Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• 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 th e 28-kDa homologous proteins of Ehrlichia canis and uses thereof.
• Canine ehrlichiosis also known as canine tropical pancytopenia, is a tick-borne rickettsial disease of dogs first de scribed in Africa in 1935 and the United States in 1963 (Donatien an d Lestoquard, 1935; Ewing, 1963). The disease became b e tter recognized after an epizootic outbreak occurred in United S tates 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 et al , 1971 ) and is transmitted by the brown dog tick, Rhipicephalus sanguineus (Groves et al , 1975).
• the progression of canine ehrlichiosis occurs in three phases, acute, subclinical and chronic.
• the acute phase i s characterized by fever, anorexia, depression, lymphadenopathy and mild thrombocytopenia (Troy and Forrester, 1990).
• Dogs typically recover from the acute phase, but become persistently infected carriers of the organism without clinical signs of disease for mo nth s 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).
• E. canis and E. chaff eensis the etiologic agent o f human monocytic ehrlichiosis (HME)
• HME human monocytic ehrlichiosis
• the prior art is deficient in the lack of cloning a n d characterization of new homologous 28-kDa immunoreactive protein genes of Ehrlichia canis and a single multigene locus containing th e homologous 28-kDa protein genes. Further, The prior art is deficient in the lack of recombinant proteins of such immunoreactive genes o f Ehrlichia canis.
• the present invention fulfills this long-standing n eed and desire in the art.
• the present invention describes the molecular cloning, sequencing, characterization, and expression of homologous mature 28-kDa immunoreactive protein genes of Ehrlichia canis (designated Eca28-1, ECa28SA3 and ECa28SA2), and the identification of a single locus (5.592-kb) containing five 28-kDa protein genes of Ehrlichia canis (ECa28SAI, ECa28SA2, ECa28SA3, Eca28-1 and ECa28-2).
• Comparison with E. chaffeensis and among E. canis 28-kDa pro tein genes revealed that ECa28-l shares the most amino acid homology with the E.
• chaffeensis omp-1 multigene family is highly conserved among E. canis isolates.
• the five 28-kDa proteins were predicted t o 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 th at these genes may be independently and differentially expressed .
• Intergenic noncoding regions ranged in size from 299 to 355-bp, a n d were 48 to 71 % homologous.
• DNA sequences encoding a 30-kDa immunoreactive pro tein of Ehrlichia canis Preferably, the protein has an amino acid s equ ence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 a n d SEQ ID No. 6, and the gene has a nucleic acid sequence selected fro m 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.
• the protein has an N-terminal signal sequence which is cleaved after post-translational process resulting in the production of a mature 28 - kDa protein.
• 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.
• an expression vector comprising a gene encoding a 28 -kDa immunoreactive protein of Ehrlichia canis and capable of expressing the gene when the vector is introduced into a cell.
• a recombinant protein comprising an amino acid s equence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 a n d SEQ ID No. 6.
• 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.
• the recombinant protein comprises four variable regions which are surface exposed, hydrophilic and antigenic. The recombinant protein may be useful a s an antigen.
• th ere is provided a method of producing the recombinant protei n , 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 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 th e 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 e subject' s body.
• Figure 1 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 s tart codon 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- l -thioredoxin fusion protein (Lane 1 , arrow) and 16-kDa thioredoxin control (Lane 2, arrow), and corresponding immunoblot of recombinant ECa28- l -thioredoxin fusion protein recognized by covalescent-phase E. canis canine antiserum (Lane 3 ) . Thiroredoxin control was not detected by E. canis antiserum ( no t shown) .
• Figure 3 shows alignment of ECa28- l protein (SEQ ID NO.
• ECa28SA2 partial sequence, SEQ ID NO. 7 and ECa28SAl (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-l amino acid sequence is presented as the consensus sequence. Amino acids not shown are identical to ECa28- l 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 (VRl , 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 ECa28SAl , 6 members o f the E. chaff eensis omp - 1 multiple gene family, and C. rumanin tium map - 1 from deduced amino acid sequences utilizing unbalanced tre e construction.
• the length of each pair of branches represents th e 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 an d hybridized with a ECa28- l 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-l (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 nm 2 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 of 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 dedu c ed 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 1 195-2031 : SEQ ID No. 5; amino acid sequence: SEQ ID No. 6) including intergenic noncoding sequences (NC2, nucleotide 850-1 194: 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 (28NC 1 -4).
• the 28-kDa protein genes shown in Locus 1 and 2 (shaded) have been described (McBride et al., 1999; Reddy et al. 1998: Ohashi et al, 1998).
• the complete sequence of ECaSA2 and a new 28-kDa protein gene designated (ECa28SA3 - unshaded) was sequenced.
• ECa28SA3 and ECa28- l were completed joining th e 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 o f branches represents the distance between amino acid pairs.
• the scale measures the distance beteween sequences.
• Figure 10 shows alignment of E. canis 28-kDa pro tein 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 th e 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.
• the present invention describes cloning, sequencing an d expression of homologous genes encoding a 30-kilodalton (kD a) protein of Ehrlichia canis.
• a comparative molecular analysis o f homologous genes among seven E. canis isolates and the E. chaffeensis omp- 1 multigene family was also performed.
• Two new 28-kDa protein genes are identified, ECa28-l and ECa28SA3.
• ECa28-I has an 834- bp open reading frame encoding a protein of 278 amino acids (SEQ ID No. 2) with a predicted molecular mass of 30.5-kDa.
• ECa28SA3 has an 840-bp open reading frame encoding a 280 amino acid protein (SEQ ID No. 6).
• Intergenic noncoding regions (28NC 1 -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 Ehrlichia 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.
• DNA sequences encoding a 30-kDa immunoreactive protein of Ehrlichia canis Preferably, the protein has an amino acid s eque nce selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 a n d SEQ ID No. 6. and the gene has a nucleic acid sequence selected fro m 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. M ore preferably, the protein has an N-terminal signal sequence which i s cleaved after post-translational process resulting in the production o f a mature 28-kDa protein.
• the DNAs encoding 28 - kDa proteins are contained in a single multigene locus, which has the size of 5.592 kb and encodes all five homologous 28-kDa proteins o f Ehrlichia canis.
• an expression vector comprising a gene encoding a 28 -kDa immunoreactive protein of Ehrlichia canis and capable of expressing the gene when the vector is introduced into a cell.
• a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 a n d SEQ ID No. 6.
• 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.
• the recombi nant protein comprises four variable regions which are surface exposed, hydrophilic and antigenic. Still preferably, the recombinant protein is an antigen.
• 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 certai n 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.
• 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 ab o u t the replication of the attached segment.
• a "DNA molecule” refers to the polymeric form o f deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix.
• This te rm refers only to the primary and secondary structure of the molecule , and does not limit it to any particular tertiary forms. Thus, this t e rm includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, a n d chromosomes.
• linear DNA molecules e.g., restriction fragments
• a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in 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 c o do n 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, an d even synthetic DNA sequences.
• a polyadenylation signal and transcription termination sequence will usually be located 3' to th e coding sequence.
• Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers , polyadenylation signals, terminators, and the like, that provide for th e 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.
• 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.
• a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding d omains (consensus sequences) responsible for the binding of RNA polymerase.
• Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT” boxes.
• Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the - 10 and -35 c o nsensu s sequ ences .
• An "expression control sequence” is a DNA sequence th at controls and regulates the transcription and translation of ano th er 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 th en 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 th e polypeptide, that communicates to the host cell to direct th e polypeptide to the cell surface or secrete the polypeptide into th e media, and this signal peptide is clipped off by the host cell before th e 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 th e probe of the present invention, is defined as a molecule comprised o f two or more ribonucleotides, preferably more than three.
• primer refers to a n oligonucleotide, whether occurring naturally as in a purifi ed restriction digest or produced synthetically, which is capable of ac ting as a point of initiation of synthesis when placed under conditions i n which synthesis of a primer extension product, which i s complementary to a nucleic acid strand, is induced, i.e., in th e presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
• the primer may b e either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in th e presence of the inducing agent.
• the exact length of the primer will depend upon many factors, including temperature, source of pri mer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, th e oligonucleotide primer typically contains 15-25 or more nucleotides , although it may contain fewer nucleotides.
• the primers herein are selected to be "substanti" 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, the primer sequence need not reflect the exact sequence of th e template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of th e 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 a n d thereby form the template for the synthesis of the extension product.
• a cell has been "transformed" by exogenous o r heterologous 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.
• the transforming DNA may b e maintained on an episomal element such as a plasmid.
• a stably transformed cell is one in which t h e transforming DNA has become integrated into a chromosome so th at it is inherited by daughter cells through chromosome replication.
• 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 mo st preferably at least 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 e 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.
• heterologous' 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.
• the gene 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 genome of the source organism.
• 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 codons different than the native gene). Allelic variations or naturally- occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
• the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, 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 th e currently available counting procedures.
• the preferred isotope may be selected from 3H, ⁇ C, 2 P, 35s, 36Q, 5i Cr , 57 o , 58 Co , 59 Fe , 90 Y> 125 I? 1 11, and i 86R e .
• Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, 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, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -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.
• 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 canis of the present invention c an be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art.
• 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.
• 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 c an 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 No. 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. More preferably, the DNA includes the coding sequence of the nucleotides of SEQ ID No. 1 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 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 -kD a immunoreactive protein of Ehrlichia canis in a human cell by a method including the steps of (a) contacting mRNA obtained from th e cell with the labeled hybridization probe; and (b) de tecting 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.
• high stringency 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.
• 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.
• substantially pure DNA DNA that is not part 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 th at 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 (PCR) or restriction endonuclease digestion) independent of o th er sequences.
• PCR polymerase chain reaction
• telomere sequence e.g., a telomere sequence which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein.
• a recombinant DNA which includes a porti on 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 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 .
• a subunit position in both of the two sequences is occupied by 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
• 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 for said protein.
• the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5.
• a "vector” may be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid. Vectors may be u 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 su ch control sequences will vary depending upon the cell selected and th e transformation method chosen.
• control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination o f transcription and translation.
• Methods which are well known to tho se skilled in the art can be used to construct expression vectors containing appropriate transcriptional 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.
• substantially pure protein a protein which has been separated from at least some of those components which naturally accompany it.
• the protein is substantially pure when it is at least 60%, by weight, free from the proteins and o th er naturally-occurring organic molecules with which it is naturally associated in vivo.
• 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 Ehrlichia canis 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 appropri ate 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 .
• substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes , or any other organism in which they do not naturally occur.
• th e invention also includes fragments (e.g., antigenic fragments) of th e 28-kDa immunoreactive protein of Ehrlichia canis (SEQ ID No. 2 o r SEQ ID No. 4 or SEQ ID No. 6).
• fragment as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues i n length, but less than the entire, intact sequence.
• Fragments of the 28 - kDa immunoreactive protein of Ehrlichia canis can be generated b y methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant 28-kDa immunoreactive 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 can be assessed by methods described herein.
• Purified 28 -kDa immunoreactive protein of Ehrlichia canis or antigenic fragments o f 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 protocol s known to those skilled in the art.
• 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.
• S tandard 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.
• fragments of the 28 - kDa immunoreactive protein of Ehrlichia canis which are encoded a t least in part by portions of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of the sequence has b e en 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.
• compositions that do not produce an allergic 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.
• su ch 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 c an also be emulsified.
• a protein may be formulated into a composition in a neutral or salt form.
• Pharmaceutically acceptable salts include th e 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, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms such as injectable solutions.
• aqueous 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.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which c an be employed will be known to those of skill in the art in light of th e present disclosure.
• one dosage could be dissolved in 1 mL of isotonic NaCI solution and either added to lOOOmL o f 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.
• a given polypeptide may vary in its immunogenicity. It is often necessary therefore to couple th e immunogen (e.g., a polypeptide of the present invention) with a carrier.
• th e immunogen e.g., a polypeptide of the present invention
• exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and human serum albumin.
• KLH keyhole limpet hemocyanin
• Other carriers m ay 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 a n d bis-biazotized benzidine. It is also understood that the peptide m ay be conjugated to a protein by genetic engineering techniques that are 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).
• 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, but precludes hybridization of random sequences.
• hybridization at low temperature and/or high ionic strength is term e d low stringency
• hybridization at high temperature and/or 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 th e sequences and to the presence of formamide in the hybridization mixture .
• engineered or "recombinant” cell is intended to refer to a cell into which a recombinant gene, su ch as a gene encoding an Ehrlichia chaffeensis antigen has b een introduced. Therefore, engineered cells are distinguishable fro m 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.
• the recombinant gene may be integrated into th e host genome, or it may be contained in a vector, or in a bacterial genome transfected into the host cell.
• Ehrlichia canis (Florida strain and isolates Demon, DJ, Jake, and Fuzzy) were provided by Dr. Edward Breithedt, (College 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 . 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- glutamine at 37°C. The intracellular growth in DH82 cells was monitored by presence of E.
• Ehrlichia canis genomic DNA was prepared by resuspending the renografin-purified ehrlichiae in 600 ⁇ l of 10 m M Tris-HCl buffer (pH 7.5) with 1 % sodium dodecyl sulfate (SDS, w/v ) and 100 ng/ml of proteinase K as described previously (McBride et al, 1996). This mixture was incubated for 1 hr at 56° C, and the nucleic acids were extracted twice with a mixture o f phenol/chloroform/isoamyl alcohol (24:24: 1 ).
• Plasmid DNA was pelleted b y absolute ethanol precipitation, washed once with 70% ethanol, dri ed and resuspended in lOmM Tris (pH 7.5). 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 Purification Kit (Qiagen, Santa Clarita, CA).
• Forward primer 793 (5-GCAGGAGCTGTTGGTTACTC-3') (SEQ ID NO. 16)
• an d reverse primer 1330 (5'-CCTTCCTCCAAGTTCTATGCC-3') (SEQ ID NO. 17) corresponded to nucleotides 313-332 and 823-843 of C.
• E. canis (a North Carolina isolate, Jake) DNA was amplified with primers 793 and 1330 with a thermal cycling profile of 95°C for 2 min, and 30 cycles of 95°C for 30 sec, 62° C for 1 min, 72°C for 2 mi n followed by a 72°C extension for 10 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 ' - ATATACTTCCTACCTAATGTCTCA-3', SEQ ID No. 18) and primer 1 330 (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 sequ enced bidirectionally with primers: Ml 3 reverse from the vector, 46f, ECa28SA2 (5'-AGTGCAGAGTCTTCGGTTTC-3', SEQ ID No. 19), ECa5.3 (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.
• the full length sequence of ECa28-l was determined using a Universal GenomeWalker 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 (Dral, EcoRV, Pvull, Seal, Stul) which produce blunt-ended DNA.
• An adapter (API) supplied in the kit was ligated to each end of E. canis DNA.
• the genomic libraries were used as templates to find th e unknown DNA sequence of the ECa28-l gene by PCR using a primer complementary to a known portion of the ECa28-l sequence and a primer specific for the adapter API .
• Primers specific for ECa28-l u s ed for genome walking were designed from the known DNA sequence derived from PCR amplification of ECa28-l with primers 793 (SEQ ID NO. 16) and 1330 (SEQ ID NO. 17).
• Primers 394 ( 5 ' -
• GCATTTCCACAGGATCATAGGTAA-3'; nucleotides 687-710, SEQ ID NO. 21) and 394C (5'-TTACCTATGATCCTGT GGAAATGC-3; nucleotides 710-687, SEQ ID NO. 22) were used in conjunction with supplied primer API to amplify the unknown 5' and 3' regions of the ECa28-l gene by PCR.
• a PCR product corresponding to the 5' region of th e ECa28-l gene amplified with primers 394C and API (2000-bp) was sequenced unidirectionally with primer 793C (5'-GAGTA ACCAACAGCTCCTGC-3', SEQ ID No. 23).
• a PCR product corresponding to the 3' region of the ECa28-l gene amplified with primers 394 an d API (580-bp) was sequenced bidirectionally with the same primers .
• Noncoding regions on the 5' and 3' regions adjacent to the op e n reading frame were sequenced, and primers EC280M-F ( 5 ' - TCTACTTTGCACTTCC ACTATTGT-3', SEQ ID NO. 24) and EC280M-R ( 5 ' - ATTCTTTTGCCACTATTT TTCTTT-3', SEQ ID NO. 25) complementary t o these regions were designed in order to amplify the entire ECa28-l gene .
• DNA was sequenced with an ABI Prism 377 DNA S equencer
• Eca28- 1 genes of seven E. canis isolates were amplified by PCR with primers EC280M-F (SEQ ID No. 24) and EC280M-R (SEQ ID No.
• the entire E. canis ECa28-l gene was PCR-amplified with primers-EC280M-F and EC280M-R and cloned into pCR2.1-TOPO TA cloning vector to obtain the desired set of restriction enzyme cleavage sites (Invitrogen, Carlsbad, CA).
• the insert was excised from pCR2.1 - TOPO with BstX 1 and ligated into pcDNA 3.1 eukaryotic expression vector (Invitrogen, Carlsbad, CA) designated pcDNA3.1/EC28 fo r subsequent studies.
• the pcDNA3.1/EC28 plasmid was amplified, a n d the gene was excised with a Kpnl-Xbal double digestion an d directionally ligated into pThioHis prokaryotic expression v e ctor (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 e 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).
• E. canis ECa28-l fusion protein was subj ected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4- 15% Tris- HCl gradient gels (Bio-Rad, Hercules, CA) and transferred to p ure nitrocellulose (Schleicher & Schuell, Keene, NH) using a semi-dry transfer cell (Bio-Rad, Hercules, CA). The membrane was incub ated with convalescent phase antisera from an E.
• the probe was produced by PCR amplification with primers EC280M-F and EC280M-R and digoxigenin (DIG)-labeled deoxynucleotide triphosphates (dNTPs) (Boehringer Mannheim, Indianapolis, IN) and digested with Asel.
• the digested probe (566-bp) was separated by agarose gel electrophoresis, gel- purified and then used for hybridization. The completely digested genomic E.
• 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-l 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).
• GenBank accession numbers for nucleic acid and amino acid sequences of the E. canis ECa28-l genes described in this study are: Jake, AF082744; Louisiana, AF082745 ; Oklahoma, AF082746 ; Demon, AF082747; DJ, AF082748; Fuzzy, AF082749; Florida,
• AF082750 Sequence analysis of ECa28-l 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 nd Oklahoma.
• chaffeensis p28 gene was accomplished with primers 793 and 1330, resulting in a 5 1 8 -bp PCR product.
• the nucleic acid sequence of the E. canis PCR produ c t was obtained by sequencing the product directly with primers 793 and 1330. Analysis of the sequence revealed an open reading frame encoding a protein of 170 amino acids, and alignment of the 5 1 8 -bp sequence obtained from PCR amplification of E. canis with the DNA sequence of E. chaffeensis p28 gene revealed a similarity greater th an 70%, 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 produ ced four PCR products (3-kb, 2-kb, 1 -kb, and 0.8-kb), and the 0.8 -bp product was sequenced bidirectionally using primers 394 and API .
• the deduced sequence overlapped with the 3' end of the 5 1 8 -bp product, extending the open reading frame 12-bp to a termination codon.
• An additional 625-bp of non-coding sequence at the 3' end o f the ECa28-l gene was also sequenced.
• Primer 394C was used t o amplify the 5' end of the ECa28-l gene with supplied primer API .
• 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-l gene and completed the 834-bp open reading frame encoding a protein of 278 amino acids.
• An additional 144-bp o f readable sequence in the 5' noncoding region of the ECa28-l gene was generated.
• Primers EC280M-F and EC280M-R were designed fro m complementary non-coding regions adjacent to the ECa28-l gene.
• the PCR product amplified with these primers was sequenced directly with the same primers.
• the complete DNA sequence (SEQ ID NO. 1) for the E. canis ECa28-l gene is shown i n Figure 1.
• the ECa28-l PCR fragment amplified with these primers contained the entire open reading frame and 17 additional ami no acids from the 5' non-coding primer region.
• the gene was directionally subcloned into pThioHis expression vector, and E. coli (BL21) were transformed with this construct.
• the expressed ECa28- l - thioredoxin fusion protein was insoluble.
• the expressed protein h ad an additional 114 amino acids associated with the thioredoxin, 5 amino acids for the enterokinase recognition site, and 32 amino acids from the multiple cloning site and 5' non-coding primer region at th e N-terminus.
• Convalescent-phase antiserum from an E. canis infected dog recognized the expressed recombinant fusion protein, but did n o t react with the thioredoxin control ( Figure 2).
• E. canis 28-kDa protein 1 gene (60%) and 28-kDa protein 2 gene (partial) (59.5%) were also homologous to ECa28-l .
• E chaffeensis omp-1 B had the least nucleic acid homology (45.1 %) with E. Ca28-l .
• Digestion with Asel produced 3 bands (approximately 566-bp, 850 - bp, and 3-kb) that hybridized with the ECa28-l DNA probe indicating the presence of multiple genes homologous to ECa28-l in the genome .
• Digestion with EcoRV and Spel produced two bands that hybridized with the ECa28-l gene probe.
• ECaSA3-2 5'-CTAGGATTA GGTTATAGTATAAGTT-3', SEQ ID No. 26
• primer 793C SEQ ID No. 23
• DNA was amplified with a thermal cycling profile of 95°C for 2 min, and 30 cycles of 95°C for 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.
• primer 46f 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, an d 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-l.
• An 800-bp PCR product was amplified which contained the 3' end of Eca28SA3, the intergenic region between ECa28SA3 and ECa28-l (28NC3) and the 5' end o f Eca28-1 , joining the previously separate loci ( Figure 8).
• the 849-bp open reading frame of ECa28SA2 encodes a 283 amino acid protein
• ECa28SA3 has an 840-bp open reading frame encoding a 280 amino acid protein.
• the intergenic noncoding region between ECa28SA3 and ECa28-l was 345-bp in length ( Figures 7 and 8)
• 28-kDa protein genes were aligned using the Clustal method t o examine the homology between these genes.
• the nucleic acid 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).
• the intergenic regions between the 28-kDa protein genes were analyzed for promoter sequences by comparison with consensu s Escherichia coli promoter regions and a promoter from E. chaffeensis
• Putative promoter sequences including RBS, - 10 and - 35 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.
• E. canis ECa28-l has a deduced molecular mass of 30.5-kDa and th e entire ECa28SA3 has a deduced molecular mass of 30.7-kDa.
• B oth proteins have a predicted N-terminal signal peptide of 23 amino acids (MNCKKIL ⁇ TALMSLMYYAPSIS, SEQ ID No. 27), which is similar to th at predicted for E. chaffeensis P28 (MNYKKEJTSALISLISSLPGV 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- l .
• An additional putative cleavage site at amino acid position 2 5 (MNCKKIL ⁇ TALISLMYSIPSISSFS, SEQ ID NO. 29) identical to th e predicted cleavage site of E. chaffeensis P28 (SFS) was also pres ent, and would result in a mature ECa28- l with a predicted molecular mas 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 ECa28SAl had an uncleavable signal sequence .
• Proteins of similar molecular mass have been identified and cloned from multiple rickettsial agents including E. canis, E chaffeensis, and C. ruminantium (Reddy et al , 1998; Jongejan et al, 1993; Ohashi et al , 1998).
• a single locus in Ehrlichia chaffeensis with 6 homologous p28 genes, and 2 loci in E. canis, each containing s o me 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.
• E.canis 28-kDa protein are homologous t o
• E. chaffeensis OMP-1 family and the MAP-1 protein of C. rumanintium The most homologous E. canis 28-kDa proteins (ECa28SA3, ECa28- l 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 ECa28SAl and ECa28SA2 were the least homologous with homology ranging from 50.9% to 59.4% and divergence of 53.3 to 69.9 % .
• 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, 1 997 ; 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 i n dogs may be related to differential expression of p28 genes resulting in antigenic changes in vivo, thus allowing the organism to evade th e immune response. 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. ruminantium map- 1 gene.
• Previous studies have identified a 30-kDa protein of E. canis th at 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 e antigenic differences between E. canis and E.
• chaffeensis P28 are located in these variable regions and are readily accessible to th e 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 e related to antigenic differences in the variable regions of P28 as well as in other immunologically important antigens of these ehrlichial species.
• E. canis 28-kDa proteins may be important immunoprotective antigens.
• 30-kDa antigen of E. canis exhibits strong immunoreactivity (Rikihisa et al , 1994; Rikihisa et al , 1992).
• Antibodies in 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 , 1997).
• antibodies to 30, 24 and 21 -kDa proteins developed early i n the immune response to E.

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