Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45
Ser Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Lys Asn
50 55 60
Pro Thr Val Ala Leu Tyr Gly Leu Lys Gln Asp Trp Asn Gly Val
65 70 75
Ser Ala Ser Ser His Ala Asp Ala Asp Phe Asn Asn Lys Gly Tyr
80 85 90
Ser Phe Lys Tyr Glu Asn Asn. Pro Phe Leu Gly Phe A1a Gly Ala
95 100 105
Ile Gly Tyr Sex Met Gly Gly Pro Arg Ile Glu Phe Glu VaI Ser
110 115 120
Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Gly Asn Tyr Lys Asn
125 130 135
Asp Ala His Arg Tyr Cys Ala Leu Asp Arg Lys Ala Ser Ser Thr
140 145 150
Asn Ala Thr Ala Ser His Tyr Val Leu Leu Lys Asn Glu Gly Leu
155 160 165
Leu Asp Ile Ser Leu Met Leu Asn Ala Cys Tyr Asp Val Va1 Ser
170 175 ~ 180
Glu Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala Gly Val Gly Thr
185 190 195
Asp Leu Ile Ser Met Phe Glu Ala Ile Asn Pro Lys Iie Ser Tyr
200 205 210
Gln Gly Lys Leu Gly Leu Ser Tyr Ser Ile Asn Pro Glu Ala Ser
215 220 225
Val Phe Val Gly Gly His Phe His Lys Val Ala Gly Asn Glu Phe
230 235 240
SEQ 12124
CA 02352466 2001-05-28
WO 00132745 PCT/US99l2$075
Arg Asp Ile Ser Thr Leu Lys A1a Phe Ala Thr Pro Ser Ser Ala
245 250 255
A1a Thr Pro Asp Leu Ala Thr Val Thr Leu Ser Va1 Cys His Phe
260 265 270
Gly Val Glu Leu Gly Gly Arg Phe Asn Phe
275 280
<210> 12
<211> 286
<212> PRT
<213> Ehrlichia chaffeensis
<220>
<223> amino acid sequence of E. chaffeensis OMP-1D
<400> 12
Met Asn Cys Glu Lys Phe Phe Ile Thr Thr Ala Leu Thr Leu Leu
10 15
Met Ser Phe Leu Pro Gly I1e Ser Leu Ser Asp Pro Val Gln Asp
20 25 30
Asp Asn Ile Ser Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45
Ser Ala Ser His Phe Gly Va1 Phe Ser Ala Lys Glu Glu Arg Asn
50 55 60
Thr Thr Val Gly Val Phe Gly Ile Glu Gln Asp Trp Asp Arg Cys
65 70 75
Val Ile Ser Arg Thr Thr Leu Ser Asp Ile Phe Thr Val Pro Asn
80 85 90
Tyr Ser Phe Lys Tyr Glu Asn Asn Leu Phe Ser Gly Phe Ala Gly
95 100 105
Ala Ile Gly Tyr Ser Met Asp G1y Pro Arg Tle Glu Leu Glu Val
110 115 120
Ser Tyr Glu Ala Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys
125 130 135
Asn Glu Ala His Arg Tyr Tyr Ala Leu Ser His Leu Leu Gly Thr
140 145 150
Glu Thr Gln Ile Asp Gly Ala Gly Ser Ala Ser Val Phe Leu Ile
155 160 165
SEQ 13!24
CA 02352466 2001-05-28
WO 00132745 PCT/US99128075
Asn Glu Gly Leu Leu Asp Lys Ser Phe Met Leu Asn Ala Cys Tyr
170 175 180
Asp Val Ile Ser Glu Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala
185 190 195
Gly Ile Gly Ile Asp Leu Val Ser Met Phe G1u A1a Ile Asn Pro
200 205 210
Lys Ile Ser Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Pro I1e Ser
215 220 225
Pro Glu Ala Ser Val Phe Ile Gly Gly His Phe His Lys Val Ile
230 235 240
Gly Asn Glu Phe Arg Asp Ile Pro Thr Met Lle Pro Ser Glu Ser
245 250 255
Ala Leu A1a Gly Lys Gly Asn Tyr Pro Ala Ile Val Thr Leu Asp
260 265 270
Val Phe Tyr Phe Gly Ile Glu Leu Gly Gly Arg Phe Asn Phe Gln
275 280 285
Leu
<210> 13
<211> 278
<212> PRT
<213> Eh.rlichia chaffeensis
<220>
<223> amino acid sequence of E. chaffeensis OMP-1E
<400> 13
Met Asn Cys Lys Lys Phe Phe Ile Thr Thr Ala Leu Val Ser Leu
10 15
Met Sex Phe Leu Pro Gly I3.e Ser Phe Ser Asp Pro Val Gln Gly
20 25 30
Asp Asn Ile Ser Gly Asn Phe Tyr Val Ser Gly Lys Tyr Met Pro
35 40 45
Ser A1a Ser His Phe Gly Met Phe Ser Ala Lys Glu Glu Lys Asn
50 55 60
Pro Thr Val Ala Leu Tyr Gly Leu Lys Gln Asp Trp Glu Gly Ile
65 70 75
Ser Ser Ser Ser His Asn Asp Asn His Phe Asn Asn Lys Gly Tyr
80 85 90
SEQ 14/24
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105
Ile G1y Tyr Ser Met Gly Gly Pro Arg Val Glu Phe Glu Val Ser
120 115 120
Tyr Glu Thr Phe Asp Val Lys Asn Gln G1y Asn Asn Tyr Lys Asn
125 130 135
Asp Ala His Arg Tyr Cys Ala Leu Gly Gln Gln Asp Asn Ser Gly
140 145 150
Ile Pro Lys Thr Ser Lys Tyr Val Leu Leu Lys Ser Glu Gly Leu
155 160 165
Leu Asp Ile Ser Phe Met Leu Asn Ala Cys Tyr Asp Ile Ile Asn
170 175 180
Glu Ser Ile Pro Leu Ser Pro Tyr Ile Cys Ala Gly Val Gly Thr
185 190 195
Asp Leu Ile Ser Met Phe Glu Ala Thr Asn Pro Lys Ile Ser Tyr
200 205 210
Gln Gly Lys Leu Gly Leu Ser Tyr Ser Ile Asn Pro Glu Ala Ser
215 220 225
Val Phe Tle Gly Gly His Phe His Lys Val Ile Gly Asn Glu Phe
230 235 240
Arg Asp Ile Pro Thr Leu Lys Ala Phe Val Thr Ser Ser Ala Thr
245 250 255
Pro Asp Leu Ala Ile Val Thr Leu Ser Val Cys His Phe Gly Ile
260 265 270
Glu Leu Gly Gly Arg Phe Asn Phe
275
<210> 14
<211> 280
<212> PRT
<213> Ehrlichia chaffeensis
<220>
<223> amino acid sequence of E. chaffeensis OMP-1F
<400> 14
Met Asn Cys Lys Lys Phe Phe Ile Thr Thr Thr Leu Val Ser Leu
10 15
SEQ 15/24
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
Met Ser Phe Leu Pro Gly IleSer PheSer Asp Ala Val Gln Asn
20 25 30
Asp Asn Val Gly Gly Asn PheTyr IleSer Gly Lys Tyr Val Pro
35 40 45
Ser Val Ser His Phe G1y Va1Phe SerAla Lys Gln Glu Arg Asn
50 55 60
Thr Thr Thr Gly Val Phe GlyLeu LysGln Asp Trp Asp Gly Ser
65 70 75
Thr Ile Ser Lys Asn Ser ProGlu AsnThr Phe Asn Val Pro Asn
80 85 90
Tyr Ser Phe Lys Tyr Glu AsnAsn ProPhe Leu Gly Phe Ala Gly
95 100 105
Ala Val Gly Tyr Leu Met AsnGly ProArg Ile Glu Leu Glu Met
110 115 120
Ser Tyr Glu Thr Phe Asp ValLys AsnGln Gly Asn Asn Tyr Lys
125 130 135
Asn Asp Ala His Lys Tyr TyrAla LeuThr His Asn Ser Gly Gly
140 145 150
Lys Leu Ser Asn Ala Gly AspLys PheVal Phe Leu Lys Asn G1u
155 160 165
Gly Leu Leu Asp Ile Ser LeuMet LeuAsn Ala Cys Tyr Asp Val
170 175 180
Ile Ser Glu Gly Ile Pro PheSer ProTyr I1e Cys Ala Gly Val
185 190 195
Gly Thr Asp Leu Ile Ser MetPhe GluAla Ile Asn Pro Lys Ile
200 205 210
Ser Tyr Gln Gly Lys Leu GlyLeu SerTyr Ser Ile Ser Pro Glu
215 220 225
Ala Ser Val Phe Va1 Gly GlyHis PheHis Lys Val Ile Gly Asn
230 235 240
Glu Phe Arg Asp Ile Pro AlaMet IlePro Ser Thr Ser Thr Leu
245 250 255
Thr Gly Asn His Phe Thr IleVa1 ThrLeu Ser Val Cys His Phe
260 265 270
Gly Val Glu Leu Gly Gly Phe Phe
Arg Asn
275 280
SEQ 16/24
CA 02352466 2001-05-28
WD 00/32745 PCT/US99/28075
<210> 15
<211> 284
<212> PRT
<213> Cowdria ruminantium
<220>
<223> amino acid sequence of C. ruminantium MAP-1
<400> 15
Met Asn Cys Lys Lys Ile Phe Ile Thr Ser Thr Leu Ile Ser Leu
10 15
Val Ser Phe Leu Pro Gly Val Ser Phe Ser Asp Val Ile Gln Glu
20 25 30
Glu Asn Asn Pro Val Gly Ser Val Tyr Ile Ser Ala Lys Tyr Met
35 40 45
Pro Thr A1a Ser His Phe Gly Lys Met Ser Ile Lys Glu Asp Ser
50 55 60
Arg Asp Thr Lys Ala Val Phe Gly Leu Lys Lys Asp Trp Asp Gly
65 70 75
Val Lys Thr Pro Ser Gly Asn Thr Asn Ser Ile Phe Thr Glu Lys
80 85 90
Asp Tyr Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala
g5 100 105
Gly Ala Val Gly Tyr Ser Met Asn Gly Pro Arg Ile Glu Phe Glu
110 115 120
Val Ser Tyr Glu Thr Phe Asp Val Arg Asn Pro Gly GIy Asn Tyr
125 130 135
Lys Asn Asp Ala His Met Tyr Cys Ala Leu Asp Thr Ala Ser Ser
140 145 150
Ser Thr Ala Gly Ala Thr Thr Ser Val Met Val Lys Asn Glu Asn
255 160 165
Leu Thr Asp IIe Ser Leu Met Leu Asn Ala Cys Tyr Asp I1e Met
170 175 180
Leu Asp Gly Met Pro Val Ser Pro Tyr Val Cys Ala Gly IIe Gly
185 190 195
Thr Asp Leu Val Ser Val Tle Asn Ala Thr Asn Pro Lys Leu Ser
200 205 210
Tyr Gln Gly Lys Leu Gly Ile Ser Tyr Ser Ile Asn Pro Glu Ala
215 220 225
SEQ 17/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
Ser Ile Phe Ile Gly Gly His Phe His Arg Val Ile Gly Asn Glu
230 235 240
Phe Lys Asp Ile Ala Thr Ser Lys Val Phe Thr Ser Ser Gly Asn
245 250 255
Ala Ser Ser Ala Val Ser Pro Gly Phe Ala Ser Ala Ile Leu Asp
260 265 270
Val Cys His Phe Gly Ile Glu Ile Gly Gly Arg Phe Val Phe
275 280
<210> 16
<211> 20
<212> DNA
<2I3> artificial sequence
<220>
<221> primer bind
<222> nucleotides 313-332 of C. ruminantium MAP-2,
also nucleotides 307-326 of E. chaffeensis P28
<223> forward primer 793 for PCR
<400> 16
gcaggagctg ttggttactc 20
<210> 17
<221> 21
<212> DNA
<213> artificial sequence
<220>
<221> primer-bind
<222> nucleotides 823-843 of C. ruminantium MAP-Z,
also nucleotides 814-834 of E. chaffeensis P28
<223> reverse primer 1330 for PCR
<400> 17
ccttcctcca agttctatgc c 21
<210> 18
<211> 24
<212> DNA
SEQ 18/24
CA 02352466 2001-05-28
WO 00132745 PCT/US99/28075
<213> artificial sequence
<220>
<221> primer_bind
<223> primer 46f, specific for ECa28SA2 gene
<400> 18
atatacttcc tacctaatgt ctca 24
<210> 19
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221> primer_bind
<223> primer used for sequencing 28-kDa protein genes
in E. canis
<400> 19
agtgcagagt cttcggtttc 20
<210> 20
<211> 18
<212> DNA
<213> artificial sequence
<220>
<221> primer_bind
<223> primer used for sequencing 28-kDa protein genes
in E. cams
<400> 20
gttacttgcg gaggacat 1g
<210> 21
<221> 24
<212> DNA
<213> artificial sequence
<220>
<221> primer band
<222> nucleotides 687-710 of ECa28-2
SEQ 19/24
CA 02352466 2001-05-28
WO 00/32?45 PCT/US99128075.
<223> primer 394 for PCR
<400> 21
gcatttccac aggatcatag gtaa 24
<210> 22
<211> 24
<212> DNA
<213> artificial sequence
<220>
<221> primer_band
<222> nucleotides 710-687 of ECa28-2
<223> primer 394C for PCR
<400> 22
ttacctatga tcctgtggaa atgc 24
<210> 23
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221> primer_bind
<223> primer 793C which anneals to a region with
Eca28-1, used to amplify the intergenic
region between gene ECa28SA3 and ECa28-1
<400> 23
gagtaaccaa cagctcctgc 20
<210> 24
<211> 24
<212> DNA
<213> artificial sequence
<220>
<221> primer band
<222>
<223> primer EC280M-F complementary to noncoding
regions adjacent to the open reading frame
SEQ 20/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99I28075
of ECa28-.Z
<400> 24
tctactttgc acttccacta ttgt 24
<210> 25
<211> 24
<212> DNA
<213> artificial sequence
<220>
<221> primer_band
<223> primer EC280M-R complementary to noncoding
regions adjacent to the open reading frame
of ECa28-1
<400> 25
attcttttgc cactattttt cttt 24
<210> 26
<211> 25
<212> DNA
<213> artificial sequence
<220>
<221> primer_bind
<223> primer ECaSA3-2 corresponding to regions within
ECa28SA3,used to amplify the intergenic region
NC3 between gene ECa28SA3 and ECa28-1
<400> 26
ctaggattag gttatagtat aagtt 25
<210> 27
<211> 23
<212> PRT
<213 Ehrlichia cani s
>
<220>
<221> PEPTIDE
<223> a predicted N-terminal signal peptide of ECa28-1
SEQ 2 I /24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
and ECa28SA3
<400> 27
Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Met Ser Leu
10 15
Met Tyr Tyr Ala Pro Ser Ile Ser
<210> 28
<211> 25
<212> PRT
<213> Ehrlichia chaffeensis
<220>
<223> amino acid sequence of N-terminal signal peptide
of E. chaffeensis P28
<400> 28
Met Asn Tyr Lys Lys Ile Leu Ile Thr Ser Ala Leu I1e Ser Leu
5 10 15
Ile Ser Ser Leu Pro Gly Va1 Ser Phe Ser
20 25
<210> 29
<211> 2G
<212> PRT
<213> Ehrlichia canis
<220>
<223> amino acid sequence of putative cleavage site of
ECa28-1
<400> 29
Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Ile Ser Leu
5 10 15
Met Tyr Ser Ile Pro Ser Lle Ser Ser Phe Ser
20 25
<210> 30
<211> 299
SEQ 22/24
CA 02352466 2001-05-28
WO 00/32745 PCT/US99/28075
<212> DNA
<213> Ehrlichia canis
<220>
<223> nucleic acid sequence of intergenic noncoding
region 1 (28NC1)
<400> 30
taatacttct attgtacatg ttaaaaatag tactagtttg cttctgtggt 50
ttataaacgc aagagagaaa tagttagtaa taaattagaa agttaaatat 100
tagaaaagtc atatgttttt cattgtcatt gatactcaac taaaagtagt 150
ataaatgtta cttattaata attttacgta gtatattaaa tttcccttac 200
aaaagccact agtattttat actaaaagct atactttggc ttgtatttaa 250
tttgtatttt tactactgtt aatttacttt cactgtttct ggtgtaaat 299
<210> 31
<211> 345
<212> DNA
<213> Ehrlichia cams
<220>
<223> nucleic acid sequence of intergenic noncoding
region 2 {28NC2)
<400> 31
taatttcgtg gtacacatat cacgaagcta aaattgtttt tttatctctg 50
ctgtatacaa gagaaaaaat agtagtgaaa attacctaac aatatgacag 100
tacaagttta ccaagcttat tctcacaaaa cttcttgtgt cttttatctc 150
tttacaatga aatgtacact tagcttcact actgtagagt gtgtttatca 200
atgctttgtt tattaatact ctacataata tgttaaattt ttcttacaaa 250
actcactagt aatttatact agaatatata ttctgacttg tatttgcttt 300
atacttccac tattgttaat ttattttcac tattttaggt gtaat 345
<210> 32
<211> 345
<212> DNA
<213> Ehrlichia cams
<220>
<223> nucleic acid sequence of intergenic noncoding
region 3 { 2 8NC3 )
<400> 32
SEQ 23/24
CA 02352466 2001-05-28
WO 00/32745 PCTIUS99/28075
tgattttatt gttgccacat attaaaaatg atctaaactt gtttttatta 50
ttgctacata caaaaaaaag aaaaatagtg gcaaaagaat gtagcaataa 100
gagggggggg ggggactaaa tttaccttct attcttctaa tattctttac 150
tatattcaaa tagcacaact caatgcttcc aggaaaatat gtttctaata 200
ttttatttat taccaatcct tatataatat attaaatttc tcttacaaaa 250
atctctaatg ttttatactt aatatatata ttctggcttg tatttacttt 300
gcacttccac tattgttaat ttattttcac tattttaggt gtaat 345
<210> 33
<211> 355
<212> DNA
<213> Ehrlichia cams
<220>
<223> nucleic acid sequence of intergenic noncoding
region 4 (28NC4)
<400> 33
taattttatt gttgccacat attaaaaatg atctaaactt gtttttawta 50
ttgctacata caaaaaaaga aaaatagtgg caaaagaatg tagcaataag 100
aggggggggg gggaccaaat ttatcttcta tgcttcccaa gttttttcyc 150
gctatttatg acttaaacaa cagaaggtaa tatcctcacg gaaaacttat 200
cttcaaatat tttatttatt accaatctta tataatatat taaatttctc 250
ttacaaaaat cactagtatt ttataccaaa atatatattc tgacttgctt 300
ttcttctgca cttctactat ttttaattta tttgtcacta ttaggttata 350
ataaw 355
SEQ 24/24
