JP2002527042A - Granulocyte aerichia gene and use thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to granulocyte Ehrlichia (GE) proteins. In particular, the invention relates to nucleic acid molecules encoding WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOVE2 proteins; purified WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOV1 and SLOV2 proteins and polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, An antibody having specific binding affinity for SWED, BOV, EQ, SLOV1 and SLOV2 proteins and polypeptides; a hybridoma containing this antibody; WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and A nucleic acid probe used for detection of a nucleic acid encoding an SLOV2 protein; WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOVE2 protein and a polypeptide; A kit containing a nucleic acid probe or antibody; a bioassay using the nucleic acid sequence, protein, or antibody of the present invention to diagnose, evaluate, or determine the prognosis of a mammal infected with Ehrlichiosis. Therapeutic uses, especially including vaccines, including proteins, polypeptides or nucleic acids; and methods for preventing or inhibiting erhrichiosis in animals.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field of the Invention The present invention relates relates to proteins commonly granulocyte Erurikia (Ehrlichia) genus (GE). In particular, the invention relates to WI1, WI2, WI3, WI4, WIC, NY1, NY
2, a nucleic acid molecule encoding NY3, SWED, BOV, EQ, SLOV1 and SLOV2 proteins; purified WI1, WI2, WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1, and slov
2 proteins and polypeptides; recombinant nucleic acid molecules; cells containing recombinant nucleic acid molecules; WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
An antibody having specific binding affinity for WED, BOV, EQ, SLOV1 and SLOV2 proteins and polypeptides; a hybridoma comprising the antibody; WI
1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, B
Nucleic acid probes used for detection of nucleic acids encoding OV, EQ, SLOV1 and SLOV2 proteins; WI1, WI2, WI3, WI4, WIC, NY1, NY
2, a method for detecting a nucleic acid encoding a NY3, SWED, BOV, EQ, SLOV1 and SLOV2 protein or polypeptide in a sample; a kit containing a nucleic acid probe or an antibody; diagnosing and evaluating a mammal infected with aerial disease. Or a bioassay using a nucleic acid sequence, protein or antibody of the invention to determine prognosis; WI1, WI2, WI3, WI4, WIC, NY1,
The present invention relates to therapeutic uses, especially vaccines, including NY2, NY3, SWED, BOV, EQ, SLOV1 and SLOV2 proteins or polypeptides or nucleic acids; and methods for preventing or preventing erhrichiosis in animals.

2. Related Art Granulocytic aerial disease is a potentially lethal mite-borne infection.
The pathogenic bacterium, granulocyte Ehrlichia bacillus (GE), is an intrinsic bacterium 16S ribosomal RNA (
rRNA) using the universal primers (P
CR) by amplifying blood DNA of infected patients (Chen, et al., J. Clin. Micro., 32: 589-595 (1994)). GE 16S ・ r
Comparison of the RNA gene sequence with other known 16S rRNA gene sequences revealed a nearly identical match for the 16S gene between Ehrlichia phagocytophila and Ehrlichia equi (Chen, et al., 1994). Another two groups of Ehrlichia species, the Ehrlichia canis group and the Ehrlichia sennetsu group, also had 16S rRNA.
Classified according to gene sequence. E. canis and E. sennetsu species mainly infect mononuclear phagocytes (Dumler et al., N. Eng. J. Med., 325: 1109-1110 (1
On the other hand, members of the E. phagocytophila group, including GE, are granulocyte-tropic (Ristic et al., In Bergey's Manual of Systemic Bacteriology, Kreig et al.
al. eds., (1984), pp. 704-709). Approximate identity of 16S rRNA gene sequences and clear sharing of antigenicity by IFA and immunoblotting (Dumler et al.
al., J. Clin. Micro., 33: 1098-1103 (1995)) are E. phagocytophila, E. equi.
And GE are closely related.

[0003] Complete classification of the E. phagocytophila species, including antigenic relationships with respect to individual isolates, was not possible because this microorganism could not be cultured by cell culture, but the human promyelocytic leukemia cell lineage Reported that GE was successfully cultured in HL60 cells (Coughlin et al., PCT Application No. PCT / US96 / 10117; Goodman et al.
al., N. Eng. J. Med., 334: 209-215 (1996)). Walker et al., PCT Applicat
ion No. PCT / US97 / 09147 taught that an isolated gene encoding a 120 kDa immunodominant antigen of E. chaffeensis promotes specific antibody production in infected humans.

[0004] The present invention relates to 13 proteins (WI1, WI2, WI3, WI4, WIC, NY
1, NY2, NY3, SWED, BOV, EQ, SLov1, and slov2)
Disclosed are GE-specific genes that encode, which can be used as diagnostics and vaccines.

SUMMARY OF THE INVENTION The present invention provides WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3.
, SWED, BOV, EQ, SLOV1 and SLOV2 isolated nucleic acid molecules encoding polypeptides comprising amino acid sequences corresponding to proteins.

The present invention further provides WI1, WI2, WI3, WI4, WIC, NY1, NY2,
Provided is a purified polypeptide comprising an amino acid sequence corresponding to NY3, SWED, BOV, EQ, SLOVE, and SLOV2 proteins.

The present invention also provides WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
Provided is a nucleic acid probe for specifically detecting the presence of Y3, SWED, BOV, EQ, SLOV1 and SLOV2 proteins or polypeptides in a sample.

The present invention further provides WI1, WI2, WI3, WI4, WIC, NY1, NY2,
Provided is a method for detecting a nucleic acid encoding NY3, SWED, BOV, EQ, SLOV1 and SLOV2 proteins in a sample.

The present invention also provides WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
Provided is a kit for detecting the presence of a nucleic acid encoding Y3, SWED, BOV, EQ, SLOV1 or SLOV2 protein in a sample.

The present invention further provides, from 5 ′ to 3 ′, a recombinant nucleic acid molecule comprising a promoter effective to initiate transcription in a host cell and a nucleic acid molecule isolated as described above. I do.

[0011] The present invention also provides a recombinant nucleic acid molecule comprising a vector and the nucleic acid molecule separated as described above.

[0012] The present invention also provides a recombinant nucleic acid molecule comprising a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the polypeptide.

[0013] The present invention also provides a cell containing the recombinant nucleic acid molecule. The present invention further provides a non-human organism containing the recombinant nucleic acid molecule.

The present invention also provides WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
Antibodies with specific binding affinity for Y3, SWED, BOV, EQ, SLOVE, or SLOV2 proteins or polypeptides are provided.

[0015] The present invention further relates to WI1, WI2, WI3, WI4, WIC, NY1,
Provided is a method for detecting NY2, NY3, SWED, BOV, EQ, SLOVE, or SLOV2 protein or polypeptide therein.

The present invention also relates to WI1, WI2, WI3, WI4, WIC, N
Y1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOVE2
Methods for determining the amount of a protein or polypeptide are provided.

The present invention further provides WI1, WI2, WI3, WI4, WIC, NY1, NY2,
Methods are provided for detecting an antibody having specific binding affinity for NY3, SWED, BOV, EQ, SLOVE, or SLOV2 proteins or polypeptides.

The present invention further provides a diagnostic kit comprising: a first container containing the antibody; and a second container containing a binding partner of the monoclonal antibody and a label.

The present invention also provides a hybridoma that produces the monoclonal antibody.

The present invention further provides a method for diagnosing Ehrlichiosis. More particularly, the present invention further provides for the use of WI1, WI2, WI3, WI4,
Methods for identifying granulocyte aleuria in an animal are provided, including analyzing for nucleic acids, proteins, polysaccharides or antibodies of WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOV1 or SLOV2.

The present invention also provides WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
Provided are methods for therapeutic use involving all or a portion of the nucleic acids or proteins of Y3, SWED, BOV, EQ, SLOVE, or SLOV2. More specifically, the present invention further relates to WI1, WI2, WI3, WI4, WIC, NY1, N
Provided is a vaccine comprising a protein or nucleic acid of Y2, NY3, SWED, BOV, EQ, SLOV1 or SLOV2 together with a pharmaceutically acceptable diluent, carrier or additive. Provided that the protein or nucleic acid is present in an animal in an amount effective to induce a useful immune response to the protein.

The present invention also provides a method for preventing or preventing errickliosis in an animal, comprising administering the vaccine to the animal.

Other objects and advantages of the present invention will become apparent from the following description.

Definitions In the following description, a number of terms used in the art of recombinant DNA (rDNA) will be utilized extensively. The following definitions are provided to provide a clear and consistent understanding of the specification and claims, including the scope given by the terms.

Isolated nucleic acid molecule: As generally understood and used herein, an isolated nucleic acid molecule refers to a polymer of nucleotides, including but not limited to DNA and RNA.

Recombinant DNA: formed by joining together DNA segments from different sources,
A DNA molecule produced using recombinant DNA technology (ie, molecular genetic engineering).

DNA segment: As generally understood and used herein, a segment of DNA refers to a molecule consisting of a linear extension of nucleotides. Here, a nucleotide is present in a sequence that can encode a molecule comprising a linear sequence of amino acids, ie, a protein, protein fragment or polypeptide, according to the genetic code.

Gene: A DNA sequence for a single polypeptide chain or protein, as used herein, includes the 5 'and 3' untranslated ends. The polypeptide can be encoded by either the full-length sequence or a portion of the coding sequence, provided that the functional activity of the protein is maintained.

Complementary DNA (cDNA): A recombinant nucleic acid molecule synthesized by reverse transcription of messenger RNA (mRNA). Structural gene: A DNA sequence that is transcribed into mRNA, which is then translated into an amino acid sequence specific for a particular polypeptide.

Open reading frame (orf): The nature of a nucleic acid sequence that encodes one or more peptides within the same sequence, which sequence comprises a series of triplets encoding amino acids, the open reading frame of which. This is possible because the stop codon is not interrupted.

Restriction endonuclease: Restriction endonuclease (also called restriction enzyme) recognizes a specific base sequence (usually 4, 5, or 6 base pairs in length) in a DNA molecule, and this sequence is Is an enzyme capable of cleaving a DNA molecule at any position where it exists. For example, EcoRI recognizes the base sequence GAATTC / CTTAAG. Restriction fragment: DNA produced by digestion with restriction endonuclease
The molecule is called a restriction fragment. Any genome can be digested by a particular restriction endonuclease into an individual set of restriction fragments.

Agarose gel electrophoresis: Determining the length of restriction fragments requires an analytical method to sort duplex DNA molecules based on size. The most commonly, but not exclusively, technique used to accomplish this separation is agarose gel electrophoresis. The principle of this method is to move DNA molecules in a gel, such as a sieve, which delays the movement of the largest molecule to the maximum and the movement of the smallest molecule to the minimum. It should be noted that the smaller the DNA fragment in the agarose gel, the greater the mobility during electrophoresis.

The DNA fragments separated by agarose gel electrophoresis can be directly visualized by a staining operation if the number of fragments contained in the pattern is small. Visualization of genomic DNA fragments may be successful. However, many genomes, including the human genome, contain too many DNA sequences to produce a simple pattern of restriction fragments. For example, the human genome is digested with EcoRI into about 1,000,000 different DNA fragments. To visualize a small subpopulation of these fragments, a strategy called the Southern hybridization operation can be used.

Southern transfer method: The purpose of the Southern transfer method (also called blotting) is to physically transfer DNA fractionated by agarose gel electrophoresis to nitrocellulose filter paper or other suitable surface; This is a method in which transcription is performed while maintaining the relative positions of the obtained DNA fragments. Strategies used to achieve transfer from agarose gels to nitrocellulose include drawing DNA from the gel onto nitrocellulose paper by capillary action or electrophoretic transfer.

Nucleic acid hybridization: Nucleic acid hybridization relies on the principle that mixing two single-stranded nucleic acid molecules having complementary base sequences under suitable conditions will reform a thermodynamically favorable double-stranded structure. I do. This double-stranded structure is formed between two complementary single-stranded nucleic acids, even if one is immobilized on a nitrocellulose filter as in the case of a Southern hybridization transfer operation. The latter case corresponds to the Southern hybridization operation. As described above, the individual DNAs to be tested are digested with restriction endonucleases, fractionated by agarose gel electrophoresis, converted to single-stranded form, and transferred to nitrocellulose paper for reannealing to hybridization probes. Make it usable. Some examples of hybridization conditions are given in Ausubel, FM, et al., Current Protocols in Molecular Biology, John W.
ily & Sons, Inc., New York, NY (1989). For example, a nitrocellulose filter with 50% formamide, high salt (5 × SS
C [20X: 3M-NaCl / 0.3M-trisodium citrate] or 5XS
SPE [20X: 3.6M-NaCl / 0.2M-NaH 2 PO 4 /0.02M-E
DTA, pH 7.7], 5X Denhardt's solution, 1% SDS and 10%
Incubate overnight at 68 ° C. in a solution containing 0 μg / mL denatured salmon sperm DNA. This is subsequently selected based on the desired stringency: 0.2 X SSC / 0.1% at room temperature (low stringency), 42 ° C (intermediate stringency) or 68 ° C (high stringency). Wash several times with SDS. The temperature selected is determined based on the melting point (Tm) of the DNA hybrid.

Hybridization probe: To visualize a specific DNA sequence by the Southern hybridization procedure, a labeled DNA molecule or a hybridization probe is reacted with the fractionated DNA bound to the nitrocellulose filter paper. The portion of the filter where the DNA sequence complementary to the labeled DNA probe is present is itself labeled as a result of the reannealing reaction. The portion of the filter showing such a label is visualized. Hybridization probes are generally specific DNA
Created by molecular cloning of the sequence.

Oligonucleotide or oligomer: A molecule comprising two or more, preferably three or more deoxyribonucleotides or ribonucleotides. Its exact size will depend on a number of factors, in turn on the ultimate function or use of the oligonucleotide. Oligonucleotides can be derived synthetically or by cloning.

Amplification of a sequence: A method for producing a target sequence in large quantities. Generally, one or more amplification primers are annealed into a nucleic acid sequence. A sequence existing adjacent to the primer or a sequence existing between the primers is amplified using an appropriate enzyme.

Amplification primer: an oligonucleotide capable of annealing adjacent to a target sequence when placed under conditions that initiate the synthesis of a primer extension product complementary to a nucleic acid strand, Serves as a starting point for

Vector: A plasmid or phage DNA or other DNA sequence into which DNA can be inserted and cloned. The vector can replicate automatically in the host cell and can be further identified by one or a few endonuclease recognition sites, at which sites the DNA sequence can be cut in a predetermined manner and into which the DNA can be inserted. The vector can further include a marker suitable for use in identifying cells transformed with the vector. For example, the marker is tetracycline resistance or ampicillin resistance. Sometimes the term "vector" is replaced by "cloning vehicle".

Expression: Expression is the process by which a structural gene produces a polypeptide. This includes transcription of the gene into mRNA and translation of such mRNA into a polypeptide.

Expression vector: A vector or vehicle similar to a cloning vector, but which is capable of expressing a cloned gene after transformation into a host. The cloned gene is usually placed under the control of (ie, operably linked to) certain control sequences, such as, for example, a promoter sequence.

The expression control sequences will vary depending on whether the vector is designed to express a gene operably linked in a prokaryotic or eukaryotic host, eg, enhancer elements, termination sequences, tissues. Specific elements and / or transcriptional elements such as translation start and stop sites may additionally be included.

Functional derivative: A functional derivative of a sequence, whether a protein or nucleic acid, is a biological activity (either functional or structural) that is substantially similar to the biological activity of a protein or nucleic acid sequence. Is a molecule having Functional derivatives of proteins can include post-transcriptional modifications, such as carbohydrates, which are covalently linked depending on the need for such modifications to achieve a particular function. The term "functional derivative" is intended to include fragments, segments, variants, congeners, or chemical derivatives of a molecule.

As used herein, a molecule is a chemical derivative of the molecule when it contains additional chemical groups that are not normally part of a molecule. Such groups can improve the solubility, absorption, biological half-life, etc. of the molecule. In addition, this group can reduce the toxicity of the molecule and eliminate or attenuate unwanted side effects of the molecule.
Groups that can mediate such effects are described in Remington's Pharmaceutical Sciences (1980
). The operation of attaching such groups to a molecule is well known in the art.

Variant: A variant of a protein or nucleic acid is meant to indicate a molecule that is substantially similar in structure and biological activity to either a protein or nucleic acid. Thus, if two molecules have a common activity and are interchangeable, the term is used herein to
Even if the secondary, tertiary or quaternary structure of one molecule is not identical to that of another molecule, or the amino acid or nucleotide sequence is not identical, they are considered to be variants.

Allele: An allele is a variant of a gene that occupies a given locus on a chromosome.

Mutation: A mutation is any change in detectable genetic material that is transmitted to a daughter cell, possibly to a later generation, to produce a mutant cell or mutant individual. Can be. If the progeny of the mutant cell occurs only in somatic cells in a multicellular organism, a mutation spot or area of the cell is observed. Mutations in the germline of a sexually reproductive organism can be transmitted to the next generation by gametes, giving both somatic and germ cells individuals with new mutational conditions. A mutation is any unnatural change (or a combination thereof) of one or more deoxyribonucleotides that is detectable, affecting chemical or physical organization, variability, replication, phenotypic function or recombination. Can also;
Nucleotides can be added, deleted, substituted, inverted or transposed to new sites with or without inversion. Mutations can occur spontaneously or can be induced experimentally by the application of a mutagen. Mutants of the nucleic acid molecule result from the mutation.
Mutant polypeptides can also be derived from mutant nucleic acid molecules.

Species: A species is a naturally occurring group of populations that can actually or potentially cross. A variant in a nucleic acid or protein species is a change in the nucleic acid or amino acid sequence that occurs in the species, which can be determined by DNA sequencing the molecule in question.

Purification: A purified protein or nucleic acid is a protein or nucleic acid that has been separated from cellular components. A purified protein or nucleic acid is one that has been purified to a level of purity that does not occur in nature.

BRIEF DESCRIPTION OF THE DRAWINGS FIG . 1 is a schematic representation of the GE160 gene (S2 clone), used for PCR amplification using DNA extracted from the blood of animals, including infected humans. The schematic position of the primer set is shown. Thin line: DNA sequence; Bar graph: coding region with nucleotide numbers indicating start codon and stop codon based on S2 clone. FIG. 2 shows representative clones WI1, WI2, WI3, WI4, WIC, N of the present invention.
Y1, NY2, NY3, SWED, BOV, EQ, SLov1, and slov2
2 shows the nucleotide sequence derived from GE160 of S2 is usg3, WIC
Is named widget, EQ is named equi, and BOV is named swedbovine. FIG. 3 shows the DNA sequence of a representative clone WIC (SEQ ID NO: 1). FIG. 4 shows the DNA sequence of a representative clone SWED (SEQ ID NO: 2). FIG. 5 shows the DNA sequence of a representative clone EQ (SEQ ID NO: 3). FIG. 6 shows the DNA sequence of a representative clone BOV (SEQ ID NO: 4). FIG. 7 shows the DNA sequence of a representative clone WI1 (SEQ ID NO: 5). FIG. 8 shows the DNA sequence of a representative clone WI2 (SEQ ID NO: 6). FIG. 9 shows the DNA sequence of a representative clone WI3 (SEQ ID NO: 7). FIG. 10 shows the DNA sequence of a representative clone WI4 (SEQ ID NO: 8). FIG. 11 shows the DNA sequence of a representative clone NY1 (SEQ ID NO: 9). FIG. 12 shows the DNA sequence of a representative clone NY2 (SEQ ID NO: 10). FIG. 13 shows the DNA sequence of a representative clone NY3 (SEQ ID NO: 11). FIG. 14 shows the DNA sequence of a representative clone SLOV1 (SEQ ID NO: 12). FIG. 15 shows the DNA sequence of a representative clone SLOV2 (SEQ ID NO: 13). FIG. 16 shows the amino acid sequence of a representative clone WIC (SEQ ID NO: 14). FIG. 17 shows the amino acid sequence of a representative clone SWED (SEQ ID NO: 15). FIG. 18 shows the amino acid sequence of a representative clone EQ (SEQ ID NO: 16). FIG. 19 shows the amino acid sequence of a representative clone BOV (SEQ ID NO: 17). FIG. 20 shows the amino acid sequence of a representative clone WI1 (SEQ ID NO: 18). FIG. 21 shows the amino acid sequence of a representative clone WI2 (SEQ ID NO: 19). FIG. 22 shows the amino acid sequence of a representative clone WI3 (SEQ ID NO: 20). FIG. 23 shows the amino acid sequence of a representative clone WI4 (SEQ ID NO: 21). FIG. 24 shows the amino acid sequence of a representative clone NY1 (SEQ ID NO: 22). FIG. 25 shows the amino acid sequence of a representative clone NY2 (SEQ ID NO: 23). FIG. 26 shows the amino acid sequence of a representative clone NY3 (SEQ ID NO: 24). FIG. 27 shows the amino acid sequence of a representative clone SLOV1 (SEQ ID NO: 25). FIG. 28 shows the amino acid sequence of a representative clone SLOV2 (SEQ ID NO: 26). FIG. 29 shows representative clones WI1, WI2, WI3, WI4, WIC of the present invention.
NY1, NY2, NY3, SWED, BOV, EQ, SLov1, and slov
2 shows the deduced amino acid sequence corresponding to 2. GE160 of S2 is usg3, W
IC is named as widget, EQ is named as equi, and BOV is named as swedbovine.

[0052] Preferred embodiments recombinant clones 13 species was confirmed by PCR amplification of blood DNA of various animals including humans infected with the detailed description GE of (WI1, WI2, WI3, WI4 , WIC, NY
1, NY2, NY3, SWED, BOV, EQ, SLov1, and slov2)
The following describes sequencing and protein analysis. These clones have been proven to encode the GE160 gene (Storey et al. Infect. Immun., 66: 1).
356-1363 (1998)).

In a previous analysis by the inventors on a genomic expression library made from DNA of the USG3 strain, a convalescent canine serum library was screened to identify three genes. It appears likely that the genes described herein encode the immunoreactive GE160 antigen. Another immunodominant rickettsia antigen has proven to be important as an important diagnostic reagent and target for vaccines, including: the outer membrane polypeptide of Anaplasma marginale (Tebele et al., Infect. Immun.
., 59: 3199-3204 (1991)), an immunogenic protein of Cowdria rumantium (Mahan et al.
al., Microbiology, 140: 2135-2142 (1994); van Vliet et al., Infect.Imm.
un., 62: 1451-1456 (1994)), 120 kDa immunodominant protein of E. chaffeensis (Yu et al., J. Clin. Micro., 34: 2853-2855 (1996)), Rickettsia prowa
zekii immunodominant surface protein antigen (Dasch et al., in Microbiology, D. Schl
essinger (ed.), American Society for Microbiology, Washington, DC (19
84), pp. 251-256), and two Rickettsia rickettsii surface proteins (Anack
er et al., Infect. Immun., 55: 825-827 (1987); Sumner et al., Vaccine, 1
3: 29-35 (1995)). Many of these proteins contain highly repetitive regions similar to those found in GE proteins. Protein repeat domains have been shown to function in ligand binding (Wren, Mol. Microbiol., 5: 797-803 (1991)),
It may serve to promote rickettsia uptake by host cell membranes.

[0054] For purposes of clarity of disclosure, not for purposes of limitation, a detailed description of the invention is provided in the following sections:

I. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
An isolated nucleic acid molecule encoding a WED, BOV, EQ, SLOVE, and SLOV2 polypeptide.

II. Recombinantly produced WI1, WI2, WI3, WI4, WIC, NY1
, NY2, NY3, SWED, BOV, EQ, SLOVE, and SLOVE2 polypeptides.

III. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3,
A nucleic acid probe for specifically detecting SWED, BOV, EQ, SLOV1, and SLOV2.

IV. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
A method for detecting the presence of WED, BOV, EQ, SLOVE, or SLOV2 in a sample.

V. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
A kit for detecting the presence of WED, BOV, EQ, SLOV1 or SLOV2 in a sample.

VI. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
DNA containing WED, BOV, EQ, SLOVE1 and SLOV2 nucleic acid molecules
Constructs and cells containing these constructs.

VII. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3,
An antibody having a binding affinity for a SWED, BOV, EQ, SLOV1 or SLOV2 polypeptide and a hybridoma containing the antibody.

VIII. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3,
A method for detecting a SWED, BOV, EQ, SLOVE or SLOVE polypeptide or antibody in a sample.

IX. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
A diagnostic kit comprising WED, BOV, EQ, SLov1 or slov2 protein or antibody.

X. Diagnostic screening. XI. vaccine.

I. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
Isolated Nucleic Acid Molecules Encoding ED, BOV, EQ, SLOV1 and SLOV2 Polypeptides In one aspect, the invention pertains to isolated amino acid molecules and includes: (a) SEQ ID NOs: 5,6 , WI1, WI2, WI3, WI4 each containing the complete amino acid sequence represented by, 7, 8, 1, 9, 10, 11, 2, 4, 3, 12, and 13
A nucleotide sequence encoding a WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOV2 polypeptide;

(B) WI1, WI2 containing the complete amino acid sequence encoded by the polynucleotide
, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ
, A nucleotide sequence encoding a SLOV1 and SLOV2 polypeptide; and (c) a nucleotide sequence complementary to any of the nucleotide sequences of (a) or (b).

In a preferred aspect, the isolated nucleic acid molecule is SEQ ID NO: 5, 6, 7, 8, 1,
WI1, WI2, WI corresponding to 9, 10, 11, 2, 4, 3, 12, or 13
3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SL
Includes OV1 or SLOV2 nucleotide sequence. In another preferred aspect, the separated nucleic acid molecule comprises SEQ ID NOs: 5, 6, 7, 8, 1, 9, 10, 11, 2, 4,
WI1, WI2, WI3, WI4, W present in each of 3, 12, and 13
IC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or S
Contains the LOV2 nucleotide sequence. In another aspect, the isolated nucleic acid molecule is SEQ ID NOs: 18, 19, 20, 21, 14, 22, 23, 24, 15, 17, 16
, WI1, WI2, WI3, WI4, WI present in each of
C, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1, and SL
Encodes an OV2 amino acid sequence.

Also within the scope of the invention are the functional equivalents of the isolated nucleic acid molecules and derivatives thereof described herein. For example, SEQ ID NOs: 5, 6, 7, 8, 1
, 9, 10, 11, 2, 4, 3, 12 or 13 can be converted by functionally equivalent substitutions, additions or deletions. Due to the degeneracy of the nucleotide coding sequence, SEQ ID NOs: 18, 19, 20, 21, 14, 22, 23, 24, 1
Other DNA sequences that substantially encode amino acid sequences corresponding to the amino acid sequences of the peptides encoded by 5, 17, 16, 25 and 26 can also be used in the practice of the invention. Without limitation, these are SEQ ID NOs: 5, 6, 7, 8, 1, 9 that have been converted by substitutions at various codons encoding functionally equivalent amino acid residues within the sequence. WI, WI2, WI3, WI4, WIC, NY depicted for each of 10, 11, 2, 4, 3, 12 or 13
1, NY2, NY3, SWED, BOV, EQ, SLOV1 and SLOV2.

In addition, the nucleic acid sequence has SEQ ID NOs: 5, 6, 7, 8, 1, 9, 10, 11,
The 5'-end and / or 3 'of the nucleic acid formula shown in 2, 4, 3, 12 and 13
-May comprise a nucleotide sequence or a derivative thereof that is the result of the addition, deletion or substitution of at least one nucleotide at the terminus. In this regard, the additions, deletions or substitutions are made according to SEQ ID NOs: 18, 19, 20, 2 encoded by the nucleotide sequence.
Any nucleotide or polynucleotide can be used so long as the amino acid sequence of 1, 14, 22, 23, 24, 15, 17, 16, 25 and 26 is not substantially changed. Furthermore, the nucleic acid molecules of the present invention can have a restriction endonuclease recognition site added to their 5'-end and / or 3'-end if necessary. Therefore, the WI1, WI2, WI3,
WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE
All variants of the nucleotide sequence of the 1 and SLOV2 genes and fragments thereof are encompassed by the present invention.

Further, codons may be deleted or codons may be deleted to produce a polypeptide that is structurally modified but has substantially the same utility or activity as the polypeptide produced by the unmodified nucleic acid molecule. One or more can be replaced by a codon other than a degenerate codon. As is recognized in the art, the two polypeptides are functionally equivalent, even as the nucleic acid molecule that causes its production, even though the difference in the nucleic acid molecule is not related to the degeneracy of the genetic code.

A. In one aspect of the invention, WI1, WI2, WI3, WI4, WIC, NY1, N
Provided is an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence corresponding to Y2, NY3, SWED, BOV, EQ, SLOV1, and SLOV2. In particular, the nucleic acid molecule can be isolated from a biological sample (preferably from mammals or ticks) containing GE RNA or DNA.

The nucleic acid molecule can be isolated from a biological sample containing GE RNA using techniques of cDNA cloning and extinction hybridization. The nucleic acid molecule can also be separated from the cDNA library using a homologous probe.

The nucleic acid molecule can be isolated from a biological sample containing genomic DNA or a genomic library. Suitable biological samples include, but are not limited to, whole organisms, organs, tissues, blood and cells. The method of obtaining a biological sample will vary depending on the nature of the sample.

Those skilled in the art will realize that the genome can have few allelic variants between individuals. Therefore, if the sequence is WI1, WI2, WI3, W
I4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1
It is intended that the separated nucleic acid molecules also include allelic variants, as long as they are functional derivatives of the sequence encoding SLOV2. WI1, WI2, W
I3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, S
SEQ ID NOS: 5, 6, 7, 8, 1, 9, 10
, 11, 2, 4, 3, 12, or 13, when it does not encode a sequence identical to that found in WI1, WI2, WI3, WI4, WIC, NY1, NY2
, NY3, SWED, BOV, EQ, SLOV1 or SLOV2 can be isolated and identified using the same techniques as used herein, and especially primers based on the sequences disclosed herein. Use PCR technology to amplify the appropriate gene to use.

Those skilled in the art will appreciate that organisms other than GE are WI1, WI2, WI3, WI
4. It will be noticed that it will contain the WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOV2 genes. Although not limited thereto, the present invention relates to WI1, WI2, WI3,
WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE
1 and SLOV2 nucleic acid molecules. Also, infected eukaryotes (eg, humans, birds and fish for mammals) are WI1, WI2, WI3, W3.
I4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1
And the SLOV2 gene.

B. Nucleic Acid Synthesis Isolated nucleic acids of the invention are also intended to include chemically synthesized ones. For example, WI1, WI2, WI3, WI4, WIC, NY1, NY2
, NY3, SWED, BOV, EQ, SLOV1 or SLOV2 nucleic acid molecules having a nucleotide sequence encoding the expression product can be designed and, if necessary, divided into appropriately small fragments. Oligomers corresponding to each of the nucleic acid molecules or split fragments can then be synthesized. Such synthetic oligonucleotides are described, for example, in Matteucci et al., J. Am. Chem. Soc., 103, 3185-319.
2 (1981), or by using an automated DNA synthesizer.

The oligonucleotide can be derived synthetically or by cloning. If necessary, the 5'-end of the oligomer can be phosphorylated using T4-polynucleotide kinase. Phosphorylation of the single strand prior to annealing or phosphorylation for labeling can be achieved using an excess of enzyme. If phosphorylation is for labeling the probe, ATP can have high radioactivity. The DNA oligomer can then be subjected to annealing and ligation with a T4 ligase, etc.

II. Recombinantly produced WI1, WI2, WI3, WI4, WIC, NY1,
NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOVE2
In another embodiment of the peptide , the invention is a purified (preferably substantially pure) polypeptide comprising WI1, WI2, WI3, WI4, WIC, NY1, NY2,
NY3, SWED, BOV, EQ, SLLV1 or SLOVE2 or a functional derivative thereof, or a variant thereof, or at least 6 amino acids (preferably at least 10, 15, 20, 25 or 50 contiguous amino acids) Amino acids)
Having an amino acid sequence corresponding to the polypeptide sequence encoded by the nucleic acid sequence corresponding to

In a preferred embodiment, the invention relates to WI1, WI2, WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1, and slov
2 epitopes. The epitope of these polypeptides is an immunogenic or antigenic epitope. An immunogenic epitope is a portion of a protein that elicits an antibody response when the entire protein is an immunogen. An antigenic epitope is a fragment of a protein that can elicit an antibody response. Methods for selecting antigenic epitope fragments are well known in the art (Sutcliffe et al., Science, 219:
660-666 (1983)). The peptides and polypeptides of the present invention having an antigenic epitope are useful for generating an immune response that specifically recognizes the polypeptide.
Peptides and polypeptides of the invention having an antigenic epitope contain at least 7 (preferably 9, 10, 12, 15 or 20 amino acids) of a protein of the invention.

WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWE
Amino acid sequence variants of D, BOV, EQ, SLOV1 and SLOV2 are DNA
Can be produced by mutation. Such variants include, for example, SEQ ID NO: 18,
19, 20, 21, 14, 22, 23, 24, 15, 17, 16, 25 or 2
6 includes deletion or insertion or substitution of residues in the amino acid sequence of the peptide encoded by the peptide. If the final construct has the desired activity, deletions, insertions and substitutions can be made to arrive at the final construct.

The site for introducing a mutation in the amino acid sequence is determined in advance, but the mutation itself need not be determined in advance. For example, random mutagenesis at the targeted codon or region to optimize the outcome of the mutation at a given site can be used to generate WI1,
WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV
, EQ, SLOV1 and SLOV2 mutants can be expressed and screened for the optimal combination of desired activities. Techniques for making substitution mutations at predetermined sites in known DNA are well known, for example, site-directed mutagenesis.

WI1, WI2, WI3, WI4, WIC, NY1, NY2 according to this specification
, NY3, SWED, BOV, EQ, SLOV1 or SLOV2 variants are preferably achieved by site-directed mutagenesis of DNA encoding a non-mutant or previously produced variant of the protein. Site-directed mutagenesis is by using specific oligonucleotide sequences encoding the DNA sequence of the desired mutant, WI1, WI2, WI3, WI4, WIC, NY1.
, NY2, NY3, SWED, BOV, EQ, SLOV1 and SLOV2. Techniques for site-directed mutagenesis include, for example, Adelman et a
l., DNA, 2: 183 (1983); Ausubel et al., "Current Protocols in Molecular
Biology ", J. Wiley & Sons, New York, NY, 1996, as is well known in the art.

As will be appreciated, site-directed mutagenesis techniques can employ phage vectors that exist in either single-stranded or double-stranded form. Typical vectors useful for site-directed mutagenesis are described, for example, in Messing et al., Third Cleveland Symposium.
on Macromolecules and Recombinant DNA, A. Walton (ed.), Elsevier, Amster
and vectors such as the M13 phage disclosed in dam (1981). These phages are readily available commercially and their use is generally well known to those skilled in the art. Alternatively, to obtain single-stranded DNA, a plasmid vector containing a single-stranded phage replication origin can be employed (Vieira et al., Meth.
Enzymol., 153: 3 (1987)).

In general, site-directed mutagenesis according to the present invention begins with obtaining a single-stranded vector containing a DNA sequence encoding a protein related to that sequence. Generally, oligonucleotide primers having the desired mutated sequence are produced synthetically, for example, by the method of Crea et al., Proc. Natl. Acad. Sci. (USA), 75: 5765 (1978). The primer is then annealed with a vector containing a single-chain protein sequence and treated with a DNA polymerase enzyme such as the Klenow fragment of E. coli polymerase I to complete the synthesis of the mutation-containing strand.
The sequence thus mutated and the second strand have the desired mutation. next,
The heteroduplex vector is used to transform appropriate cells and a clone containing the recombinant vector having the mutated sequence is selected.

After selecting such a clone, the mutated protein region was removed and
It is introduced into a suitable vector for protein production, generally an expression vector of a type that can be employed for transformation of a suitable host.

[0086] Amino acid sequence deletions generally comprise from about 1 to 30 residues, more preferably 1 residue.
In the range from 10 to 10 and is typically continuous.

For amino acid insertions, an amino-terminal and / or carboxy-terminal fusion from one residue to a polypeptide of essentially unlimited length, and a sequence of one or more amino acid residues Includes internal insertion. Intra-sequence insertion (ie, WI1, W
I2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV,
The insertion into the complete sequence of the EQ, SLOV1 or SLOV2) can generally range from about 1 to 10 residues, preferably 1 to 5 residues.

The third group of variants is WI1, WI2, WI3, WI4, WIC, NY1, NY.
2, NY3, SWED, BOV, EQ, SLOV1 or SLOV2, wherein at least one, and preferably only one, amino acid residue is removed and another residue is inserted in its place. Such replacements are WI1, WI2, WI3, WI4
, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOV2.

Table 1

[Table 1]

Substantial changes in functional or immunological identity may result in the selection of less conservative substitutions than those described in Table 1, ie, (a) the structure of the polypeptide backbone in the region of the substitution, eg, a sheet Or (h) maintaining the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain; by selecting residues whose effects are more distinctly different. Commonly contemplated substitutions are (a) replacing glycine and / or proline with other amino acids, or deleting or inserting; (b) replacing a hydrophilic residue, eg, ceryl or threonyl, with a hydrophobic residue, eg, leucyl. , Isoleucyl, phenylalanyl, valyl or alanyl and the like; (c) replacing a cysteine residue with another residue;
d) replacing residues having an electrically positive side chain, such as lysyl, arginyl or histidyl, with residues having an electrically negative charge, such as glutamyl or aspartyl; or (e) bulky side chains , For example, phenylalanine, etc., are substituted with those without such side chains, such as glycine.

Certain deletions and insertions and substitutions are made in WI1, WI2, WI3, WI4, WI
C, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SL
It is not expected to cause drastic changes in the properties of OV2. However, the substitution,
When it is difficult to predict the exact effect of a deletion or insertion before making it, one skilled in the art will evaluate the effect by routine screening assays. For example, variants are typically native WI1, WI2, WI3, WI4,
Performing site-directed mutagenesis of nucleic acids encoding WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1, or SLOV2, expressing mutant nucleic acids in recombinant cell culture, and, if desired, cell culture From the product, for example by immunoaffinity adsorption onto a column (the variant is adsorbed by binding to at least one remaining immune epitope). Then cell lysate or purified WI1, W
I2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV,
The activity of the EQ, SLOV1 or SLOV2 molecule variants is screened for the desired properties by a suitable screening assay. For example, WI1, WI2,
WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ,
Changes in the immunological properties of the SLOV1 or SLOV2 molecule, such as affinity for a given antibody, are measured by a competitive immunoassay. Changes in immunomodulatory activity are measured by a suitable assay. Modifications of protein properties such as redox or thermal stability, hydrophobicity, receptivity to proteolysis or propensity to aggregate into carriers or complexes are assayed using methods well known to those of ordinary skill in the art. .

[0092] Various strategies known in the art can be used to obtain the peptides of the present invention. In one embodiment, the peptide is purified from tissues or cells that naturally produce the peptide. Alternatively, WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED can be prepared using the separated nucleic acid fragments.
, BOV, EQ, SLOV1 or SLOV2 protein can be expressed in any organism. Samples of the present invention include cells, protein extracts, or membrane extracts of cells, or body fluids. This sample will vary based on the assay format, detection method and the nature of the tissue, cells or extracts used as the sample.

[0093] Any prokaryote (preferably granulocyte Ehrlichia) can be used as a source of the peptide of the present invention as long as the organism naturally contains this peptide. Eukaryotes infected with granulocyte alericia can also be used as source organisms.
As used herein, "source organism" refers to whether the organism expresses a subunit of the amino acid sequence, and whether the subunit is ultimately separated from the organism.
Shows the organism from which the subunit of the amino acid sequence is derived.

Those skilled in the art can readily use known methods for separating proteins to obtain proteins free of natural contaminants. These methods include, but are not limited to, immunochromatography, size exclusion chromatography, HPLC, ion exchange chromatography and immunoaffinity chromatography.

III. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
For specifically detecting WED, BOV, EQ, SLOVE1 and SLOVE2
In another aspect of the nucleic acid probe , the present invention provides WI1, WI2, WI3, WI4, WIC, NY1
, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOVE2 nucleic acid probe for specifically detecting the presence of a nucleic acid in a sample,
I2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV,
It relates to a nucleic acid probe comprising said nucleic acid molecule or at least a fragment thereof which binds under stringent conditions to an EQ, SLOV1 or SLOV2 nucleic acid.

In one preferred embodiment, the present invention relates to an isolated nucleic acid probe, wherein the probe comprises 10 to 1000 nucleotides (preferably 10 to 500,
10 to 100, 10 to 50, 10 to 35, 20 to 100
0, 20 to 500, 20 to 100, 20 to 50 or 20
From 35), which preferentially hybridize with RNA or DNA of granulocyte Ehrlichia, but which are not granulocyte Ehrlichia (e.g., humans).
It does not hybridize with NA or DNA, and the nucleic acid probe has the following sequence: (a) SEQ ID NOs: 5, 6, 7, 8, 1, 9, 10, 11, 2, 4, 3, 12, or 13, WI1, WI containing the complete amino acid sequence corresponding to the amino acid sequence encoded by
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
A nucleotide sequence encoding a Q, SLOV1 or SLOV2 polypeptide; (b) WI1, WI2 comprising the complete amino acid sequence encoded by the polynucleotide
, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ
, A nucleotide sequence encoding a SLOV1 and SLOV2 polypeptide; (c) a nucleotide sequence complementary to any of the nucleotide sequences described in (a) or (b); and (d) a nucleotide sequence described so far; At least 10 (preferably 15, 20, 25 or 30) from nucleic acids comprising a polynucleotide sequence that is at least 90% identical to the selected sequence
Are contiguous nucleotides or nucleotides complementary thereto.

This nucleic acid probe can be used to probe a suitable chromosomal or cDNA library by using conventional hybridization techniques to obtain another nucleic acid molecule of the invention. Chromosomal DNA or cDNA libraries can be prepared from appropriate cells according to art-recognized methods (Molecular Cloning: A La
boratory Manual, 2nd edition, edited by Sambrook, Fritsch, & Maniatis,
Cold Spring Harbor Laboratory, 1989).

Separately, SEQ ID NOS: 5, 6, 7, 8, 1, 9, 10, 11, 2, 4, 3,
Chemical synthesis is performed to obtain a nucleic acid probe having a nucleotide sequence corresponding to the amino and carboxy terminal portions of the amino acid sequence corresponding to the amino acid sequence of the peptide encoded by 12 or 13. Thus, the synthesized nucleic acid probes are essentially PCR Protocols, A Guide to Methods and Applications, edited b
y Using the appropriate chromosome, cDNA or cell line library to obtain a fragment of the invention, using as primers in the polymerase chain reaction (PCR) according to the recognized PCR technique by Michael et al., Academic Press, 1990. Can be.

Those skilled in the art can readily design such probes based on the sequences disclosed herein using the methods of computer-based alignment and sequence analysis known in the art. Yes (Molecular Cloning: A Laboratory Manual, 2nd
edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor La
boratory, 1989).

The hybridization probes of the present invention can be labeled by standard labeling techniques such as, for example, radiolabeling, enzymatic labeling, fluorescent labeling, biotin-avidin labeling, chemiluminescence, and the like. After hybridization, the probes can be visualized using known methods.

The nucleic acid probe of the present invention includes an RNA probe and a DNA probe,
Such probes can be made using techniques known in the art.

[0102] In one embodiment of the method, the nucleic acid probe is immobilized on a solid support. Examples of such solid carriers include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylics such as polyacrylamide, and latex beads. Including. Techniques for attaching nucleic acid probes to these solid supports are well known in the art.

Test samples suitable for the nucleic acid probe method of the present invention include, for example, cells or nucleic acid extracts of cells or biological fluids. The sample used in the above method is an assay method,
It will vary based on the method of detection and the nature of the tissue, cell or extract. Methods for producing nucleic acid extracts of cells are well known in the art and can be readily adapted to obtain a sample that is compatible with the method used.

IV. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
Detect the presence of ED, BOV, EQ, SLov1 and slov2 in the sample
In another aspect of the method , the invention relates to a method for providing WI1, WI2, WI3, WI4, W
IC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or S
The present invention relates to a method for detecting the presence of LOV2, the method comprising: (a) contacting a sample under specific hybridization conditions such that a hybrid forms with the nucleic acid probe; and (b) detecting the presence of the probe bound to the nucleic acid molecule. Detecting the presence. Alternatively, in another preferred aspect, WI1, WI2, WI3, W
I4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1
And a method for detecting the presence of a SLOV2 nucleic acid in a sample comprises: (a) amplifying the nucleic acid in the sample by a PCR technique using a nucleic acid probe; and (b) detecting the presence of the amplified nucleic acid molecule. Including. One skilled in the art will select nucleic acid probes according to techniques known to those skilled in the art. Samples to be tested include, but are not limited to, RNA samples obtained from human tissues.

V. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
Detect the presence of ED, BOV, EQ, SLov1 or slov2 in the sample
In another aspect, the present invention provides a kit comprising WI1, WI2, WI3, WI4, WI
C, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SL
A kit for detecting the presence of an OV2 nucleic acid, the kit comprising at least one container containing the nucleic acid probe. In a preferred embodiment, the kit further comprises a container containing one or more of the following: a detergent and a reagent capable of detecting the presence of the bound nucleic acid probe. Examples of detection reagents include, but are not limited to, radiolabeled probes, enzyme-labeled probes (horseradish peroxidase, alkaline phosphatase) and affinity-labeled probes (biotin, avidin or streptavidin).

In particular, compartmentalized kits include any kit in which the reagents are contained in separate containers. Such containers include small glass containers, plastic containers, or strips of plastic or paper. This container allows for the efficient transfer of reagents from one compartment to another, but avoids cross-mixing of samples and reagents, and ensures that the reagents or solutions in each container are in one compartment in a quantitative manner. Can be added to another compartment. This container contains the sample to be tested, the container containing the probe or primer used for the assay, the container containing the detergent (phosphate buffered saline, Tris buffer, etc.), and the probe forming the hybrid,
A container containing reagents used to detect bound antibody, amplified product, etc. would also be included.

[0107] Those skilled in the art will readily recognize that the nucleic acid probes described herein can be introduced into one of the well-known kit formats in the art.

VI. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
DNA structures containing ED, BOV, EQ, SLOVE, or SLOVE2 nucleic acid molecules
In another aspect of the constructs and cells containing these constructs , the present invention provides a recombinant DNA molecule comprising a promoter effective to initiate transcription in a 5 'to 3' host cell and said nucleic acid molecule. About. In another aspect, the invention relates to a vector and a recombinant DNA molecule comprising said nucleic acid molecule.

In another aspect, the present invention comprises a transcription control region that functions in a cell, a sequence that is complementary to an RNA sequence encoding an amino acid sequence corresponding to the polypeptide, and a transcription termination region that functions in a cell. Related to nucleic acid molecules.

Preferably, said molecule is an isolated and / or purified DNA molecule. In another aspect, the invention relates to a cell or a non-human organism comprising said nucleic acid molecule.

In another aspect, the peptide has been purified from cells that have been converted to express the peptide.

As used herein, a cell that does not normally produce or normally produces only low levels of a protein, and that has been genetically engineered to produce that protein, is referred to as the desired peptide. Cells transformed to express the ". Those skilled in the art can readily adapt the techniques for introducing and expressing genomic, cDNA or synthetic sequences into eukaryotic or prokaryotic cells.

A polypeptide is described as “expressible” if a nucleic acid molecule, such as DNA, contains a nucleotide sequence containing transcriptional and translational control information, and such a sequence is referred to as a “nucleotide sequence encoding a polypeptide. Operatively associated with the device ". An operable linkage is a linkage in which the regulatory DNA sequence and the DNA sequence to be expressed are related in such a way as to express the gene sequence. The precise nature of the regulatory regions required for gene sequence expression varies from organism to organism, but generally in prokaryotes, both a promoter (which directs the initiation of RNA transcription) as well as a DNA sequence which, when transcribed into RNA, initiate synthesis. The promoter region should be included. Such regions normally include 5'-noncoding sequences involved in transcription initiation and translation, such as, for example, TATA boxes, capping sequences, CAAT sequences, and the like.

If desired, WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY
The non-coding region for 3 'of the 3, SWED, BOV, EQ, SLOVE, or SLOV2 coding sequence can be obtained by the method described above. This region can carry its transcription termination regulatory sequences, such as termination and polyadenylation. Therefore, WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
The DNA sequence encoding the Y3, SWED, BOV, EQ, SLOV1 or SLOV2 gene can maintain a naturally contiguous 3'-region to provide a transcription termination signal. When the transcription termination signal does not work well in the expression host cell,
It can be replaced with a 3 'region that functions in the host cell. If the bond between two DNAs is
(1) its introduction causes a frame shift; (2) WI1, WI2, WI3, WI4
Inhibits the ability of the promoter region sequence to direct transcription of the WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOV2 coding sequence; and (3) WI1, WI2 to be transcribed by the promoter region sequence.
WI3, WI4, WIC, NY1, NY2, SWED, BOV, EQ, SLOVE
1 or SLOV2 coding sequence; if not, two DNA sequences (eg, promoter region sequence and WI1, WI
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
Q, SLOV1 or SLOV2 coding sequence) are described as operably linked. Thus, if a promoter is capable of effecting transcription of a DNA sequence, it can be said that the promoter region is operably linked to the DNA sequence.

The present invention relates to WI1, WI2, WI3, WI4, in prokaryotic or eukaryotic cells.
Includes expression of the WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE, or SLOV2 coding sequence (or a functional derivative thereof). Prokaryotic hosts are generally the most efficient and convenient for the production of recombinant proteins and are therefore WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
It is suitable for the expression of WED, BOV, EQ, SLOVE or SLOVE2 coding sequences.

Prokaryotes are most often represented by various strains of E. coli. However, other microbial strains can be used, including other bacterial strains. In prokaryotic systems, plasmid vectors containing replication sites and control sequences derived from species compatible with the host cell can be used. Examples of suitable plasmid vectors include pBR322, pU
C18, pUC19, pUC118, pUC119, etc .; examples of suitable phage or bacteriophage vectors include λgt10, λgt11, etc .; examples of suitable viral vectors include pMAM-neo, pKRC, etc. Selected preferred vectors of the present invention have the ability to replicate in selected host cells.

Recognized prokaryotic hosts include, for example, E. coli, Bacillus, Streptomyces
, Pseudomonas, Salmonella, Serratia and others. However, under these conditions, the peptide is not glycosylated. Prokaryotic hosts must be compatible with the replicon and control sequences in the expression plasmid.

In prokaryotic cells, WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
In order to express Y3, SWED, BOV, EQ, SLOVE1 or SLOV2, WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
It is necessary to operably link the ED, BOV, EQ, SLOVE1 or SLOV2 coding sequence to a structural prokaryotic promoter. Such a promoter can be either constitutive or, more preferably, regulatable (ie, inducible or activating). Examples of constitutive promoters include bacteriophage λ i
nt promoter, bla promoter of β-lactamase gene sequence of pBR322, and CAT promoter of chloramphenicol acetyltransferase gene sequence of pBR325, and the like. Examples of inducible prokaryotic promoters include the major right and major left promoters of bacteriophage λ.
(P _L and P _R), E. Coli of trp, recA, lacZ, lacI, and g
al promoter, B-subtilis α-amylase (Ulmanen et al., J. Bacteri
ol., 162: 176-182 (1985)) and the ζ-28-specific promoter (Gilman et al.
., Gene Sequence, 32: 11-20 (1984)), Bacillus bacteriophage promoter (Gryczan et al., In: The Molecular Biology of the Bacilli, Acade
mic Press, Inc. NY, (1982)), and the Streptomyces promoter (Ward et al.,
Mol. Gen. Genet., 203: 468-478 (1986)). Prokaryotic promoters include Glick (J. Ind. Microbiol. 1: 277-282 (1987)); Centatiempo (Biochimie, 6
8: 505-516 (1986)); and Gottesman (Ann. Rev. Genet., 18: 415-442 (1984)).
) There is a comprehensive review.

[0119] Proper expression in prokaryotes also requires the presence of a ribosome binding site upstream of the sequence encoding the gene sequence. Such ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol., 35: 365-404 (1981)).

[0120] The choice of control sequences, expression vectors, transformation methods, and others, will depend on the type of host cell used to express the gene. As used herein, “cell,” “cell line,” and “cell culture” may be used interchangeably, but all such terms include progeny. Thus, the term "transformants" or "transformed cells" includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or accidental mutations. However, the progeny of the mutant as defined herein has the same function as that of the originally transformed cell. Host cells that can be used in the expression system of the present invention include WI1, WI2, WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1 or SLov
It is not strictly limited as long as it is suitable for use in expressing two peptides. Suitable hosts include eukaryotic cells.

Suitable eukaryotes include, for example, yeast, mold, insect cells in vivo or in tissue culture,
Includes mammalian cells. Suitable mammalian cells include HeLa cells, eg, VERO
Or cells of fibroblast origin, such as CHO-K1, or cells of lymphocyte origin and derivatives thereof.

In addition, plant cells can also be used as hosts, and regulatory sequences compatible with plant cells, such as cauliflower mosaic virus 35S and 19S, and the nopaline synthase promoter and polyadenylation signal sequence are also available. It is possible.

Other suitable hosts are insect cells such as, for example, Drosophila larvae. To use insect cells as a host, a Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science, 240: 1453-1459 (1988)). Separately, if the baculovirus vector is processed, a large amount of WI1, WI2, WI3, WI4,
WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOV1 or SLOV2 can be expressed in insect cells (Jasny, Science 238: 1653 (1987); Mill)
er et al., In: Genetic Engineering (1986), Setlow, JK et al. eds., Pl
enum, Vol. 8, 277-297).

Different host cells are responsible for the translation and post-translational processing and modification of proteins (
It has characteristic and specific mechanisms for, for example, glycosylation, cleavage).
Selection of the appropriate cell line or host system can ensure the desired modification and processing of the foreign protein expressed.

[0125] A series of yeast gene sequence expression systems, into which a promoter and a termination element obtained from a gene sequence encoding an actively expressed glycolytic enzyme are introduced, can be used. This enzyme is produced in large quantities when yeast grows in a glucose-rich medium. Known glycolytic gene sequences also provide very efficient transcription control signals.

Yeast offers the substantial advantage that post-translational peptide modifications can be made.
There are a number of recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids available to produce the desired protein in yeast.
Yeast recognizes leader sequences in cloned mammalian gene sequences and secretes leader sequences with peptides (ie, pre-peptides). WI1, WI2,
WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ,
Several possible vector systems are available in mammalian hosts for the expression of SLOV1 or SLOV2.

A wide variety of transcriptional and translational regulatory sequences can be employed, depending on the nature of the host.
Transcriptional and translational control signals can be derived from viral sources such as, for example, adenovirus, bovine papilloma virus, simian virus, etc., but the control signals are associated with specific gene sequences with high levels of expression. Alternatively, promoters for mammalian expression products such as, for example, actin, collagen, myosin, etc. can be employed. The transcription initiation control signal can be selected to control or activate such that the expression of the gene sequence can be modulated. Of interest are those temperature-sensitive regulatory signals that repress or initiate expression by altering the temperature, or those that are subject to chemical regulation (such as metabolites).

As described above, WI1, WI2, WI3, WI4, WIC, in eukaryotic hosts.
NY1, NY2, NY3, SWED, BOV, EQ, SLov1 or SLov
Expression of 2 requires the use of eukaryotic regulatory regions. This region will generally include sufficient promoter region to direct initiation of RNA synthesis. Suitable eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen., 1: 273-288 (1982)); the herpesvirus TK promoter (McKnight , Cell, 31: 355-365 (1982)); SV40 early promoter (Benoist et al., Nature (London), 290: 304-310 (1981)); yeast gal4 gene sequence promoter (Johonston et al., Proc. Natl. Acad. Sci.
(USA), 79: 6971-6975 (1982); Silver et al .: Proc. Natl. Acad. Sci. (USA)
, 81: 5951-5955 (1984)); and the CMV immediate-early gene promoter (Thomsen et al.).
Natl. Acad. Sci. (USA), 81: 659-663 (1984)).

As is widely known, translation of eukaryotic mRNA begins at the first methionine-encoding codon. Eukaryotic promoters and WI1, WI
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
It is preferred to ensure that the binding between the Q, SLOV1 or SLOV2 coding sequence is not interrupted by a codon capable of encoding methionine (AUG). The presence of this codon indicates the formation of a fusion protein (AUG codon is WI1, WI2
, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ
, SLOV1 or SLOV2 coding sequence (if in the same open reading frame) or frameshift mutation (AUG codon is WI1, WI2, WI3, WI4, W
IC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1, and S
(If not in the same open reading frame as the LOV2 coding sequence).

WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWE
The D, BOV, EQ, SLOV1 or SLOV2 nucleic acid molecule and operably linked promoter can be formulated as either linear molecules or more preferably as covalently closed circular molecules as either non-replicating DNA (or RNA) molecules. It can be introduced into prokaryotic or eukaryotic cells of the ent. Since such molecules cannot replicate autonomously, gene expression can occur by transient expression of the introduced sequence. Alternatively, permanent expression can occur by integrating the introduced DNA sequence into the host chromosome.

In one embodiment, a vector is employed that can integrate the desired gene sequence into the chromosome of the host cell. Cells into which the introduced DNA has been stably integrated into the chromosome can be selected by introducing one or more markers that enable selection of host cells containing the expression vector. This marker provides prototrophy to an auxotrophic host,
It can provide resistance to biocides such as antibiotics or heavy metals such as copper, and the like. The selectable marker gene sequence may be directly linked to the DNA gene sequence to be expressed, or may have been introduced by co-transfection into the same cell. Additional elements are also required for optimal synthesis of single-stranded protein mRNA. These elements can include splicing signals as well as transcription promoter, enhancer signal sequences and termination signals. CDNA expression vectors for introducing such elements include Okayama, Molec. Ce.
II. Biol., 3: 280 (1983).

In a preferred embodiment, the introduced nucleic acid molecule is introduced into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a wide variety of vectors can be employed for this purpose. Important requirements for selecting a particular plasmid or viral vector include the following: ease of recognizing recipient cells containing the vector and selecting from recipient cells not containing the vector; The desired number of copies of the vector; and whether it is desirable for the vector to be able to "shuttle" between host cells of different species. Suitable prokaryotic vectors include plasmids capable of replication in E. coli (eg, pBR322, ColE1, pS
C101, pACYC184, such as πVX). Such plasmids are described, for example, in Sambrook (Molecular Cloning: A Laboratory Manual, Second edit
ion, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laborat
ory, 1989). Bacillus plasmids include pC194, pC22
1, pT127 and others. Such a plasmid is Gryczan (In: The Mole
Molecular Biology of the Bacilli, Academic Press, NY (1982), pp 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall
et al., J. Bacteriol., 169: 4177-4183 (1987)) and Streptomyces bacteriophages such as φC31 (Chater et al., In: Sixth Internatio).
nal Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hun
gary (1986), pp. 45-54). A review has been published on Pseudomonas plasmids (John et al., Rev. Infect. Dis., 8: 693-704 (1986); and
Izaki (Jpn. J. Bacteriol., 33: 729-742 (1978)).

Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-
Including micron circles and others. Such plasmids are well known in the art (Botstein et al., Miami Wintr. Symp. 19: 265-274 (1982); Broac
h, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and
Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 4
45- 470 (1981); Broach, Cell, 28: 203-204 (1982); Bollon et al., J. Clin.
Hematol. Oncol., 10: 39-48 (1980); Maniatis, In: Cell Biology: A Compr.
ehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY,
pp. 563-608 (1980)).

After a vector or nucleic acid molecule containing the construct has been prepared for expression, its DNA
The construct is introduced into the appropriate host cell by any suitable method, including transformation, gene transfer, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. After the introduction of the vector, the recipient cells are grown in a selection medium to select for the growth of vector-containing cells. Expression of the cloned gene molecule results in WI1, WI2, WI
3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SL
Causes production of OV1 or SLOV2. This expression occurs in the transformed cells themselves or after induction of differentiation of these cells (eg, administration of bromodeoxyuracil to neuroblastoma cells, etc.).

VII. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
Binding to WED, BOV, EQ, SLOVE1 or SLOVE2 polypeptide
Antibodies with Affinity and Hybridomas Containing the Antibodies In another aspect, the invention relates to the aforementioned WI1, WI2, WI3, WI4, WIC, N
Y1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOVE2
A polypeptide or its WI1, WI2, WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1 or SLov
(2) An antibody having specific binding affinity for the polypeptide-binding fragment.
Some antibodies are non-WI1, WI2, WI3, WI4, WIC, NY1, NY2, N
If it does not bind to the Y3, SWED, BOV, EQ, SLOVE, or SLOV2 polypeptides, WI1, WI2, WI3, WI4, WIC, NY1, NY
2, NY3, SWED, BOV, EQ, SLOVE1 or SLOVE2 polypeptide or WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3,
Specific binding if specifically bound to a consensus sequence or binding fragment thereof described herein corresponding to the amino- and / or carboxy-terminal regions shared by SWED, BOV, EQ, SLOVE1 and SLOV2 It is. WI1, WI
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
Q, selectively couples to SLOV1 or SLOV2, or WI1, WI2
, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ
, SLOV1 and SLOV2 may selectively bind to, but are not limited to, the consensus sequences described herein corresponding to the amino- and / or carboxy-terminal regions shared by SLOV1 and SLOV2. WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1 or SLov
WI1, WI2, WI3, WI4, WIC, NY in organizations including
1, NY2, NY3, SWED, BOV, EQ, SLOV1 or SLOV2.

WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWE
D, BOV, EQ, SLOV1 or SLOV2 proteins, or proteins containing a consensus sequence corresponding to the amino- and / or carboxy-terminal regions shared by the proteins of the present invention can be used, for example, for the production of antibodies, the validation of pharmaceutical compositions and the DN.
It can be used for various procedures and methods, such as studying A / protein interactions.

WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWE
D, BOV, EQ, SLOV1 or SLOV2 proteins, or proteins containing a consensus sequence corresponding to the amino- and / or carboxy-terminal regions shared by the proteins of the present invention can be used for the production of antibodies or hybridomas.
One of skill in the art will recognize that if an antibody is desired, such a peptide would be made as described herein and used as an immunogen.

The antibodies of the present invention include monoclonal and polyclonal antibodies and fragments of these antibodies. The invention further includes single chain antibodies. Antibody fragments which contain the idiotype of this molecule can be produced by known techniques. Without limitation, for example, such fragments include F (ab ') ₂ fragments;
Fragments; Fab fragments; and Fv fragments.

Of particular interest to the present invention are WI1, WI2, WI3, WI4, WI
C, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1, and SL
OV2 or WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY
3, amino-shared with SWED, BOV, EQ, SLOVE1 and SLOVE2
And / or antibodies to one or more proteins that contain a consensus sequence corresponding to the carboxy-terminal region, produced in the human body or "humanized" by recombinant or other techniques (i.e., No immunogenicity in humans). Humanized antibodies are produced, for example, by replacing the immunogenic portion of the antibody with the corresponding non-immunogenic portion (i.e., a chimeric antibody) (Robinson et al.
, PCT Application No. PCT / US86 / 02269; Akira et al., European Patent No.,
184,187; Taniguchi, European Patent No., 171,496; Morrison et al., Euro
pean Patent No., 173,494; Neuberger et al., PCT Application WO86 / 01533;
Cabilly et al., European Patent No., 125,023; Better et al., Science, 24
0: 1041-1043 (1988); Liu et al., Proc. Acad. Sci. (USA), 84: 3439-3443 (
1987); Liu et al., J. Immunol., 139: 3521-3526 (1987); Sun et al., Proc.
Acad. Sci. (USA), 84: 214-218 (1987); Nishimura et al., Canc. Res., 47:
999-1005 (1987); Wood et al., Nature, 314: 446-449 (1985); Shaw et al.,
J. Natl. Cancer Inst., 80: 1553-1559 (1988)). A general review of "humanized" chimeric antibodies can be found in Morrison (Science, 229: 1202-1207 (1985)) and Oi et al. (Bi
oTechniques, 4: 214 (1986)). Suitable "humanized" antibodies can be prepared separately by CDR or CEA substitution (Jones et al., Nature, 321: 522-525).
(1986); Verhoeyan et al., Science, 239: 1534 (1988); Beidler et al., J.
Immunol., 141: 4053-4060 (1988)).

In another aspect, the present invention relates to a hybridoma producing said monoclonal antibody. Hybridomas are immortalized cell lines capable of secreting specific monoclonal antibodies.

In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, "Monoclonal Antibody Technology
: Laboratory Techniques in Biochemistry and Molecular Biology ", Elsevier
Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al.
, J. Immunol. Methods, 35: 1-21 (1980)).

Any animal (mouse, rabbit, etc.) known to produce antibodies can be immunized with the selected polypeptide. Methods of immunization are well-known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. Those skilled in the art will recognize that the amount of polypeptide used for immunization will vary depending on the animal being immunized, the antigenicity of the polypeptide and the site of injection.

The polypeptide can be modified to increase the antigenicity of the peptide or administered with an adjuvant. Methods for increasing the antigenicity of a polypeptide are well known in the art. Such procedures include binding of the antigen to a heterologous protein (eg, globulin or β-galactosidase) or mixing of adjuvants during immunization.

For the monoclonal antibody, spleen cells of the immunized animal are collected, fused with myeloma cells, and allowed to stand to produce monoclonal antibody-producing hybridoma cells.

A number of methods well known in the art can all be used to identify hybridoma cells that produce antibodies with the desired properties. These include screening of hybridomas by ELISA, Western blot analysis or radioimmunoassay (Lutz et al., Exp. Cell Res., 175: 109-124 (1988)).

After cloning the hybridoma secreting the desired antibody, the procedures known in the art (Campbell, "Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology ", supra (1984)), to determine its class and subclass.

For polyclonal antibodies, antiserum containing the antibody is collected from the immunized animal and screened for the presence of an antibody with the desired specificity using one of the procedures described above.

In another aspect of the invention, the antibody is detectably labeled. Detectable labels for antibodies include radioisotopes, affinity labels (e.g., biotin, avidin, etc.), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase,
Others), fluorescent labels (eg FITC or rhodamine, etc.), diamagnetic atoms,
Use other. Procedures for achieving such labeling are well known in the art, see, for example, the following references (Sternberger et al., J. Histochem. Cytochem.
, 18: 315 (1970); Bayer et al., Meth.Enzym. 63: 308 (1979); Engval et a.
l., Immunol., 109: 129 (1972); Goding, J. Immunol. Meth. 13: 215 (1976))
. Using the labeled antibodies of the present invention, in vitro, in vivo and in situ assays for identifying cells or tissues expressing a particular peptide can be performed.

In another embodiment of the present invention, the antibody is immobilized on a solid carrier. Examples of such solid carriers include, for example, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylics such as polyacrylamide, and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experiment-
ental Immunology ", 4th Ed., Blackwell Scientific Publications, Oxford, E
ngland, Chapter 10 (1986); Jacoby et al., Meth.Enzym., 34, Academic Pre
ss, NY, (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, in situ assays and for immunochromatography.

Further, those skilled in the art will be able to adapt the currently available procedures and techniques, methods and kits disclosed for the above antibodies to specific peptide sequences in order to generate rationally designed anti-peptide peptides. It can easily be adapted to produce peptides capable of binding. For example, Hurby et al., "Application of Synthetic Pe
ptides: Antisense Peptides ", in Synthetic Peptides, A User's Guide, W. H
Freeman, NY, PP. 289-307 (1992); and Kaspczak et al., Biochemistry, 28.
: 9230-8 (1989).

The anti-peptide-peptide can be made in two ways. First, the anti-peptide
The peptides were WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3
, SWED, BOV, EQ, SLOV1 and SLOV2 peptide sequences or basic amino acid residues found in the consensus sequences described herein are replaced by acidic residues, while hydrophobic and uncharged electrode groups are Maintain. For example, a lysine, arginine and / or histidine residue is replaced with aspartic acid or glutamic acid, and a glutamic acid residue is replaced with lysine, arginine or histidine.

VIII. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, S
A WED, BOV, EQ, SLOVE or SLOVE polypeptide or antibody.
Methods for Detecting in a Sample In another aspect, the invention provides a method for detecting WI1, WI2, WI comprising a consensus sequence corresponding to an amino- and / or carboxy-terminal region shared by a polypeptide in a sample.
3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SL
A method for detecting an OV1 or SLOV2 polypeptide, comprising: (a) contacting a sample with said antibody (or protein) under conditions that form an immune complex; and
(b) detecting the presence of an antibody bound to the polypeptide.
In particular, the method comprises incubating a test sample with one or more antibodies of the invention; and assaying whether the antibodies bind to the test sample. WI1, WI2, WI3, WI4, WIC in the sample compared to normal levels
, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE, or SLO
Changes in V2 peptide levels can be indicative of a particular disease.

In yet another aspect, the invention relates to WI1, WI2, WI3, WI4, WIC
, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE, or SLO
A method for detecting a V2 antibody in a sample, comprising: (a) separating the sample from the WI1, WI2, W
I3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, S
A LOV1 or SLOV2 polypeptide, comprising WI1, WI2, WI3, W
I4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1
And amino- and / or carboxy- shared with SLOV2 polypeptides
Contacting those containing a consensus sequence corresponding to the terminal region under conditions that form an immune complex; and (b) detecting the protein bound to the antibody or the antibody bound to the protein. In particular, the method comprises incubating a test sample with one or more proteins of the invention and assaying whether the antibody has bound to the test sample. WI1, WI2, WI3, WI4
The presence of antibodies to WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOV2 indicates contact with GE, may require treatment as an infected individual, or May indicate GE contamination of the target sample.

Incubation conditions between the test sample and the antibody are not constant. Incubation conditions will depend on the format employed in the assay, the detection method employed, and the type and nature of the antibody used in the assay. One of ordinary skill in the art will appreciate that any one of the commonly used immunoassay formats (e.g., radioimmunoassays, enzyme-linked immunosorbent assays, diffusion-based Oakterlonie or rocket immunofluorescence assays) ) Can be readily applied to the antibodies of the present invention. Examples of such assays include Chard, An Introduction to Radio-immunoassay and Related Techniques
, Elsevier Science Publishers, Amsterdam, The Netherlands, (1986); Bullo
ck et al., Techniques in Immunocytochemistry, Academic Press, Orlando, F
L, Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice and Th
eory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and M
olecular Biology, Elsevier Science Publishers, Amsterdam, The Netherland
s (1985).

The immunoassay test samples of the present invention include cells, membrane extracts of proteins or cells, or biological fluids such as blood, serum, plasma or urine. The test sample used in the method will vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample to be assayed. Methods for preparing protein extracts or cell membrane extracts are well known in the art and can be readily adapted to obtain samples that can be employed in the system employed.

IX. WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
Contains ED, BOV, EQ, SLov1 or slov2 protein or antibody
Diagnostic Kit In another aspect of the present invention, there is provided a kit including all of the reagents necessary for performing the above-described detection method.

The kit can include (i) a first container containing the antibody described above; and (ii) a second container containing a binding partner of the antibody and a label.

The kit comprises (i) a first container containing the protein described above; and preferably (ii)
A) a second container containing a conjugate comprising a protein binding partner and a label. More specifically, the diagnostic kit comprises WI1, WI2, WI3, WI4, WIC for detecting antibodies in the serum of potentially infected animals or humans.
, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE, and SLO
V2 or the aforementioned WI1, WI2, WI3, WI4, WIC, NY1, NY2,
Includes peptides having a consensus sequence corresponding to the amino and / or carboxy terminal regions shared with NY3, SWED, BOV, EQ, SLOVE, and SLOV2 proteins.

In another preferred embodiment, the kit further comprises one or more additional containers containing one or more washing reagents and one or more reagents capable of detecting the presence of bound antibody. Without limitation, examples of detection reagents include a labeled secondary antibody, or alternatively, a chromogenic, enzymatic or antibody-binding agent that can react with a labeled antibody if the primary antibody is labeled. Of the reagent. The kit separated into compartments can be as described above for the nucleic acid probe kit.

Those skilled in the art will readily recognize that the antibodies described in the present invention can be introduced into kits in formats well known in the art.

X. Diagnostic Screening Although the following description is specifically directed to human patients, it should be understood that the teachings can be applied to any animal that can be infected with GE.

The diagnostic and screening methods of the present invention are particularly useful for patients suspected to be at risk for developing Ehrlichiosis.

According to the present invention, WI1, WI2, WI3, WI4, WIC, NY1 of the present invention
, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOVE2 protein or fragment thereof or WI1, WI2, WI3, WI4, WIC of the present invention
, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE, or SLO
The use of DNA encoding a protein having a consensus sequence corresponding to the amino and / or carboxy terminal regions shared with V2 allows pre- and post-symptomatic screening of individuals in need of such screening. This screening method of the present invention employs WI1, WI2, WI3, WI4,
Allows pre-symptomatic diagnosis of the presence of WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOVE2 protein or DNA,
Therefore, it allows an expression of opinion as to whether or not the individual develops aerial disease. Early diagnosis is desirable for maximal appropriate timely treatment.

In a preferred embodiment of the present screening method, a tissue sample is collected from an individual, and (1) W
I1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED,
The presence of the coding sequence of the BOV, EQ, SLOVE or SLOV2 DNA; (2) W
I1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED,
(3) WI1, WI presence of BOV, EQ, SLOV1 or SLOV2 mRNA;
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
Q, the presence of a SLOV1 or SLOV2 protein; and / or (4) WI1
, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BO
V, EQ, the presence of antibodies against the SLOV1 or SLOV2 protein.

WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWE
Presence of D, BOV, EQ, SLOVE1 or SLOV2 protein and / or WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWE
A preferred method for detecting the presence of antibodies to D, BOV, EQ, SLOVE, or SLOV2 proteins is to use (a) a sample with WI1, WI2, WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1 or SLov
2) contacting an antibody against a polypeptide having the amino acid sequence of the polypeptide or a fragment thereof under conditions for forming an immune complex; and (b) detecting the antibody and polypeptide that have become an immune complex; including.

Individuals not infected with GE are WI1, WI2, WI3, WI4, WIC, NY
1, No NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOV2 DNA, mRNA or protein.

In the screening and diagnostic methods of the present invention, WI1, WI2, WI3, WI4,
It is not necessary that all of the WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1, or SLOV2 coding sequences be used for the probe.
On the contrary, WI1, WI2, WI3, WI4,
It is only necessary to use a fragment of sufficient length to detect the presence of WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOV2 nucleic acids.

Analysis of nucleic acids specific for GE can be by PCR technology or by hybridization technology (division of spotted fever groups of Rickettsia spp. By analysis of polymorphisms for restriction fragment length of PCR amplified DNA). Molecular Cloning that states:
A Laboratory Manual, 2nd Edition, edited by Sambrook, Fritsch & Maniati
s, Cold Spring Harbor Laboratory, 1989; Eremeeva et al., J. Clin. Microb
iol., 32: 803-810 (1994)). Nucleic acid probes used to analyze GE genomic DNA by PCR analysis are described in Chen et al., J. Clin. Microbiol., 32
: 589-595 (1994).

XI. In another aspect of the vaccine , the invention relates to WI1, WI2, WI3, WI4, WIC, NY1.
, NY2, NY3, SWED, BOV, EQ, SLLV1 or SLOV2 protein or fragment thereof, or a protein having a consensus sequence corresponding to a shared amino- and / or carboxy-terminal region (preferably an immunologically active fragment) And a pharmaceutically acceptable diluent, carrier or excipient. Here, however, the protein is present in an amount effective to produce an advantageous immune response of the animal to GE. WI1, WI2, WI3, WI4,
WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE, or SLOV2 proteins or proteins having a consensus sequence corresponding to a shared amino- and / or carboxy-terminal region can be obtained as described above and described in the art. It may be obtained using methods well known in the art. Immunologically active fragments include the epitope-containing portion of the protein.

In yet another preferred aspect, the invention relates to WI1, WI2, WI3, WI4, WI
C, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SL
A composition comprising an OV2 protein or fragment thereof, or a protein having a consensus sequence corresponding to a shared amino- and / or carboxy-terminal region, together with a carrier.

In another aspect, the invention relates to WI1, WI2, WI3, WI4, WIC, NY1,
NY2, NY3, SWED, BOV, EQ, SLOVE, or SLOVE2 nucleic acids (
Preferably DNA) or a fragment thereof (preferably a fragment encoding an immunologically active protein or peptide), or a polypeptide, or a consensus sequence corresponding to a shared amino- and / or carboxy-terminal region. A vaccine comprising a nucleic acid encoding a protein having a pharmaceutically acceptable diluent, carrier or excipient, wherein the nucleic acid is present in an amount effective to produce an advantageous immune response of the animal to GE. I do. WI1, WI2, WI3, WI4
, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 or SLOV2 nucleic acids may be obtained as described above, and may be obtained using methods well known in the art. Immunologically active fragments include the epitope-containing portion of the nucleic acid.

In a further preferred aspect, the invention relates to WI1, WI2, WI3, WI4, WIC,
NY1, NY2, NY3, SWED, BOV, EQ, SLov1 or SLov
2 a nucleic acid (preferably DNA) or a fragment thereof (preferably one encoding an immunologically active protein or fragment-an antigenic epitope) and a carrier comprising a carrier.

In a further preferred embodiment, the present invention relates to a method for raising an immune response recognizing GE in a host, comprising administering the composition to the host.

In a preferred aspect, the animal to be protected is selected from a human, horse, deer, cow, pig, sheep, dog, and chicken. In a more preferred aspect, the animal is a human or a dog.

In yet another aspect, the present invention relates to a method of preventing aerial disease in an animal, comprising administering the vaccine to the animal. The vaccine of the present invention is used in an effective amount depending on the route of administration. While the intranasal, subcutaneous or intramuscular route of administration is preferred, the vaccines of the present invention can also be administered by the oral, intraperitoneal or intravascular route. Those skilled in the art will recognize that the dosage for a particular treatment protocol can be readily determined without undue experimentation. An appropriate amount is WI1, per kg of body weight,
WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV
, EQ, SLOV1 and SLOV2 proteins or proteins having a consensus sequence corresponding to the amino- and / or carboxy-terminal region up to 100 μg / kg body weight (preferably from 2 μg to 50 μg, from 2 μg to 25 μg, from 5 μg to 50 μg, or 5 μg to 10 μg).

Examples of vaccine formulations include Kensil, Therapeutic Drug Carrier Systems, 13: 1-55 (1996); Livingston, including amount of antigen, route of administration and adjuvant addition.
et al., Vaccine, 12: 1275 (1994); and Powell et al., AIDS RES., Human
Retroviruses, 10, 5105 (1994).

The vaccines of the present invention may employ, for example, capsules, solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. . Preferably, any inert carrier is used, for example, saline, phosphate buffered saline, or a carrier in which the vaccine has suitable solubility. The vaccine may be in a single dose form or may be a multidose flask which can be used in a mass vaccination program. How to make and use vaccines
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, Os
ol (ed.) (1980); and New Trends and Developments in Vaccines, Voller e
See al. (ed.), University Park Press, Baltimore, MD (1978).

The vaccine of the invention may further contain an adjuvant that enhances the production of antibodies and immune cells. Such adjuvants include, but are not limited to, various oil formulations such as Freund's Complete Adjuvant (CFA), a dipeptide called MDP, saponins (eg, QS-21, US Patent No. 5,047,540).
), Aluminum hydroxide or lymphoid cytokines. Freund's adjuvant is an emulsion of mineral acid and water, which is mixed with the immunogen. Freund's adjuvant, while powerful, is not usually administered to humans. Instead, alum adjuvant (aluminum hydroxide) is used for human administration. The vaccine may be adsorbed to the aluminum hydroxide and may be slowly released from the aluminum hydroxide after injection. The vaccine is Fullerton, US Patent No. 4,235,877
May be encapsulated in a liposome.

[0179]

【Example】

The present invention is further described in the following non-limiting examples. EXAMPLES The following protocols AG and detailed experimental descriptions are referred to in the non-limiting Examples 1-6.

Protocol A: Culture of GE in HL60 Cells The USH3, a HL60 cell line infected with GE, is a HL60 cell line (ATCC.
CCL240) and blood collected from dogs inoculated with the tick Ixodes scapularis collected from the field. After the deformed cell morphology becomes apparent, the infected cells are passed through fresh, uninfected HL60 cells to maintain the culture. USG3 is grown in RPMI 1640 containing 10-20% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and fresh HL60 cells are added a few times a week. Distribute to This operation is outlined in Coughlin et al., PCT Application No. PCT / US96 / 10117;
oodman et al., N. Eng. J. Med., 334: 209-215 (1996).

Protocol B: Purification of GE A USG3 culture (microscopic examination) containing 80% lysed cells was 840 min at 4 ° C. for 840 minutes.
Centrifuge at xg to remove debris of host HL60 cells. Supernatant 47m in diameter
m through a 5 μm polycarbonate membrane (Livermore, CA) followed by filtration through a 3 μm Poretics filter at negative pressure. The USG3 filtrate is centrifuged at 9460 × g for 30 minutes at 4 ° C. in a Sorvall centrifuge. Following centrifugation, the GE pellet was washed with 25 mM Tris, pH 8.0,
10 mM-MgCl _2, and resuspended in 5mL of 0.9% NaCl. DNase I (Life Technologies, Gaithersburg, MD) is added at a final concentration of 9 μg / mL and the solution is incubated for 15 minutes at 37 ° C. Following the incubation, the DNase was inactivated by adding 0.5 mL of 0.5 M-EDTA,
Pellet GE at 14000 × g for 30 minutes at 4 ° C. using a val centrifuge.

Protocol C: Construction of GE Genomic Library For production of experiments (Stratagene, La Jolla, CA) QIAmp Genomic DNA Kit (Qiage
n, Chatsworth, CA) from the purified GE. This DN
A is mechanically sheared to a size in the range of 4-10 kb and linked to an EcoRI linker. The ligated fragment is ligated into the EcoRI site of Lambda ZapII and this library is amplified in E. coli XL1-Blue MFR 'strain to titer 10 ¹⁰ Pfu / mL.

Protocol D: Preparation of Screening Serum Canine Serum: Mature mite Ixodes scaplularis collected from the eastern United States, where human Lyme disease prevalence is high, is described in the literature (Coughlin et al., J. Infect. Dis., 171: 1049
-1052 (1995)). Serum from the dog is tested for immunoreactivity to E. equi by immunofluorescence. Positive sera from infected dogs are collected and used for immunoscreening of the GE genomic library.

Mouse serum: The protein contained in all GEs disrupted in SDS was
Separate by PAGE and cut out 46 bands from two 10% and 15% gels respectively. The fragments of each gel are crushed, added to buffer and Ribi adjuvant, and used to immunize two mice. Sera having a similar immunoreactivity pattern to GE antigens determined by Western blotting are collected and divided into groups.

Goat serum: Immunize goats with a mixture of 100 μg of purified heat-inactivated USG3 antigen. Goats are subcutaneously administered antigen three times at two week intervals. Two weeks after the third immunization, serum is collected and used for immunoscreening of a GE genomic DNA library.

Protocol E: Screening of GE Genomic DNA Library Bacteriophages are diluted and spread on NZY agar plates with XL1-Blue MFR 'strain cells. Make a plate showing about 50,000 plaques per plate. 10
Phage is induced with mM-IPTG (Sigma, St. Louis, Missouri) to express the cloned protein and transferred to a nitrocellulose filter. For immunoscreening, 0.1% polyoxyethylene 20 cetyl ether (Brij.
58) containing TBS (25 mM Tris HCl, pH 7.5, 0.5 M NaCl)
Block the filter with and incubate with preserved dog serum, preserved mouse serum, or preserved goat serum. The filters are washed and then reacted with an anti-dog HRP conjugated antibody, an anti-mouse HRP conjugated antibody, or an anti-goat HRP conjugated antibody. The filters are washed again and developed with 4-chloronaphthol (BioRad).

[0187] Positive plaques are separated, re-plated, and rescreened twice to reach purity. Plasmid DNA containing the putative recombinant clone is obtained by the plasmid rescue method (Stratagene, La Jolla, CA).

Protocol F: DNA analysis Restriction enzyme analysis: Sambrook et al., Molecular Cloning (2nd ed.), Cold Sprin
g Performed with standard techniques according to the protocol of the Harbor Laboratory Press, New York (1989).

DNA Sequencing and Sequence Analysis: DNA sequencing of recombinant clones was performed using the primer walking method and the ABI 373A DNA sequencer (ACGT, Northbrook,
IL; Lark Technologies, Houston, TX; and Sequegen, Shrewsbury, MA) and the like. The sequence is MacVector (Oxford
(Molecular Group) sequence analysis program, version 6.0 or by using the GCG package. A search for homologous nucleic acid and protein sequences available from the server at the National Center for Biotechnology Information (NCBI) was performed using the BLAST algorithm, D, version 1.4.

PCR Amplification of Target Sequences: DNA oligonucleotide primer sets are designed based on sequence information obtained from individual clones. PCR primers are synthesized by standard oligonucleotide synthesis methods or, for example, Life Technologies (
Gaithersburg, MD). The template for PCR is purified plasmid DNA
Purified GE or HL60 genomic DNA, genomic DNA isolated from infected blood,
Alternatively, it is a phage degradation product. All reactions were Gene Amp 9600 thermal cycler (Perk
in-Elmer, CT), Gene Amp reagent from Perkin-Elmer, and TaqStart antibody (Clonte
ch, CA). The cycling program consists of 30 cycles,
Each is 30 seconds 94 ° C, 30 seconds 48-55 ° C, and 1 minute 72 ° C, with an additional cycle of 10 minutes 72 ° C. Apart from this, some of the PCR amplifications are
g et al., J. Clin. Micro., 36: 1090 (1998), using a nested reaction. The PCR product was a 4% Nusieve (3: 1) agarose gel (FMC Bio
products, Rockland, ME).

Protocol G: Protein Separation and Analysis An overnight culture of each clone was treated with TP broth (20 bactotryptone / liter).
g, ₂ g of Na ₂ HPO ₄ , 1 g of KH ₂ PO ₄ , 8 g of NaCl, 15 g of yeast extract) and grow at 37 ° C. at 37 ° C. until the OD ₆₀₀ reaches 0.5 to 1. Collect a 1.5 mL volume of culture. IPTG is added to a concentration of 5 mM and growth is continued at 37 ° C. for a further 3 hours. Measure the OD ₆₀₀ and pellet each culture. 5% Laemmli buffer (12% glycerin, 0.2 M-Tris-HCl, pH 6.8, 5% SDS, 5 μL Laemmli buffer (200 μL / unit)
(5% β-mercaptoethanol). Alternatively, the collected GE protein product is pelleted and 0.4% SDS, 12.5 mM Tris, pH 6.8.
And heated to 90-100 ° C for 20 minutes. GE5m for cell lysis
Add 50 μL of cocktail containing RNaze (33 μg / mL) and aprotinin (0.2 mg / mL) and 9 μL of DNaze (0.17 mg / mL) per g. 25X Boehringer / Mannheim protease inhibitor cocktail (cat. # 1697498) 2
0 μL is added to each 0.5 mL of the cell suspension, and the PMSF solution (DMSO
1M-) is added. The cells are disrupted at 30-minute intervals for a total of 3 minutes using a BX-4 mini-bead beater cell disrupter (BioSpec), stirred at room temperature for 30 minutes, and at 15,000 × g for 10 minutes.
Centrifuge for minutes. The pellet is suspended in Laemmli sample buffer and adjusted to 1.4 mg SDS per mg protein. Boil samples and add 10 μL each to SDS
-Perform electrophoresis on PAGE.

For Western blot analysis, the gel is transferred to a nitrocellulose filter, the filter is blocked with TBS / Brij58, and the blot is probed with antiserum. The blot is then washed and incubated with the HRP-conjugated secondary antibody. After the final washing step, blots were blotted with 4-chloronaphthol (Bio-Rad,
(Hercules, CA) or detected using enhanced chemiluminescence (Pierce, Rockford, IL).

Example 1 GE clones WI1, WI2, WI3, WI4, WIC, NY1, N
Y2, NY3, SWED, BOV, EQ, SLOV1 and SLOV2 isolated infected animals (1 dog, 1 horse and cow) or 10 humans (4 from Wisconsin, 3 from New York, 1 from Sweden, And two patients from Slovenia) were confirmed by PCR amplification of the GE160 gene. USG3 GE1 isolated from experimentally infected dog blood
Based on the 60 nucleotide sequence (designated S2), a nested primer combination was designed to cover the entire coding region. FIG. 1 shows the positions of the primer sets. (A
QUA1 (SEQ ID NO: 27) and AQ1R2 (SEQ ID NO: 28);
9) and AQ1R1 (SEQ ID NO: 30); AQ1F (SEQ ID NO: 31) and AQ1R (
AQ2F (SEQ ID NO: 33) and AQ2R (SEQ ID NO: 34); AQ
3F (SEQ ID NO: 35) and AQ3R (SEQ ID NO: 36); AQ4F (SEQ ID NO: 37) and AQ4R (SEQ ID NO: 38); AQ4F1 (SEQ ID NO: 39) and AQ4R1 (SEQ ID NO: 40); AQ4F2 (SEQ ID NO: 41) and AQD2 (SEQ ID NO: 42); and AQ4F3 (SEQ ID NO: 43) and AQD1 (SEQ ID NO: 44)) and the corresponding PCR amplified fragments obtained. G listed using the primer set shown in Table 2
E clones WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3
, SWED, BOV, EQ, SLOV1 and SLOV2 regions were amplified. Each oligonucleotide sequence is shown in a 5 'to 3' orientation. Figure 2 shows the amplified GE
The DNA sequences of the 13 genes of 160 genes are illustrated by comparison with the S2 · G160 gene (shown in the second column of the sequence as USG3).

Preparation of Blood Samples One of ordinary skill in the art will appreciate that DNA isolation for PCR amplification is described, for example, in Massung et al., J. Am.
It is envisioned that this can be accomplished using a variety of protocols known in the art as described in Clin. Microb., 36 (4): 1090-1095 (1998).

50 μL of each PCR amplification reaction was 0.5 μM of each primer, 1 × PCR Supermer
ix (Life Technologies, Gaithersburg, MD) and 2 μL of a blood mixture corresponding to approximately 100 ng of DNA prepared from individual blood samples. PCR amplification was performed as described in Protocol F. Table 2

[Table 2] (*) The approximate position of each primer is shown in FIG. 1 (names are based on numbers in S2 clones obtained from USG3).

Separate PCR reactions using each primer pair resulted in 3 infected animals (1 dog, 1 horse and cow) and 10 humans (4 from Wisconsin, 3 from New York, 1 from Sweden, and Slovenia). From two patients) were amplified. Appropriate amounts from each PCR reaction were run on a 1% agarose gel and stained with ethidium bromide to confirm the approximate size of the PCR amplified fragments. In most cases, the size of the PCR product matched that predicted based on the nucleotide sequence of S2 (data not shown). In some samples, the PCR product fragment contains nucleotides spanning the deletion (14
38-1518), which corresponds to a deletion of a nucleotide encoding a total of 27 amino acid repeats.

Example 2 DNA Sequencing and Sequence Analysis Sequencing was performed by the primer walking method. Both strands of each insert were sequenced as described in Protocol F. The sequences of the 13 clones shared considerable homology. The nucleotide sequences of all 13 clones are shown in FIGS. GE clones are as follows: 1 dog (named WIC; see FIG. 3), horse 1
5 (named EQ; see FIG. 5) and one cow (named BOV; see FIG. 6), and 4 patients from Wisconsin (named WI1, WI2, WI3, WI4, respectively; see FIGS. 7-10), Three patients from New York (named NY1, NY2, NY3, respectively; see FIGS. 11-13) and one patient from Sweden (named SWED; FIG. 4)
, And two patients from Slovenia (designated SLov1 and SLov2, respectively;
10 humans including Figs. The amino acid sequences deduced from all 13 clones are shown in FIGS. The amino acid sequences deduced from this representative clone are as follows: one dog (named WIC; see FIG. 16), one horse (named EQ; see FIG. 18), one cow (named BOV) ; See FIG. 19) and four patients from Wisconsin (named WI1, WI2, WI3, WI4, respectively; see FIGS. 20-23) and three patients from New York (named NY1, NY2, NY3, respectively; FIG. 24).
-26), one patient from Sweden (named SWED; see Figure 17), and two patients from Slovenia (named SLOVl and SLOV2, respectively; Figure 2).
10 humans, including 7-28). The amino acid sequence derived from all 13 of the GE clones was compared with the amino acid sequence of S2 · GE160 protein shown in FIG.

Sequence analysis (MacVector, Oxford Molecular Group) showed that each clone contained a single large open reading frame encoded by the plus strand of the insert,
They also showed that each seemed to be a complete gene. WI1, WI of the present invention
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
Q, SLOV1 and SLOV2 clones contain S2 GE1 containing three repeats.
It was found to encode 60 proteins (160 kDa). The first set is 33
The second set is made up of 27 amino acid repeat units, and the third set is made up of 11 amino acid repeat units, comprising a number of ankyrin-like repeat units containing amino acids. This ankyrin repeat was found by searching the BLAST database for protein homology. Ankyrin repeats exist as at least four consecutive copies and are found in yeast, plants, bacteria and mammals.

Example 3 GR-WI1, WI2, WI3, WI4, W in HL60 cells
IC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1, and S
Culture of LOV2 The HL60 cell line infected with the GE isolate of the present invention comprises HL60 cells (ATCC.
CCL 240) with the following various animals and patients: 1 dog (designated WIC), 1 horse (designated EQ) and 1 cow (designated BOV), and 4 patients from Wisconsin (WI1, WI2, WI3, WI4), Patient 3 from New York
Obtained by co-culturing with blood cells from humans (named NY1, NY2, NY3), one patient from Sweden (named SWED) and two patients from Slovenia (named SLOVE1 and SLOV2). After confirming the altered morphology, the infected cells are maintained in culture over fresh, uninfected HL60. Next, GE was added to 10-20% heat-inactivated fetal bovine serum, RMPI164 containing 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM MEM non-essential amino acids.
0 and split into fresh HL60 cells two to three times a week. This procedure is outlined in Coughlin et al., PCT Application No. PCT / US96 / 10117, and is also shown in Goodman et al., N. Eng. J. Med., 334: 209-215 (1996). I have.

To confirm that the isolate is GE, the 16S ribosomal gene is amplified and analyzed as follows. Prepare cell extract by lysis protocol as described above. The PCR primer (specific for 16S ribosomal DNA) is modified to introduce a restriction enzyme recognition site as follows: Forward primer: 5'-CTGCAGGTTTGATCCTGG-3 '(PstI site) (SEQ ID NO: 45); reverse Direction primer: 5'-GGATCCTACCTTGTTACGACTT-3 '(BamHI site) (SEQ ID NO: 46
).

These primers (0.5 μM) were combined with 1 × PCR buffer II (Perkin-Elmer Corp.
), 1.5 mM MgCl ₂ (Perkin-Elmer Corp.), dATP, dGTP, dCT
Add 200 μM each of P and dTTP, 2.5 U Amplitaq DNA polymerase, and 100 μL of reaction mixture containing 20 μL of USG3 DNA. Amplification was performed as described in Protocol F. This amplification is expected to give a 1500 bp fragment which was digested with PstI and BamHI and pUC linearized with the same enzyme.
Connect to 19. The resulting clone is then sequenced.

Example 4 Isolation of Clones Using Serum GE from the culture was purified according to Protocol B and a genomic library was constructed according to Protocol C. The library is screened using sera prepared according to Protocol D or sera derived from the blood of the infected animals or patients described above. Screening is performed as per Protocol E. The confirmed clone is then purified as a single plaque by a third immunoscreen. The plasmid is removed according to the protocol of Stratagene and the DNA is purified using a plasmid purification kit of Qiagen. Next, EcoRI, HindIII, Ba
A single enzymatic digest may be performed using mHI, HincII, Xbal, PstI and Alw26I, and optionally a certain number of double digestions. Based on these digests, restriction maps are generated and compared to those deduced based on the PCR-derived nucleotide sequence of Example 1.

Example 5 Clones WI1, WI2, WI3, WI4, WIC, NY1, NY2
, NY3, SWED, BOV, EQ, SLOVE1 and SLOVE2 are derived from GE
Validated PCR primer sets by PCR analysis are listed in Table 2. Clones WI1, WI2, WI3, WI4 displayed using the sequences of the respective primer sets shown in Table 2.
, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOV2. Each oligonucleotide sequence is shown in the 5 'to 3' direction. 50 μL of each reaction was 0.5 μM of each primer, 1 × PCR Su
permix (Life Technologies, Gaithersburg, MD) and 6E (WI1, WI
2, WI3, WI4, WIC, NY1, NY2, NY3, SWED, BOV, E
Q, 100 ng of purified GE DNA of various types of SLOV1 and SLOV2),
Contains either 100 ng of HL60 DNA or 200 ng of plasmid DNA. Perform PCR amplification as described in Protocol F. These experiments ensure that the sequence gene is derived from GE DNA and not HL60 DNA or other animal or patient DNA. The size of the PCR product using genomic DNA as a template is generated by the purified plasmid and the first PC
It is expected to be the same as that observed with R amplification (see Example 1).

Example 6 Other Characteristics of the Isolated GE Clones The isolated clones were derived to express the encoding protein, and bacterial extracts were prepared and used for SDS-PAGE as abbreviated in Protocol G. SDS-PAG
E and Western blot analysis (all GEs interrupted with SDS as a positive control and non-protein expressing clones as a negative control) are expected to confirm immunoreactive proteins for each clone using the serum described in Protocol D Is done.
Scrutiny with serum from human GE patients is expected to detect the same protein. Based on the amino acid sequences of these proteins, the calculated molecular weights are expected to be significantly lower than those revealed by SDS-PAGE. The calculated molecular weight (based on amino acid sequence) for the protein encoded by the open reading frame of each clone is expected to be approximately 78 kDa, while the apparent (based on SDS-PAGE mobility) value is 160 kDa. Is expected.
This phenomenon has also been observed with other proteins. (Barbet et al., Infect. Immu
n., 59: 971-976 (1991); Hollingshead et al., J. Biol. Chem., 261: 1677-1.
686 (1986); Yu et al., Gene, 184: 149-154 (1997)).

[0205] Those skilled in the art will recognize that the isolation of purified protein samples obtained from isolated clones is well known in the art. Similarly, separation of individual protein products could proceed to the preparation of polyclonal and / or monoclonal antibodies according to any of a number of methods available in the art.

Although the present invention has been described in detail for clarity and understanding, those of ordinary skill in the art will appreciate, upon reading this disclosure, the type and details without departing from the scope of the invention and the appended claims. It will be appreciated that various changes can be made to.

[Brief description of the drawings]

FIG. 1 is a schematic representation of the GE160 gene (S2 clone),
1 shows the schematic location of primer sets used for PCR amplification using DNA extracted from the blood of animals, including infected humans. Thin line: DNA sequence; Bar graph: coding region with nucleotide numbers indicating start codon and stop codon based on S2 clone.

FIG. 2 shows representative clones WI1, WI2, WI3, WI4,
1 shows nucleotide sequences derived from WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOV2. GE160 of S2 is usg
3. WIC for wide, EQ for equi, and BOV for swedbovine
It is named.

FIG. 3 shows the DNA sequence of a representative clone WIC (SEQ ID NO: 1).

FIG. 4 shows the DNA sequence of a representative clone SWED (SEQ ID NO: 2).

FIG. 5 shows the DNA sequence of a representative clone EQ (SEQ ID NO: 3).

FIG. 6 shows the DNA sequence of a representative clone BOV (SEQ ID NO: 4).

FIG. 7 shows the DNA sequence of a representative clone WI1 (SEQ ID NO: 5).

FIG. 8 shows the DNA sequence of a representative clone WI2 (SEQ ID NO: 6).

FIG. 9 shows the DNA sequence of a representative clone WI3 (SEQ ID NO: 7).

FIG. 10 shows the DNA sequence of a representative clone WI4 (SEQ ID NO: 8).

FIG. 11 shows the DNA sequence of a representative clone NY1 (SEQ ID NO: 9).

FIG. 12 shows the DNA sequence of a representative clone NY2 (SEQ ID NO: 10).

FIG. 13 shows the DNA sequence of a representative clone NY3 (SEQ ID NO: 11).

FIG. 14 shows the DNA sequence of a representative clone SLOVE (SEQ ID NO: 12).
Is shown.

FIG. 15 shows the DNA sequence of a representative clone SLOVE2 (SEQ ID NO: 13).
Is shown.

FIG. 16 shows the amino acid sequence of a representative clone WIC (SEQ ID NO: 14).

FIG. 17 shows the amino acid sequence of a representative clone SWED (SEQ ID NO: 15).
Is shown.

FIG. 18 shows the amino acid sequence of a representative clone EQ (SEQ ID NO: 16).

FIG. 19 shows the amino acid sequence of a representative clone BOV (SEQ ID NO: 17).

FIG. 20 shows the amino acid sequence of a representative clone WI1 (SEQ ID NO: 18).

FIG. 21 shows the amino acid sequence of a representative clone WI2 (SEQ ID NO: 19).

FIG. 22 shows the amino acid sequence of a representative clone WI3 (SEQ ID NO: 20).

FIG. 23 shows the amino acid sequence of a representative clone WI4 (SEQ ID NO: 21).

FIG. 24 shows the amino acid sequence of a representative clone NY1 (SEQ ID NO: 22).

FIG. 25 shows the amino acid sequence of a representative clone NY2 (SEQ ID NO: 23).

FIG. 26 shows the amino acid sequence of a representative clone NY3 (SEQ ID NO: 24).

FIG. 27 shows the amino acid sequence of a representative clone SLOV1 (SEQ ID NO: 2).
5) is shown.

FIG. 28 shows the amino acid sequence of a representative clone SLOV2 (SEQ ID NO: 2).
6) is shown.

FIG. 29 shows representative clones WI1, WI2, WI3, and WI of the present invention.
4, shows deduced amino acid sequences corresponding to WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1 and SLOV2. GE160 of S2 is usg3, WIC is widget, EQ is equi, and BOV is swedbov.
ine.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/12 C12N 1/15 4C085 C12N 1/15 1/19 4H045 1/19 1/21 1/21 C12Q 1/68 A 5/10 G01N 33/50 N C12Q 1/68 33/53 D G01N 33/50 N 33/53 33/569 F 33/577 B 33/569 C12P 21/08 33/577 C12N 15/00 ZNAA // C12P 21/08 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL , PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, W), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (71) Applicants Centers for Diseases Control and Prevention Clifton Road 1600, North East, Atlanta, Georgia, USA (72) Inventor Sheryl I. Murphy United States 01748 Joseph Road, Hopkinton, Mass. 7, United States 7472 (72) Inventor Robert F. Massang United States 30047 Georgia Hunting Ridge Drive, Lillburn, Oregon 3865 No.F Term (Reference) 2G045 AA34 AA35 DA13 DA36 FB02 FB03 4B024 AA01 AA13 BA31 CA02 HA14 4B063 QA00 QA19 QQ08 QQ42 QR32 QR39 QR55 QR62 QR77 QS25 QS34 Q15Q024B01A AB01 CA24 CA45 4C085 AA03 BA47 4H045 AA10 AA11 BA10 CA11 DA75 DA86 EA29 EA31 EA52 FA74

Claims (25)

[Claims] 1. SEQ ID NOs: 5, 6, 7, 8, 1, 9, 10, 11, 2, 4, 3
WI1, WI2, WI3, W containing the complete amino acid sequence of each of
I4, WIC, NY1, NY2, NY3, SWED, BOV, EQ, SLOVE1
Or an isolated nucleic acid molecule comprising a nucleotide sequence encoding a SLOV2 polypeptide. 2. An isolated nucleic acid molecule comprising a nucleotide sequence complementary to the isolated nucleic acid molecule of claim 1. 3. The method according to claim 1, wherein the molecule is SEQ ID NO: 5, 6, 7, 8, 1, 9, 10, 11,
2. The isolated nucleic acid molecule of claim 1, which encodes a polypeptide comprising the complete amino acid sequence set forth in 2, 4, 3, 12, or 13. 4. An isolated nucleic acid molecule composed of from 10 to 50 nucleotides that hybridizes preferentially with granulocyte Ehrlichia RNA or DNA but does not hybridize with human RNA or DNA. Wherein the nucleic acid molecule is a nucleotide sequence consisting of at least 10 contiguous nucleotides from the nucleotide of claim 1 or a nucleotide complementary to the nucleotide sequence. 5. The isolated nucleic acid molecule of claim 4, wherein said molecule is comprised of from 10 to 35 nucleotides. 6. The isolated nucleic acid molecule of claim 4, wherein said molecule is comprised of from 18 to 35 nucleotides. 7. The isolated nucleic acid molecule of claim 4, wherein said nucleic acid molecule is a nucleotide sequence composed of at least 18 contiguous nucleotides or is complementary to said nucleotide sequence. 8. A method comprising: (a) contacting the sample with the nucleic acid molecule of claim 4 under conditions in which hybridization occurs; and (b) WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SW
WI, WI2, WI3, WI4, WIC, NY1, NY2, NY3, WI, WI2, WI3, WI4, WIC,
A method for detecting SWED, BOV, EQ, SLOVE, or SLOV2 nucleic acid in a sample. 9. A method comprising: (a) amplifying a nucleic acid in said sample with the nucleic acid molecule of claim 4 using polymerase chain reaction (PCR) technology; and (b) said amplified WI1, WI2, WI3, WI4, WIC, NY1, NY2,
Detecting the presence of NY3, SWED, BOV, EQ, SLOVE, or SLOV2 nucleic acids; WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3,
A method for detecting SWED, BOV, EQ, SLOVE, or SLOV2 nucleic acid in a sample. 10. A container comprising at least one container containing the nucleic acid molecule of claim 4.
WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, SWED
Kit for detecting the presence of a nucleic acid, BOV, EQ, SLOVE, or SLOV2 nucleic acid in a sample. 11. A recombinant nucleic acid molecule comprising a promoter effective to initiate transcription in a host from 5 ′ to 3 ′ and a nucleic acid molecule of claim 1. 12. A recombinant nucleic acid molecule comprising a vector and the nucleic acid molecule of claim 1. 13. A cell comprising the recombinant nucleic acid molecule of claim 11 or 12. 14. A non-human organism comprising the recombinant nucleic acid molecule of claim 11 or 12. 15. WI1, WI2, WI3, WI4, WIC, NY1, NY
2, containing the amino acid sequence corresponding to NY3, SWED, BOV, EQ, SLOV1 or SLOV2, wherein the polypeptide is SEQ ID NO: 18, 19, 20,
A purified polypeptide comprising the amino acid sequence shown in 21, 14, 22, 23, 24, 15, 17, 16, 25 and 26. 16. A vaccine comprising the polypeptide of claim 15 or an immunoactive fragment thereof together with a pharmaceutically acceptable diluent, carrier or excipient, wherein said polypeptide or fragment thereof is granulocyte in an animal. Those present in an amount effective to produce a favorable immune response to Ehrlichia. 17. A method for generating an immune response recognizing granulocyte aerichia in a host, comprising administering the vaccine of claim 16 to the host. 18. A vaccine comprising the nucleic acid molecule of claim 1 or a fragment thereof encoding an immunologically active polypeptide, together with a pharmaceutically acceptable diluent, carrier or additive. The fragment is present in an amount effective to produce an advantageous immune response against granulocyte aerichia in the animal. 19. A method for preventing errickliosis in an animal, comprising administering an effective amount of the vaccine of claim 16 or 18 to the animal for preventing the disease. 20. An antibody having a binding affinity for the polypeptide of claim 15. 21. A hybridoma that produces the antibody of claim 20. 22. The method according to claim 15, wherein (a) the sample and the immunocomplex are formed under the conditions.
WI1, WI2, WI3, WI4, WIC, NY1, NY2, NY3, comprising: contacting the polypeptide of any one of the above; and (b) detecting the presence of the polypeptide bound to the antibody.
A method for detecting an antibody against SWED, BOV, EQ, SLOVE1 or SLOVE2 polypeptide in a sample. 23. A diagnostic kit comprising: (a) a first container containing the antibody of claim 20; and (b) a second container containing a complex containing a binding partner of the monoclonal antibody and a label. 24. A diagnostic kit comprising a container containing the polypeptide of claim 15 or an immunologically active fragment thereof. 25. A method of inhibiting ehrlichiosis in an animal, comprising administering to the animal an amount that inhibits the ehrlichiosis of the vaccine of claim 16 or 18.