JPH04504422A - Rickettsial antigens for vaccines and diagnostics - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Rickettsial Antigen Amounts for Vaccines and Diagnostics Cross Reference for Applications This application is part of Application No. 253143 filed on October 4, 1988. This is a continuation application. This application is also sequentially filed on March 25, 1985. Partial Continuation of Application No. 715,528 [(now abandoned)] Partial continuation of Application No. 761178 filed on July 31, 1985 ( Application No. 1 filed on January 7, 1988 (currently abandoned) This is a continuation-in-part of No. 41505. This application was further filed on September 16, 1988. This is a continuation-in-part of application number 245855.

field of invention The present invention mainly relates to the order Rickettsiales, Rickettsia parasites of the Rickettsia family, more specifically, Ana Rickettsia of the genus Anaplasma, more specifically, Anaplasma Ricketts of Anaplasma marginale species Reduce the severity of infection or induce an immune response that prevents and protects against infection about antigenic polypeptides and proteins, related vaccines and methods useful for Ru.

Background of the invention Rickettsia are very small parasitic microorganisms of the order Rickettsia, family Rickettsiae ( approximately 2μ). Rickettsial diseases caused by these parasites affect humans and animals. Both have been of great importance throughout history. Typhoid fever and tsugae The number of human deaths caused by the disease outbreak reached into the millions. Typhoid fever is a ricketts Rickettsia prowazeki (R1ckettsia prowazeki) ) is the cause. Tsutsugamushi disease is caused by Rickettsia tsutsugamushi (Ric kettsiatsutsugamushi) and is the cause in eastern Asia and It is still endemic in many regions of Japan. Rickettsia rickettsii (R1cke) Rocky Mountain spotted fever (Rocky Mountain spotted fever) is caused by 　Mountain 5potted　fevar) is widespread in eastern America. It is dangerous in many other parts of the country as well.

Animal diseases caused by Rickettsia are Ehrlichi canis (Ehrlichi Rocky Mountain spotted fever caused by A. canis and Canine Ehrlich's disease canine ehrlichiosis, and dogs are susceptible to both diseases. I'm concerned. Rickettsial disease in horses is caused by Ehrlicia ehuis. equine ehrlichiosis caused by hia equis) and ehrlichiosis Bottle caused by Ehrlicbta risticii Includes Tomac fever (Potoa + ac fever). Significant losses It occurs in cattle from the Chia genus Anaplasma marginale. An animal ricketts diseases such as Q fever, canine Ehrlichosis and Botomac fever are contagious to humans. Can be dyed. Although rickettsial diseases are of great importance, rickettsial molecules Little is known about biology.

Anaplasmosis is a disease caused by Anaplasmoma marginale in cattle and other animals. It is an arthropod-borne hemoparasitic disease of the insects. Anaplasmosis occurs all over the world. Severely puts pressure on livestock production in tropical and subtropical regions. This Rickettsia is Transmitted to susceptible animals by ticks, biting flies, and blood contamination vectors. and infect red blood cells (red blood cells) there. anaplasma marginer A single Anaplasma marginae in the mature infectious stage of the microbial life cycle It occurs in red blood cells such as early bodies in red blood cells, which are microorganisms. Early stage of infection The body is regenerated by binary fission within red blood cells, forming an initial body of 2 to 8 fa; is then released and infects accompanying red blood cells.

During acute infection, levels of these parasites increase exponentially, leading to severe extravascular anemia. cause symptoms. Significant weight loss, miscarriage and death are associated with this parasitic infection and its consequences. It can occur during acute onset due to the parasitemia. From the acute infection Animals that recover remain persistently infectious and serve as a reservoir of transmission for susceptible animals. Ru.

Recent immunoprophylaxis against anaplasmosis is based on the less toxic Anaplasma margi. Nale isolate or Anaplasma cen trals), including infection immunization using less virulent Anaplasma species. Feeling Immunization is typically done by tetracycline treatment and has been shown to be effective in several species of animals. control severe infections. Another method of immunoprophylaxis is to kill all anaplasma. Vaccine treatment with vaccines containing Marginale microorganisms and host erythrocyte stroma It is. When cattle are challenged with virulent isolates, infection immunity successfully induces severe clinical disease. control. However, due to infection and immunity from the inoculum, weight loss and Clinical morbidity may result, including birth and possibly death. This inoculum also , Babesia, Theileria 8 and other hemoparasites such as T rypanosoma, and They may transmit viruses, such as leukemia virus, to the animals being treated. death The challenge of cattle immunized with the anti-anaplasma marginale erythrocyte stromal vaccine is , resulting in moderate clinical disease and persistent infection; Due to the presence of stroma, it can be transferred to nursing calves through acid bovine colostrum, leading to the development of new autoimmune diseases. It has been shown that it induces anti-erythrocyte antibodies that may cause allohemolysis in newborns. Ta.

Therefore, effective remedies against these and other rickettsial diseases. There remains a strong need for immunization methods. Furthermore, detection of rickettsial parasites There remains a continuing need for relatively simple diagnostic tests for carriers. Ru.

Brief description of the drawing The drawings (FIGS. 1-16) relating to preferred embodiments of the invention are attached and are as follows: Write simply.

Figure IA shows the results of experiments on nitrocellulose using four different base antibodies or antisera. Four radiographs showing the detection of native Anaplasma marginale protein ( This is a copy of 1) to (4).

Figure IB shows recombinant plasmid-containing E. coli (E. Figure 2 is a reproduction of a radiograph showing the detection of proteins from E. coli. rabbit anti Screened for reaction with serum R873, native Anaplasma margina Proteins that are reactive with surface protein complexes are separately isolated from MSP-1 or MSP-1. is called Am105.

Figure 2 shows Anaplasma margi encoding the expression of the protein recombinant Am105. This is a restriction enzyme map showing related parts of the Nale gene.

Recombinant plasmids pAM22, pAM25, pAM97 and pAM113 The relative orientation and relationships of the incorporated genes are also indicated.

Figure 3 shows electrophoretically separated Anaplanuma marginale protein, Plasma Protein from recombinant E. coli with sumid pAM25, plasmid pf Proteins from E. coli with 3R322 and molecular weight standard proteins are shown. This is a copy of the radiograph.

Figure 4A shows electrophoretically separated recombinant Aml05 and native Aml05 ( Aml05L and Am105U), recombinant protein E. coli cells containing lasmid pAM25 and plasmid pBR322 Figure 2 is a reproduction of a radiograph showing E. coli cells containing.

Figure 4B shows recombinant Am105 and purified native Am105 after treatment with protease. Electrophoretically obtained by treatment of proteins Am105L and Am105U Figure 2 is a reproduction of a radiograph showing isolated polypeptide fragments.

Figure 5 shows monoclonal antibodies IE and 22Bl, and rabbit antiserum R9. Recombinant Am105, native Am105 after immunoprecipitation reaction with 11 and R907 Shows electrophoretically separated proteins containing L and native Am, 105 U. This is a copy of the radiograph.

Figure 6 shows monoclonal antibodies IE1 and 22Bl and rabbit antiserum R9. Surface radiolabeling of Anablasma marginale early bodies using 11 and R907. and radio showing electrophoretically separated proteins obtained from immunoprecipitation reactions. It is a copy of the graph.

Figure 7A shows recombinant plasmid DNA and analytical results using Southern blotting method. Electrophoretically separated DNA comparing genomic DNA of Blasma marginale This is a copy of the radiograph showing A.

Figure 7B shows bovine leukocyte DNA and Anaplasma margina concatenated with restriction enzymes. Radiograph showing electrophoretically separated DNA to compare the genomic DNA of This is a copy of F.

Figure 8 corresponds to Am105U in the Florida (F1orida) isolate. Anabras shows the relevant parts of the genome that contain genes encoding protein expression Four restriction enzyme maps for four different geographical isolates of M. marginale ( ], ) to (4) are shown. Genetic regions identified in each map are indicated by diagonal lines.

Below the restriction map, five plasmid diagrams indicate the gene parts or genes indicated by thick lines. indicates the entire plasmid part. Incorporates recombinant base gene DNA Parts of the plasmid that are not present are indicated by thin lines.

Figure 9 shows the type II conversion. sumid pVA1, pWAOl, pID6 and pFL Electrophoretic expression expressed by a recombinant E. coli cell line incorporating lO This is a reproduction of a radiograph showing the proteins separated. Furthermore, the corresponding address Also shown is the native protein from the Naplasma marginale isolate.

Figure 10 shows the gene encoding the expression of MSP-1a or Am105U protein. 70 including F 1 orida, Virginia (Irgjnia), D of the Idaho and Washington isolates FIG. 2 is a sequence diagram showing the NA nucleotide sequence.

Figure 11A shows transcription initiation in the 70 Lida isolate MSP-1a gene. Figure 2 is a reproduction of a radiograph showing electrophoretic separation of NA7 fragments.

Figure 11B shows the presence of Anablasma marginale in four geographical isolates and E. The DNA nucleotide sequence shown in Figure 10 for the promoter region of FIG.

Figure 12 shows the results produced by four different geographical isolates of Anaplasma marginale. Sequence diagram showing the amino acid sequence of the expressed MSP-1a (Am105U) protein It is.

FIG. 13 is a sequence diagram comparing the amino acid sequence portions shown in FIG. 12. Figure 13 42 The sequences shown show repeating patterns of five different types of A-E labels. each The chart shown on the right of Figure 13 shows the number of times a repeating pattern is contained in a protein. It is written in

Figure 14 shows some synthetic polypeptide sequences and shows that such sequences are It shows whether it reacts with monoclonal antibody 22B in ro.

Figure 15 shows Anaplasma marginale protein Am for Florida isolates. The DNA nucleotide sequence containing the gene encoding the expression of 105L contains eight sequences. This is a restriction enzyme map showing the cleavage site of Kuraka's wI@enzyme.

@16 encodes the expression of Anap, Lasma marginale protein Am105L. FIG. 2 is a sequence diagram showing the DNA nucleotide sequence of the gene coded. Furthermore, Flori The corresponding amino acid sequence of the protein Am105L of the Da isolate is also shown.

1 skin 0! sex The present invention relates to detection of Rickettsial parasites, particularly Anaplasma marginale. and by providing improved antibodies and immunogens for immunization. We strive to overcome these limitations.

The present invention binds serum antibodies and, in at least some cases, Nale and other rickets with epitopes of the same or sufficiently similar nature Immunogenic to reduce the severity or prevent infection caused by microorganisms Including the appropriate purified antigen.

Furthermore, the present invention provides antigenic components of Rickettsiae such as Anablasma marginale. selectively binds and provides detection and other useful screening and diagnostic applications. This includes certain monoclonal antibodies that can be used.

Isolation and purification of selected natural proteins from Anaplasma marginale microorganisms or processed to produce one or more purified immunogenic polypeptides or proteins. can produce parka. The present invention provides at least one surface-exposed epitope. Another natural antigen is conserved; Including findings that are common to multiple geographic isolates of Lasma marginale. Ru. This protein complex has been identified and purified, and has been isolated from major surface proteins. (major 5 surface protein 1) (MS P-1) and Aml 05. The two component proteins of this complex are AmJ , 05U and Am105L. Anaplasma marginale small In at least one geographically distinct isolate, these complex proteins are approximately 1 It has an electrophoretic mobility corresponding to an approximate molecular weight of 05,000 Daltons. separate isolation In the body, the electrophoretic mobility and apparent molecular weight of these two complex proteins are , specifically with respect to one of the two complex proteins.

Other antigenic proteins have been identified from the Anaplasma margiculae microorganism, approximately 860 00 Dal door (Am8B); 61000 Dal door (Am61); 36000 Dal door ton (Am36); and 15,000 daltons (Am15) are relative to the molecular weight. It is characterized by its electrophoretic mobility. Further, as identified here, Other antigenic proteins are also useful in the present invention.

In addition to the natural protein isolated and purified from Anaplasma marginale, Antigens and immunogens according to the invention include one or more such proteins, polypeptide fragments of proteins, or polypeptide synthesis or genes. one or more immunologically similar proteins produced by engineering or Active agents formed from polypeptides can be constructed.

Several forms of novel antigens of the invention code for the expression of recombinant antigens that exhibit immunogenic activity. produced by recombinant DNA techniques. At least some of the advantages of the present invention Amino acid sequences characterizing the effective antigen and immunogen have been identified.

The antigen protein of the present invention is derived from cellular components of infected red blood cells such as Anaplasma and Margina. It is partially purified by removing or isolating the initial product. After that, as appropriate Significantly purified initial Crush the body. Pass the destroyed initial body through an antibody that selectively binds to the desired antigen Alternatively, the desired antigen can be added to crushed Anagrasma marginale microorganisms. Can be purified from living organisms. Such purification involves the production of an aqueous mixture containing crushed incipients. through an insoluble matrix such as an affinity chromatography column. can be achieved. The insoluble matrix contains the desired antigen protein or PEG. is specific for the compound and recognizes one or more epitopes on it. , with monoclonal antibodies adsorbed onto an insoluble matrix. adsorbed anti- Hara further analyzes the non-adsorbed substances of the aqueous initial temperature mixture by affinity chromatography. purified by washing through an e-column and bound to the matrix. Obtain adsorbed antigen. The adsorbed antigen is recovered from the matrix, and the purified antigen according to the present invention is Get the original.

Preferably, the novel monoclonal antibody used to prepare the purified antigen is by injecting Anaplasma marginale-infected bovine red blood cells into and then vaccinate the mice with the appropriate rickettsial parasite or otherwise It is conveniently prepared by inoculating with. Lymphocytes in the pancreas of infected mice? Also collected.

Lymphocytes collected from the mice are fused with immortal cells such as myeloma cells and then Hybridoma cells are obtained and cloned to propagate hybridoma cell lines.

Some of the hybridoma cell lines contain monochrome cells that selectively bind to the desired antigen. It produces natural antibodies. Then, collect the hybridoma cell line of interest. Screening uses a novel method to identify certain hybridomas.

The hybridoma screening method advantageously firstly comprises Anaplasma margi may include a procedure for detecting hybridomas that produce antibodies that bind to Nale. stomach. This advantageously applies to blood smears infected with Anaplasma marginale. This is accomplished by an indirect immunofluorescence assay. Then, the hybridoma were subjected to rough screening, and cell line supernatants containing monoclonal antibodies were used. The immunoprecipitation reaction of Anaplasma marginale proteins selected by and monoclonal against specific Anaplasma marginale proteins. Hybridomas producing antibodies are measured. In particular, surface-exposed evitopes, In detail, epitopes bound to the immune serum of animals previously infected with the parasite An antibody that selectively binds to the Anaplasma marginale protein that has a Select producing hybridomas.

Add amount of desired monoclonal antibody to inoculate the selected hybridoma cell line Advantageously, it is obtained by collecting ascites fluid from infected mice. rat ascites Such monoclonal antibodies collected from It is purified as appropriate by the Fee method. Then, the purified monoclonal antibody is bound to an insoluble matrix such as Sepharose. and prepare an immunoaffinity matrix. Next, Anaplasma Ma Partially purified crushed rickettsial microorganisms such as M. luginare were added to the immune system. the antigen protein of interest is selected onto the matrix. Adsorb selectively. Next, the purified protein adsorbed onto the matrix is Removed or otherwise recovered from the matrix, as appropriate, in a sufficiently purified form. of the desired antigen and effectively serve the intended use of the present invention.

The purity of the proteins obtained by the present invention depends on the production method used. natural The purity of proteins and polypeptides derived from them is determined by the purity of the antigen in its native state. significantly higher purity than that of For example, Am105 in its native state is found in the initial body. It is believed that it is present in an amount of about 0.1-1% of the total protein present in the world. Heaven In its natural state, it contains approximately 200 other proteins, carbohydrates, red blood cells, glycoproteins, and Many other impurities are present, such as nucleic acids. However, Am105 is here They can be purified to significantly higher levels of purity using the methods taught. about 10 It is believed that weight percent purity or higher is possible. 20% by weight or Higher purity is more preferred. More preferably 50% by weight or more purity.

The purification methods taught herein require at least 90% by weight, preferably at least 95% It has the ability to obtain a purity of % by weight, most preferably at least 98% by weight.

The purified Am105 has approximately 105,000 Daltons as determined by electrophoretic mobility analysis. However, according to the NA and amino acid sequence information as shown here, When measured, the molecular weight is significantly lower than that. Other antigens according to the invention are also considered to show a significant difference between the molecular weight measured by sequence information and electrophoretic mobility. available. Nevertheless, electrophoretic mobility information identifies the antigen according to the invention. and provide an effective method for isolating it.

One of the immunogenic antigens measured in the present invention, Am105 (Florida isolate), was Monochromes produced by the hybridoma cell lines ANA15D2 and ANA22Bl Reacts with local antibodies. Deposit of cell lines ANA15D2 and ANA22Bl , ATCC number in American Type Culture Collection, respectively. No. HB9046 and HB9047.

Am105 and recombinant or artificially synthesized peptides as taught herein. The immunoaffinity-purified antigens of the present invention, such as Most preferably, it is free of contaminants, red blood cells, and nucleic acids.

Active fragments or subunits of identified antigenic polypeptides of the invention is immune to Anaplasma marginale or other rickettsial organisms. It can be said that inducing it is effective. of at least some antigens Efficacy was demonstrated in cattle. The size of active fragments ranges from 6 to 20 or could possibly be as large as 6 to 10 amino acids.

Antigens according to the invention have immunoaffinity as described above and elsewhere herein. by chromatography or by using artificial methods of polypeptide synthesis, Or a desired peptide or protein.

can be obtained using genetic engineering for the expression of Possible by artificial synthesis Various methods are known in the art for producing repeptides, including known amino acid Since the effectiveness of such methods for producing polypeptides having acid sequences is unknown, Not listed here. As an illustration of one of the many suitable techniques for such synthesis, Sources include professional services and many scientific works. Such a technique , which requires knowledge of the desired amino acid sequence for the required polypeptide; The novel technique described here allows for a variety of different synthetic antigens and proteins to be produced for use in the present invention. and immunogen.

The present invention further provides certain novel genetically engineered DNA and RNA sequences and and for the purpose of analyzing and expressing the novel antigen according to the present invention. This includes microorganisms that have incorporated sequences. Such recombinant microorganisms can be used to - Create a pseudo-random genome library of recombinant bacteria such as E. coli and generate novel D. It was produced by integrating the NA plasmid. Linear plasmid is anap DNA fragments from the genomes of suitable rickettsial parasites such as Lasma marginale. Incorporate fragments. Recombinant ff1 DNA plasmid consists of plasmid DNA It was produced by cutting with an appropriate restriction enzyme.

Similarly, Anaplasma marginale DNA was cut with an appropriate restriction enzyme, and a large number of A variety of Anaplasma marginale DNA fragments are produced. anaplus Mix the DNA strands from M. marginale and the plasmid and make 7 strands of each. The components were combined and then ligated to form a recombinant plasmid vector. wire fiber Transplant the recombinant plasmid vector into a suitable host, E. coli, and clone it. A collection of recombinant bacterial cell lines was obtained. The obtained recombinant base cell line was treated as described above or monoclonal selected against parasites as described elsewhere herein. For screening to express the desired antigen, such as by using antibodies. did. In addition, a virus such as a vaccine virus can be used to control the expression of a desired antigen. Recombinant viral vectors can be produced that have nucleic acid sequences encoding the nucleotide sequence.

Recombinant RNA can also be produced encoding the expression of the desired antigen. Ru.

A surface-exposed protein contained in a protein complex also known as MSP-1 and Am105. Recombinant Streptomyces Cells Expressing Antigenic Recombinant Deaf Proteins that Mimic Native Proteins system was developed. The present invention further provides that the natural protein contained in this complex is Various geographical units of Anaplasma marginale differing in size and amino acid sequence. It also includes the knowledge that there is polymorphism between the disparities. anaplasma marginer The recombinant plasmids containing the DNA were analyzed to identify their associated polymorphic proteins. The DNA sequence related to the expression of was determined. Expressed by these four geographical isolates The derived antigen was also analyzed and its amino acid sequence determined. The antigen is a polypeptide It has a hypervariable domain with a variable number of tandem repeat sequences at its N-terminus. Found. The tandem repeats are polypeptides of 28 or 29 amino acids. Consists of C. The number of repeats was 2 and 3 among the groups of 4 different isolates analyzed. and 8. However, all tandems of the four isolates Peat was found to have a conserved amino acid sequence. Anaplasma margina It binds to the surface-exposed shrimp of parasites and is effective in neutralizing the infectivity of such parasites. Monoclonal antibodies that are contained in the tandem repeat region of the protein also contain binds to at least some conserved epitopes.

Novel recombinant cells expressing proteins containing tandem repeat polypeptide sequences A cell system was developed. Such novel recombinantly produced proteins with conserved polypeptide sequences whether or not it alleviates the degree of overlap caused by Anaplasma marginale. to generate an immune response in cattle that is effective in preventing acute infections. It was measured.

Reacts with Anazurisma marginale, especially causing a neutralizing reaction of the parasitic infection. Combined with a selected monoclonal antibody that is reactive with an epitope known to be Other compatible antigens are also within the scope of the invention.

The immunogenic antigen according to the present invention can be used in vaccines to reduce the severity of rickettsial microorganisms. Can elicit an immune response that is effective at alleviating or preventing infection Wear. About 1-1000 micrograms of such antigen in a single dose of the vaccine, Preferably 5-400 micrograms, most preferably 10-200 micrograms. Present in an amount of rum. A single injection dose usually has a volume of approximately 1-2 milliliters. have a quantity. Therefore, the concentration of purified surface antigen in injectable vaccine compositions typically In the range of about 1 to 500 micrograms per milliliter, preferably about 5 to 2 00 micrograms, ram/ml, most preferably 10-500 micrograms It is ram/ml.

The antigen is advantageously dissolved or otherwise 70 indica complete and/or Administered with an adjuvant such as incomplete adjuvant.

In addition to the purified antigen and optional adjuvant, the vaccine may further contain any other It may also contain a suitable pharmaceutically acceptable carrier or diluent. The pharmaceutical The acceptable carrier or diluent is preferably a non-toxic sterilizer useful for administration of the active antigen. a compound that increases the effectiveness of the bacterial liquid or, in some cases, the inoculation process; composition or solvent.

To immunize ruminants, young animals are inoculated with purified antigen and any desired adjuvants. It is preferred to administer a vaccine consisting of a bunt and a diluent. The antigen is Is it produced to express a polypeptide or protein from recombinant cells? or produced by artificial polypeptide synthesis, purified from parasites. Can be done.

Such purified antigen may be carried in a suitable pharmaceutically acceptable carrier or diluent, and any Preferably, the desired adjuvant is added. Preferably, the animal is aged between 1 and 6 weeks. about 2 to 8 times, preferably 3 to 5 times, at intervals, preferably 2 to 4 weeks apart. Inoculate sequentially in one dose. The purified protein or polypeptide can be used as a vaccine. present in an effective amount to induce an immune response in the animal to be treated. Such exemption The epidemic response was later attacked by virulent rickettsiae such as Anaplasma marginale. where the severity of acute infection is substantially reduced or even prevented. It is preferred to adequately protect vaccinated animals. The injection is usually intramuscular (j , m, ) or subcutaneous (s, c,) administration.

Certain purified recombinant synthetic or natural polypeptides and proteins may also be infected by suitable rickettsial parasites such as Anaplasma marginale It is useful in diagnostic tests to determine whether Conversely, such diagnostic tests and one or more monoclonal monoclonals specific for infection by such microorganisms. Antibodies may also be incorporated. The diagnostic test is advantageously an immunoassay, e.g. , one or more types of solid-phase enzyme immunization, such as serologic diagnosis of anablasmosis. It is a measurement method. The assay can also be performed using radioimmunoassay or purified antigen in infected animals. specific for antibodies produced by or antigens associated with the particular parasitic microorganism of interest. Other assays that utilize selective binding to binding monoclonal antibodies may also be used. stomach. testing samples from the subject animal with such antigens and/or antibodies; , different results are obtained in infected and uninfected animals.

Using the determined DNA sequence information, the labeled and hybridized DNA or RNA Novel nucleic acid sequences useful as nucleic acid probes that can be used to detect the presence of can provide highly sensitive diagnostic tests.

Details of the invention Detailed explanations are provided to assist you in understanding the large amount of complex material contained herein. It is divided into different sub-parts.

Bart ■ - Monoclonal antibodies and immunoaffinity purified antigens Preparation of monoclonal antibodies against Anaplasma marginale - origin of the antigen, Mouse immunization protocols, myeloma cell lines used, culture media and conditions, and Cell fusion and cloning methods are described by Davis et al. Regarding the development of monoclonal antibodies against Ma marginale and their applications. Development of monoclonal ant ibodies to Anaplasma marginal and pr Eliminary 5 studies or + their applicat ion). Proceedindus of the Seventh ・Anaplasmosis Conference (Proc, 5evanth Natio nal　Anaplas+aosisConf,), October 21-2, 1981 30, Mississippi State University (“Press”) i5tate University Press).

The procedure can be summarized as follows.

Animals - Juvenile Hereford and Holstein (Ho) In this experiment, cattle of the Ilstein variety were used. Infection with Anaplasma marginale The animals underwent pneumonectomy. Two types of mice, BALB/c and BI0 -A(3r) was used as the source of cells for producing hybridomas. child These lines and additional lines, B101A, Bl0-A (5R) and B 10-A(2R) was supplied together with hybridoma cells as a source of thymocytes. It was used as a cell for symbiotic culture.

Preparation of red blood cells infected with Anaplasma marginale - Davis W. ・Davis, W, C, et al., Infective Immunization El. Infect, Immun-22: 597-802 (1978) A. marginale idaho in pneumonectomized cows as described in isolates were infected. Blood was collected every 2-4 days after infection for tissue culture studies. Collected in heparin. A blood smear was prepared and stained using the Leith-Giemsa staining method. We investigated the anaplasm body. When parasitemia reaches 20-50% The blood is collected, centrifuged, removed from the buffy coat, and then For use, it was frozen in 30% dimethyl sulfoxide (DMSO). Additional Blood smears were prepared and kept at -70°C until used for detection of monoclonal antibodies. It was frozen. .

Antigen Preparation and Immunization - Two preparations were used as a source of antigen. first Frozen cells with 30% parasitemia (backed cell volume is 50%) was dissolved and then immediately mixed with tris (0°01M) ammonium chloride (0.87M). %) solution (pH 7.2). The lysate was incubated at 800 rpm for 30 minutes. Centrifuged; the resulting pellet was washed three times in Hank's Balanced Salt Solution (HBSS). Washed and resuspended in 10ml HBSS. Six mice [BIO,A( 3R)] was injected intraperitoneally (i, p-) with 11 m lysate. for cell fusion Three days before lung removal, mice were given an intravenous booster injection of 2 ml of lysate of Q. Ta. The second preparation, a lysate of infected red blood cells, was prepared by Dav. is, N, C, et al., Infect Immunization , In+mun, ), Vol. 22, pp. 597-602 (1978). Renogra in density gradient (25-55 %) purified above. Band containing anagrasma bodies and contaminated with red blood cell matrix were collected as above, washed and then resuspended in 10ml HBSS. 5 B-ALB/c mice were immunized intraperitoneally with 1rrxl as described above. and 0.2 mt was injected intravenously as a booster. Perform booster antigen injection All mice were immunized at least 3 weeks prior to treatment.

Myeloma cell lines used to produce hybridomas - several HAT (human Voxanthine, Aminopterin and Thymidine) Sensitive Tissue Culture Compatible Mouse Bone Marrow Use of tumor cell lines as fusion partners in the production of antibody-producing ipridomas there was. NS1, a cell line that produces but does not secrete K-type light chains [Oi et al. ・V-T., “In Selected Methods of Cellular Immunology (In 5e Lected Methods in Ce1lular Immunology ) J ($1) B, B-Mishell and varnish M Shiigi (SM, Shiigi), W H Freeman A WHFreeman and Co, pp. 351-372 (1980) 1 and SP210-Ag14, both light chain and heavy chain were synthesized. A cell line derived from the N5t-BALB/c hybrid [Schulman M. chul+man, M. ) et al., Nature, 276: 2 79 pages (1978)] was published by Salk Institute. Itute). X63-Ag3.653, both light chain and heavy chain Another cell line that does not arise [Kaarney, J.F. et al., Journal of Immunology (J.

Immunol, ), 123: pp. 1548-1550 (1979) 1. 7 Red Hutchinson (F) by M Lostrum (L Lostrum) Red Hutchinson Cancer Research Center (Seattle, WA). It was.

Culture media and conditions - 13% calf lysis (FCS), 5xlO-'M 2-Me, +17') ztanol (2ME), penicillin (100 units/m Rio Dulbecco's modified Eagle's medium containing and streptomycin (100 g/ml) Earth (Dulbacco’s Modified Eagle Medium%DM EM) to maintain myeloma cell lines and new hybridoma cell lines. The next culture medium was If this medium is used 14 days after preparation, glutamine (2mM) was added. The fusion medium and transplantation medium were from Littlefield (Li monoclonal antibody-hybridoma: "biological component" A new dimension of biological analysis al Analysis) J, (ed.) Kenn. ett, R, H, et al., Plenun+ Press, 3 63-416 (1981)], HAT and HT (1981), respectively. Hypoxanthine and thymidine). 15%FC5 and RPMI-1640 containing penicillin/streptomycin, Red blood cells infected with Anaplasma were cultured. Davis Duburi 1-Shi (Da vis W, C, et al., In7 Effective Immunology (Infect fm) Un, ), 22.597-602 (1978). All cultures were grown at 5% C It was held at 37°C in an O2 gas-air mixture.

Cell fusion and cloning - How to fuse lung cells and myeloma cells , similar to the method described by Ol et al., supra. Myeloma cells are in T757 lasks by removing excess cells and feeding every 3-4 days. maintained in logarithmic growth phase. The day before fusion, cells were harvested and washed by centrifugation. Plated in T757 flasks at 1X10' cells per flask.

On the day of use, cells were collected, centrifuged, and resuspended in DMEM without FCS. . by injecting crushed bovine red blood cells containing early Anaplasma marginale. immunize mice. Lung cells are grown from freshly killed immunized mice into their lungs. Inject DMEM, gently tear apart using forceps, then remove with 100 mesh Obtained by pressing through a clean into a 50 ml test tube. granular crushed material After removal, the cells were pelleted by centrifugation and the medium was removed.

Contaminated mouse red blood cells were selected by briefly contacting them with distilled water (2 ml). After selective lysis, the lung cells were quickly diluted in DMEM. as a supply cell Collect the thymocytes to be used as described above (but without lysis with water). 1X10? Cells/ml were suspended in DMEM-Fe2-HAT.

Count myeloma cells and lung cells, then add 5 lung cells for every myeloma cell. They were mixed at a ratio of 100% or 2.5%. Typically, 1Oa of lung cells are added 2 or 4X Mixed with 10' myeloma cells. The cell mixture was sedimented and the supernatant was removed. Next Polyethylene glycol [PEG1540, Baker Chemical Company Add 1 ml of a 50% solution of Ni (Baker Chemical Co.) to the cell base. to form a slurry consisting of small (1 mm”) clumps of cells. Mix slowly with the cells until the mixture is completely mixed. After mixing for 3 minutes, add DMEMl for 10 minutes. Gradually dilute the cells by adding 0-l and then 20 ml over 5 min. I interpreted it.

The resulting mixture of fused cells was pelleted by centrifugation, and the cells were placed in DMEM-Fe. Resuspend in 2-HAT. Then add the thymocytes to give 108 lung cells, A mixture containing ms cells and lXl0' thymocytes was obtained. 10 cells Distribute into 96-well culture plates and place in incubator. Half of the dark culture medium was replaced every 3-4 days. 300 hybridoma clones When the size of ~1.000 cells is reached (usually by day 12-18), remove the supernatant. were collected and tested by indirect immunofluorescence on smears of infected red blood cells. positive well cells were identified and transferred to a 24-well (2-1) culture grate. 3~5X10@ 2 thymocytes were added as feed cells to maintain growth. On day 14 or culture The medium was replaced with DMEM-Fe2-HT when dilution was required. ,each Remove excess cells and feed every 2-4 days, depending on the replication rate of the clone. Cells were incubated for 2 weeks (1 week in DMEM-Fe2-HT and then in D on MEM-FC5) and maintained in static culture. At this time, duplicate plates prepared and overgrown. The top groove of this plate was tested for antibody activity.

All cell lines identified as antibody producers by this procedure are then transferred to the master - Remove each strip from the plate and place each strip in a 6-well plate (each well has a volume of 5 ml). Expanded as single cultures with 3 wells per cell line. Cells were harvested twice and placed in liquid nitrogen. (3-10XIO' cells in 10% DMSO),! Itchy cells increase It was allowed to grow and die. The upper groove was then collected (approximately 15 m1) for the next analysis. Frozen.

If you want to identify cell lines that produce antibodies of direct interest, remove them from the freezer. Remove, thaw, and clean immediately or after 24 hours of incubation by limiting dilution. I turned it into a loan. Usually, hybridoma cells are placed in two to six 96-well plates. Plate at 3 cells per well in the presence of 10 thymocytes.

Wells containing single colonies were identified microscopically and the superior grooves were collected for antibody activity. Tested. Cells from positive wells were transferred to a 24-well plate as described above and then The colonies were expanded and preserved by transferring to a 6-well plate. I got used to each strain, Cloned lines 4-6111 were conserved.

In a preliminary study, we compared several methods of preparing hybridomas and Using clonal antibody technology to study Anaplasma marginale; The suitable conditions have been determined. The results obtained showed that SP210-ag14 and X63-Ag 3.653 myeloma cells, their growth characteristics, the outg of hybridomas rowth) and the inability to secrete light or heavy chains. also proved to be a useful fusion partner. I know it's the most effective The fusion ratio was between 5 and 1 for SP210-Ag14 and for X63-Ag14. For 3.653, it is 2.5-1. Under the culture conditions adopted, in the culture medium It is not necessary to have 2-ME in the cells and to have thymocytes as feeding cells. It was essential. If these criteria are not adhered to, hybridoma disease may occur prematurely. However, if the described criteria were adopted, each fusion A hybrid of 600 to 600 t, o o o s was obtained.

There is no problem with using Anaplasma marginale red blood cell-containing preparations as antigens. did not pose a problem. Both the crude lysate and the Regulafin purified formulation were highly It had strong immunogenicity. The majority of hybrids detected in primary cultures were Anagras produced antibodies against M. marginale. However, the lysate is the source of the antigen. When used as an anti-red blood cell antibody, more anti-erythrocyte antibody-producing hybrids were observed.

When collecting and storing a sufficient number of hybridomas, a clone of the antibodies being produced is required. To determine the ras, they were tested by immunodiffusion. In addition, Anapra To determine their binding pattern to Suma marginale or erythrocytes on cultures of infected monocytes grown in short-term culture and from infected cattle. Indirect detection on smears of infected red blood cells with respect to the fixed portion of the sampled tissue. They were tested by immunofluorescence.

Selection of cell lines producing anti-Am105 monoclonal antibodies - Hybridoma cells The cell supernatant was first prepared from an acetone-fixed smear of Anaplasma marginale infected blood. Anti-Anaplasma marginale antibodies were detected by indirect immunofluorescence as described above. Clean.

Positive (antibody producing) cell lines were then radiolabeled with [3″S]methionine. Immunization of [12611 surface radiolabeled Anaplasma marginale] Screen again for specific production of anti-Am105 antibodies using the precipitation method. Ru. The exact procedure for this can be found in Palmer, C.H. , H, et al. [Journal of Immuno1ogy) , 133:1010 (1984)] with a slight modification of the procedure reported by There are, but they are as follows.

Immunoprecipitation of surface radiolabeled early bodies and erythrocyte matrix was performed using Shapiro Nis Zet. (Shapiro, S., Z.) et al. [Journal of Immunological Medicine] Sods (J, Immunol, Methods), 13:153 (197 It was carried out by using a modification of the technique described by [6]. Initial body or red blood cells with 1% (v/v) Nonidet P-40 and Disintegrated with 0.1% (w/v) SDS for 30 min at 4°C, 135.000x Centrifuge for 60 minutes at Millipore Corp, ), Bed 7 Ord, Pass MAI, 50 Ultrasonication was performed with W for 15 seconds. 200.00Qcpm of trichloroacetic acid precipitate The silicone-treated tube was added to 50 liters of hybridoma supernatant. Add to 10 microliters of rabbit anti-mouse immunoglobulin in a tube and incubate at 4°C. and incubated for 30 minutes. 10% (V/,/) protein A producing staphylo Coccus aureus (Staphylococcus aureus) Calbiocham-Beh ring Corp, ), U Hoya (La Jolla), CAI's 100 My Add Chloritol to each tube, incubate for 30 minutes at 4°C, and add 0.1% Nonidet P-40 and Teene containing 2M NaC+ for the first four washes. n) Buffer (20mM Tris-HC1, 5mM EDTA, 0.1M NaCl % 5mM NaN, pH 7,6) and centrifuge at 1.250XG. It was washed six times by doing this. 50 liters of Staphylococcus binding complex Boil for 3 minutes in 5DS-PAGE buffer and centrifuge at 1,000 x c. The precipitated labeled substance was eluted.

Immunoprecipitate (2,000-10,000 cpm) and radio51'7 primed first 7.5-17°5% (W/V ) subjected to electrophoresis on a gradient polyacrylamide gel.

The gel was fixed in glass-distilled water: methanol: acetic acid (62:4:1) and dried. After drying, Kodak X-OmatAR film [Eastman Kodak (Eas tman-Kodak), Rotinister, NYI for 48 hours at room temperature; identified radioiodinated early body and erythrocyte polypeptides, and also identified Cronex ・Quanta (Cronex Quanta) I [I intensifying screen [Dupont ( DuPont), Wilmington, DEI at -70°C. Immunoprecipitates were identified by time exposure.

14c-labeled protein used for molecular weight comparison [Amersham (An+ersham) , Arlington Heights, IL] myosin, molecular weight 200.000; Lylase b, 92,500, bovine serum albumin, 69,000 two oval albumins Min, 46.000; carbonic anhydrase, 30.000; and lysozyme, 14. It consisted of 300. Hybridoma supernatant is monochrome against Am105. If it contains null antibodies, the band on the X-ray film (autoradiograph) will be The molecular weight is approximately 10s, ooo.

Two cell lines producing anti-Am105 antibodies (ANA L5D2 and ANA 2 2Bl) was identified. These cell lines were doubly cloned and isotype After determining (all IgG3), the superior groove is determined to be O1lmg immunoglobulin/ml. Concentrated. Concentrated superior grooves were used for all subsequent tests. ”J-Am Immunoprecipitation of l05 was performed using double-cloned hybridoma supernatant. repeated.

Evidence that Am105 is an early body protein and not the origin of red blood cells includes immunological evidence. using the immunofluorescence method; if ANA 15D2, ANA 22B1 or rabbit anti- Aml O5 antibody (all positive for infected red blood cell membranes) indicates non-infection that these antibodies do not react with red blood cells or infected red blood cell membranes, and that these antibodies This includes not immunoprecipitating radioactively labeled red blood cell ghosts. Furthermore, ANA 1 5D2 and ANA22Bl were isolated in vitro during short-term red blood cell culture. Metabolically radiolabeled Am105 was immunoprecipitated. 3sS induction during short-term culture It has been shown that entry occurs exclusively in the initial field. Am105 is 3IS Doublet bars most clearly seen with labeled Am105 or silver-stained Am105 immunoprecipitate as a host. This doublet is consistently present, with surface-exposed epitopes It shows a complex of two Anaplasma marginale proteins with Something has been discovered. The two proteins as a complex are defined here as time and It is designated major surface protein 1 (MSP-1) and the term Am105.

The two proteins that make up the doublet are Am105U and Am10 It is called 5L. The protein Am105U is sometimes called MSP-1a. Am105U is sometimes referred to as MSP-1b. Proteins forming complexes have electrophoretic mobilities that are almost the same. Additional tests include Am10 5L has an electrophoretic mobility corresponding to a molecular weight of approximately 105 kilodaltons. It shows. Some measurements have shown that the electrophoretic mobility of Am105L is almost 10 This shows that it is 0 kilodaltons.

These mobilities are related to the Florida geographic isolate of Anabrasma marginale. It is related to MSP-1, a protein of the AM105 complex. More below Pairs regarding other geographical isolates of Anaplasma marginale as described. The corresponding proteins exhibit a high degree of polymorphism in the proteins that make up the MSP-heavy complex. show. Therefore, the electrophoretic mobility of the proteins forming this complex changes .

However, the antigenic properties of these various isolates are similar, as explained below. are doing.

Monoclonal antibodies are generally used against other antigenic antibodies of Anaplasma marginale. Prepare using the same or similar procedure as described above for surface proteins. be able to. To date, hybridoma cells that produce monoclonal antibodies have , for additional antigenic surface proteins of Anablasma marginale, created by It is.

Am105 (complex) ANA 15D2 (1502), ANA 22Bl (2 2B1) Am105 (complex) F34CI Am86 AM075C2 Am36　F19E1, ANAPRA using ANAO-58A2 monoclonal antibody Test on neutralization of Suma Marginale - Test for neutralization of early bodies using two anti-Am105 monoclonal antibodies. In order to perform B1 ascites and the mixture was injected into pancreatectomized cows. . Although complete neutralization was not observed, a significant prolongation of the detection latency occurred (Table 2). Experiments show extended detection latencies observed using 1010 initial bodies. showed partial neutralization, and complete neutralization probably occurs at lower infectious doses. It was repeated under the assumption that it was likely to be. In the second experiment, the initial infectivity was completely Total neutralization occurred with the initial form of lO7 bonds, but with extended detection incubation period 101.10', and the initial form of the 101a bond. was generated using

The mechanism of neutralization by anti-Am105 monoclonal antibody is currently unknown. do not have. Indeed, it is directed against early body determinants required for red blood cell receptor binding. Monoclonal antibodies neutralize infectivity. ANA 15D2 and ANA 22Bl showed that in competitive radioimmunoassays they were may recognize the same or overlapping determinants that mutually inhibit binding to. neutralization Other ways of Includes interaction with effector cells and complement fixation with early body lysates.

Effective use of Am105 as a protective immunogen to prevent anaplasmosis in cattle The determinants recognized by the neutralizing monoclonal antibody are A requirement that is common to all isolates. Anaplasma Marginale Similarities and differences in protein and antigenic composition between various isolates of , both are shown. A geographically remote region of the United States (Florida: Oka) Bustard, Washington; South Idaho; North Texas; Clarkston; Six isolates from Washington; Virginia) were found on acetone-fixed blood smears. Using on-board indirect immunofluorescence, ANA, 15D2 and ANA 22B1 The presence of determinants recognized by The determinants are all six 100% of the initial bodies in the isolate (Wright in adjacent parts of the smear) ) was determined by comparison with the chromosomal initial bodies of ). Furthermore, the determinant from 1% parasitemia to a peak of parasitemia with acute hemolytic symptoms. and were present at all stages of early acute infection. The existence of determinants is currently has been identified as a protective antigen for numerous isolates and is present at all stages of infection. Am105 or its fragments currently meet important criteria for use as a vaccine. do. Am, 105 or and Am105 polypeptides are effective immunoprophylactic agents against anaslama disease in cattle. It has been tested as.

%/Krona A[:Body (ANA l 5D2/22B1)t,:Dol l　Ol.

Partial neutralization of infectivity of Anaplasma marginale early bodies - BALB/e X　BIOA (3r) mouse, 1.0ml of Pristan 7 (Pristan e) was injected intracavitally (i, p), and one week later, anti-Am105 antibodies were produced. Approximately 2-3X10 doubly cloned hybridoma cells were injected into the peritoneal cavity. I made a target injection. 10 days after injection of /% ibridoma cells, ascites was removed from mice. is taken out, centrifuged to make a pellet-like fragment, and passed through a glass cool column. did. Determine the instantaneous fluorescent antibody (IFA) titer. Strong titer is 1:16.00 It seemed like 0. 1o10 initial bodies were converted to Balmer C.H. (Palm er, G, H, et al., Journal of Immunology (J, Immunolo gy), 133:1010 (1984). Purified from Suma marginale (Florida isolate) infected red blood cells, 2.5 ml of R Resuspend in PMI 1640 (2mM L-glutamine, 25mM HEPES). Ru. Add the progenitor to 2.5 ml of ascites fluid.

The initial body-ascites suspension was briefly placed in a portex mixer and incubated for 45 minutes at room temperature. The cells were incubated and injected into the left semimatumoid muscle of each cow. Post-inoculation (DPI) 75 Blood samples were taken every day for a day, and backed cell performance (pcv) and contributions were collected. Determine the biothermia rate (count 1,000 red blood cells). The probability value (p) is Calculated using a pooled t-test. p-value less than 0.05 is significant It is considered that

NS is not significant. ND is monochrome for Am105, significance cannot be determined. Two antibodies called ANA 15D2 and ANA22B1 that produce natural antibodies Cell lines and Trypanosoma blueeei ) using a monoclonal antibody against the one-time cell line TRYP IEI. The results of the test are reported in Table 2.

Number of infections/number of attacks 6/6 4/4 1% parasitemia 31 (29-37) 24 (23-25) p≦0.01 average to DPI* (range) Mean parasitemia 51 (30-58) 71 (69-71) p≦0. Ol Peak rate (%) (range) Average low PCV* (range) 15 (13-17) 11 (10-11) NS death Number/Number of attacks 0/6 3/4 ND*DP I means the number of days after vaccination.

*pcv means packed cell volume.

These results demonstrate that the 10” This shows that partial neutralization was observed by using the initial form of .

Initial impression of grade number using monoclonal antibody (ANA 15D2/22B1) Neutralization of dyeing property - The experiment was partially due to the extended detection latency observed using the 1016 side initial body. complete neutralization probably occurs at lower infectious doses. It was repeated with the assumption that it was true.

The protocol used was similar to that described in Bart a. a certain amount Ascites should be purified with 1010.10”, 1Ol, or 107% Anabrasma mal. Guinare (Florida) isolate was inoculated with the initial body and injected into cattle, and infection was previously confirmed. Monitored as described.

The results are reported in Table 3.

Parameter Number of initial fields ANA 150222BI TRYP IEI Significant sex Number of infections/number of attacks 1011'3/4 3/3 ND10' 4/4 3/3 N D 10' 4/4 3/3 ND 10' 0/4 313 ND 1% parasitemia 101633 (32-33) 26 (25-28) p≦0 ．． Average for 01 10@35 (33-39) 2B (27=29) p≦0 ．． 01DPI* (range) to' 37 (36-38) 30 (26-32)p ≦0.0110' neg・75 34 (34-36) ND These results are Complete neutralization of infectivity occurred with the initial form of 10' bonds, partial neutralization occurred with prolonged to', 10' and 101a, as determined by the extended detection latency period. This shows that it was generated using initial fields of

Purification of monoclonal antibodies - The steps to purify monoclonal antibodies for binding to immunosorbent rams are: 1. BA, LB/c X B I OA (1.0 ml blister for 3r mouse) [Aldrich Chemical-Cumber =, -(Aldrich Chemi cal　Co.

), Milk Oakey, WI] was injected intraperitoneally, and one week later, 2 to 3 x 106 The doubly cloned hybridoma cells were injected intraperitoneally. After 10 days, Ascites was removed from the mouse and centrifuged at 1.675XG to remove pellet-shaped insoluble debris. It was crushed and passed through a glass cool column.

2.50% (v/v) N H4S O4 precipitate into the wool column effluent. and resuspended in 0.032M Tris. Dialyze for 24 hours. The dialyzed sample was chromatographed on a DE-52 cellulose column. A continuous gradient of O to 0.2M NaCI in 0-032M1-lith was applied to Elute using a solution. Acetone fixation of Anaplasma marginale infected blood Titrate the eluted fraction using indirect immunofluorescence on the smear. The purity of the antibody , protein was determined using Coomassie Blue staining method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis method ( Measured using SDS-PAGE).

Preparation of monoclonal immunoaffinity chromatography column − Purified monoclonal antibody, ANA 1 5D2 (on SDS-PAGE) pure by Coomassie blue staining) was added to 0.1M NαHCOs , 0.5M NaCl pH 8.3.

The dialyzed protein was transferred to CNBr-activated Sepharose 4B [7 Armacia Pharmacia Fine Chemicals, Scarlataway, N, J,] by stirring at 4°C overnight. Coupling is carried out at a ratio of 100 mg/ml of precipitated Sepharose 4B.

The bound immunoaffinity matrix (ANA 15D2-Sepha 4B) Engineering: Econo column [11.5ml column, Piorad Labs (Bio-Rad Lab, Richmond, CA)] Store at 4°C until use.

Origin of Anaplasma marginale early form - Anaplasma marginale is Long-term in vitro culture never resulted in successful growth. Therefore The origin of Anaplasma marginale is the blood of infected cows whose pancreas has been dissected. It is. Cattle were inoculated intramuscularly with tolO progenitors and then smeared with Wright's stained blood. Check daily for evidence of parasitemia using smear specimens. of infected red blood cells When the percentage reaches 40-95%, add 4-7 liters of blood to 4 units/ml sulfuric acid. Collect during parin. Red blood cells were washed three times with phosphate buffered saline, pH 7-2, and then Resuspend 1:1 in 31.2% dimethyl sulfoxide in phosphate buffered saline. Ru. This suspension was frozen in liquid nitrogen and constitute the source.

Crushing and processing of early bodies of Anabrasma margina - Anabrasma margina Red blood cells (1012 cells give approximately 1°Qmg of pure Am105) ) was thawed from liquid nitrogen at 37°C and using centrifugation at 27,000xc. Wash 5 times in phosphate buffered saline. The sediment was placed in 35ml of phosphate buffered saline. Resuspend and sonicate for 2 min at 50 watts [127 x 4 mm titanium probe, Braun-sonic l 510, Braun In Braun Instruments, San Francisco, C Disintegrate with Al and then wash twice for 15 minutes at 1.650XG.

The purified progenitor was dissolved in 5 ml of 50 m, M Tris-HCI (pH 8,0,1,0 % Nonidet P40, 0.1% sodium dodecyl sulfate, 5mM EDTA, 5 mM iodoacetamide, lmM7znealmethylsulfonylfluoro ANA15 D Sepharose 4B monoclonal immunoaffinity column.

Column chromatography ANA 15D2-Sepharose 4B monoclonal immunoaffinity lamb , 100 column volumes of TEN buffer (20mM Tris-HCl, 5mM ED TA, OlLM NaCl, 25mM NaN, pH 7,6), 1% NP-40 , washed with 0.1% SDS, then approximately 101! 25m1 of crushed initial bodies The column was applied at a rate of /hr. Contains 1.0% NP-40 and 0.1% SDS Wash out unbound proteins using 100 times the volume of TEN, then remove the Remaining unbound tags are removed by 100 column volumes of TEH without activator. remove protein and NP-40,5DS (non-dialyzable surfactant). 0. 50mM Tris-HCl with 5% deoxycholate and 2MKSCN % pH 8.0 is used to elute the specifically bound AM105.

Recovery of purified surface antigen - The eluted protein (Am105) was dialysed against phosphate buffered saline and purified with KSCN and removes deoxycholate. Am105 is a modified Lowry Quantitate using a protein analysis method and freeze at -70°C until use.

Preparation of vaccine - Am105 was thawed and suspended in 70 India incomplete adjuvant. such purified antigen in an amount of 10.25 or 100 micrograms per milliliter. Manufacture existing vaccines.

Immunization research A group of five Holstein cows weighing 100 kg was Ovalbumin emulsified in complete adjuvant (group l), 70 int incomplete Am105 emulsified in total adjuvant (group 2), 70 int incomplete adjuvant Immunized four times with 100 g of Am36 (group 3) emulsified in Juvant. Ta. Immunizations were performed on days 1, 17, 35 and 59. 41[ Group 4, consisting of 300 cows, was not immunized. For four groups of Am105 Antibody responses were determined using a radioimmunoassay based on l-Am105. Ta.

On day 83, 10' purified 70 Lida isolates of A. margi were administered to the cows. Trying out the initial version of Nare. Daily clinical tests, hematocrit determination, and parasitism Infect cattle by testing Wright's stained blood smears for the presence of carcasses. I monitored it. The results are listed in Table 4.

Am105L: Against t 6 titer 10" 10' 10" 0 Number of infections/Number of attacks 51 5 315 315 4/4 Days until infection 5 33 39 (p\<0.01) 138 291 Significance = p-value was calculated by pooled t-test method.

Probability values smaller than O,OS were considered significant.

ゝPCv-backed cell volume.

Significantly longer detection incubation period (number of mouths until infection is detected), presence of parasitemia 5 for significant decreases, significant differences in PCV, and bovine immunized ovalbumin Complete protection in two of the Am105 vaccinators was due to Am105 Having the ability to induce significant protection against attack by Lasma Marginale It shows.

General Discuss Seal - The major Anaplasma marginale early body surface proteins identified to date. and protein complexes (Am105 complex, Am105U, Am105L, Am8 6, Am61, Am36, Am31-Am15) are each exposed on the surface in the initial body. Has epidogue. Submembrane-permeable radiolabeling technology (lactoperoxidase) ( Palmer, GH, McGuire TC (McGuireTC): Journal of Immunology (J, I+amun ol) 133: 1010-1015.1984) on an intact initial body using Am105, Am86, Am61. A'm 36. Evidence regarding the surface properties of Am31 and Am15 proteins was obtained.

By using ultrasonic disruption and differential centrifugation, parasitized red blood cells can be removed. The initial form of Naplasma marginale was purified. The initial body was measured using an electron microscope. When tested, it is intact, not agglutinated by anti-bovine red blood cell serum, and is infectious. La The initial protein surface treated with radioactive iodine using cutoperoxidase was m220, Am 105 complex, Am105U, Am105L% Am86, Am 61, Am56, Am42, Am36, Am31 and Am25.

Among these, Am105 and Am105U.

Aml05L, Am86, Am6L Am36 and Am31 are neutralizing antibodies is precipitated by The latter group of proteins are the major surface proteins; One or more of these proteins may be present in the vaccine, either in the nest or in combination. can be incorporated in clinical trials or diagnostic tests. In fact, the data shown in Table 4 Either Am105 or Am36 produced toxic anaplasmosis in cattle. It has been shown that it will induce protective immunity against Marginale challenge. child The fact that a purified protein of consisting of six or more amino acids that mimic the antigenic structure of a sexual epitope. Synthetic peptides encode biologically active epitopes on surface proteins. or by an antigen expressed in a heterologous bacterium containing a gene that A cow containing a gene encoding a biologically active epitope of a surface protein. by one or more antigens expressed in the viral vector. It shows that you can get results.

Major Anaplasma marginale early body surface proteins (AmlO5, Am105 U, Am105L, Am86, Am61. Am36 and Am31) are neutralizing antibodies It has an epitope recognized by Early purified Anaplasma marginale Antiserum prepared by immunization of rabbits with a pancreas The infectivity of 101 purified initial organisms was completely neutralized. Using immunoprecipitation reaction technology These proteins can then be recognized by neutralizing antibodies, either side by side or in combination. Its potential role in inducing neutralizing antibodies in either Their use as good vaccines is demonstrated.

Recognition of these surface proteins can be used to immunize rabbits and prepare antiserum. Agreement was obtained regardless of the isolate used (Florida, Washington-01 Virginia).

Am105, Am105U, Am105L and Am36 were each tested Anaplasma marginale detachment (Florida, Washington-〇, Washington- C1 Virginia, North Texas, South, Idaho, Kansas, Oklahoma , Kavichi (Kenya), O and Israel-Round and Israel-Tales ) and are capable of inducing neutralizing antibodies. against Ana-plasma centrale (Ana-plasma centraleX, a less toxic species) It was found that it has an epitope that Monoclonal antibody immunoaffinity Purification of Am105 or Am36 by chromatography and protein The demonstration of its ability to induce protection in cattle immunized with alone or in combination with other surface proteins as an improved vaccine against naplasmosis. Together they show their importance.

Am105, Am105U, Am105L or Am36x pitopes are completely It is short (at least 6 amino acids) by a synthetic peptide or by a gene encoding an epitope by a polypeptide expressed in a foreign bacterium or virus containing can be easily imitated. The availability of monoclonal antibodies allows for synthetic peptides and and gene cloning methods, while others are described more fully below. A vaccine will be developed.

Omoteran protein Aml identified to date 05. Aml 05U.

Am105LSAm86, Am61, Am3.6, Am31, Am13 are infected After that, it was specifically recognized by serum taken from cows for 30 to 255 days. be recognized. This recognition is based on the isolate used to infect cattle (Florida, Virgin Islands). (near, north texas). This specific recognition Individual as an antigen in an improved serological assay for diagnosing rasmanosis Regarding the selection of Anaplasma marginale proteins used in or in conjunction with is required. These supporting data support this for diagnosing anaplasmosis. ” points out the use of these proteins for isolation and diagnostic purposes. Incorporation into serological assays is a simple technical method. The knowledge to date is that 6 or more amino acids as immunologically equivalent drugs for the purpose of a synthetic peptide consisting of a polypeptide or a polypeptide expressed in a vector microorganism. Showing possible uses.

isolated by monoclonal immunoaffinity chromatography, and I　Am1 coated in the wells of the microtiter plate with OOn9/well 05 and Am3.6 as solid-phase enzymes for serological diagnosis of anaplasmosis. The test was based on an immunoassay (ELISA). By each assay, after infection, 30 Anaplasma marginale or Anaplasma st. It was found that it is possible to distinguish between Rare infected cattle and non-infected cattle, and this test isolates used to infect cattle (Florida, North Texas, Virgina) A, Washington-01 Washington-01 Idaho, Kenya, Israel-Roun (Israel-Tilt). This serological assay is a simple Based on all Am105 or Am36 separated. However, these The findings suggest that immunologically similar synthetic peptides of 611 or more amino acids suggest possible uses of polypeptides expressed in host or vector microorganisms. Ru.

105.000 daltons (AmlO5), all identified as surface proteins, 86. 000 Daltons (Am86), 61.000 Daltons (Am61), 31. ．． 0 00 daltons (Am31) and 15,000 daltons (Am15) proteins as evidenced by antibodies in Anaplasma 1 luginale infected cattle. It is highly antigenic. In addition, diluted solutions of post-infectious serum can be used during infection. 86 and Am15, indicating a favorable response. diagnosis Am86 alone or Am86 and Am105, Am61 as antigens in the test ．． These findings suggest use in combination with Am31 or Am45. be done. This phase of the study will focus on the use of antigens in diagnostic tests for anaplasmosis. Synthetic peptides of six or more immunologically similar amino acids as or in vector microorganisms with epitopes recognized by post-infectious serum. The authors also point out potential uses for polypeptides expressed in

Bart n - Size in different geographical isolates of Anaplasma marginale Further research on size polymorphisms ・The formation of MSP-1 protein complexes among different geographical isolates of M. marginale showed that there was significant size polymorphism in the two proteins. Table 5 below is Florida (F), Southern Idaho (■), Northern Texas (T), Barge Anaplasma Ma on Near (v) and Clarkston, Washington (W) Corresponding component proteins Am105U and A for geographical isolates of L. luginale The size range of m105t is shown. These molecular weight evaluations are included in this specification. Quoted by Oberle, Infection and Im Infection and Immunity, Volume 56, No. 6 ( (1988) using polyacrylamide electrophoresis as described by This was done by electrophoretic mobility analysis.

The apparent molecular weight (kDa) of the main complex component “isolate” is ITVW Am105L 105 98 97 100 100Am100A 100 9 5 8'9 70 86 Recognized 100 98 97 100 100 ・Isolates: F Florida, I Soza?・Idaho, “T Northern Texas, ■ Virginia, W Clarkston, Washington Bart ■ - Recombinant Am105L In this case, Am105 is linked to two non-covalently bonded polyamides with the same molecular weight. Indicates that it consists of a peptide complex. Referred to as Am105U and Am105L to determine whether these two polypeptides are the products of separate genes. and to test the structural and antigenic relationships between the polypeptides. Am105L in Escherichia coli! The gene encoding bitope was cloned and expressed. In this report, A. m105U and Am105L, each a separate gene with a surface-exposed epitope. Identify the gene product. By cloning and expressing Am105L, foci Its efficacy as a non-complex immunogen will be determined.

Preparation of antiserum - Mouse monoclonal antibodies were prepared as described above (14, 17,) and as follows. Named: Trypanosoma brucei (Trypanosoma brucei) Control antibodies IEt and 24AI against the surface glycoproteins of i); Antibody that immunoprecipitates F19Ei (19); immunoprecipitates Am105 and in vitro Antibodies 15D and 22 that neutralize the infectivity of Anaplasma marginale in ro B+; as well as antibody F34G, which immunoprecipitates Am105.

In rabbits, antiserum against Am, 105 (17), isolated Anaplasma ・Antiserum against the early form of Marginale (19) and pBR322”*f- are p Against E. coli containing AM25 zurasmid DNA An antiserum was prepared. Rabbits were immunized four times with lysed bacteria (for the first immunization microbial 2XlO' in a complete 70-indoor adjuvant, and the other three times. is 10 microorganisms in incomplete adjuvant. Enzyme-linked immunosorbent assay (ELIS) A), radioimmunoassay (3), or Anaplasma marginale [3 1S] The titer was evaluated by immunoprecipitation reaction (2) of the methionine-labeled extract. child These rabbit antisera are named as follows: surface sugar protein of T. brucei. Control antibody R612 prepared against Park; isolation of Anaplasma marginale Antibody R781 prepared against the initial form of immunoaffinity chromatography on Antibodies R873 and R874 prepared against Am105 thus isolated ( 17) (purified Am105 consists of Am105U and Am105L); Antibody R907 prepared against E. furi (pBR322); Antibody R911゜nitrocellulose film prepared against E. coli (pAM25) Anti-I detection by Anaplasma marginale or recombinant E. coli protein was bound to a nitrocellulose filter, and Young and Two modifications: (i ) After dissolution in chloroform, filter in 10% acetic acid-25% inpropertool. and (Li) 1% hemoglobin in buffer instead of bovine serum albumin. Add bottle l! to the filter! Blocks non-specific binding of Jll recognition protein A and detected by reaction with specific antiserum and l■■ labeled protein A.

ELISA - The ELISA uses Am105 bound to the plate at 50n9 per well. , Ellens and Gielkens 6) according to the disclosure. Enzyme label is horseradish peroxidase protein A, and the substrate was recrystallized 5-aminosalicylic acid. monoclonal anti 15Dz-Sepharose 4B by immunoaffinity chromatography. Am105 was isolated from Anaplasma marginale (17). The Am1 05 consisted of Am105U and Am105L. In rabbits, Am 105 and containing pBR322 or pAM25. Serum against E. coli was prepared.

Immunoprecipitation reaction - Due to the short-term metabolic uptake of [3$3]medionine in in vitro culture, (2) or by Table I radioiodine treatment using lactoperoxidase. (19) to radiolabel Anaplasma 75 luginale microorganisms. 250C Medium containing [”S]methionine and ampicillin 359/+112 of E. coli microorganisms were also labeled with 315 during logarithmic growth in nutrient solution ld. Unincorporated release After removing the radiation label, 50mM Tris hydrochloride (pH 8,0), 5mM EDTA, 5mM iodoacetamide, 1mM phenylmethylsulfonyl fluoride, O, 1mM N-tosyl-lysine chloromethyl ketone, 0°1% (vt/vol) dodecyl Sodium sulfate (SDS) and 1% (vol/vol) Nonidet (Nonidet) Microorganisms were lysed by sonication in a lysis buffer consisting of P-40 at 4°C. I understand. The lysed extract was centrifuged at 130,00089 for 1 hour at 4°C and Heron or mouse antibody and protein A producing Staphylococcus aureus ( S taphylococcus aureus (Calbichem) 0.2 millipore size filtrate before use in immunoprecipitation reaction with oche + s)). Luther (Centrex): Schleicher &amp; Schleicher & 5chuell.

Inc,) (9.17.23). The precipitated radiolabel was eluted at 7°5 ~17.5% polyacrylamide-5DS gel, containing 4MJR element 7, 5% polyacrylamide gel or 5% polyacrylamide containing 4M Analyzed on Dogel. 14 (The labeled standard proteins were as follows (molecular weight ): myosin (200,000), phosphorylase b (92,500>, bovine blood clear albumin (59,000), ovalbumin (46,000), carbonyl quanhydrase (30,000), and lysozyme (14,300).

For the following experiments (see Figure 5), immunoprecipitated recombinant Am105, Am1 Dry the 05U and Am105L protein bands with f-7, 5% polyacrylate. Mido-4M urea gel was excised and then rehydrated separately and treated with 5QmM Tris. ・Hydrochloride (pH 8,0), 0.1% (it/vol) S D S and 1% Electroelution (vol/vol) into a mixture of Nonidet P-40 elute) L/ta. Polyacrylamide was removed by centrifugation and 31S Labeled proteins were again immunoprecipitated from electroelution buffer.

Peptide mapping − Immunoprecipitated 31S-labeled proteins were excised from dried polyacrylamide gels. and compared for sequence homology by peptide mapping according to (5) above. Ta. Restriction by S. aureus V8 protease Radiolabeled peptides produced by proteolysis were dissolved in 15% polyacrylamide. -Separated on a 3DS gel and detected by fluorography (4).

Isolation of Anaplasma marginale DNA - less than 50% erythroparasitemia Blood from cows infected with Anaplasma marginale was treated with phosphate-buffered saline. Washed 4 times. After each wash, the upper layer containing white blood cells and red blood cells was removed. . Next, the remaining red blood cells were added to 50% packed red blood cell volume in phosphate-buffered saline. It froze inside. Thaw 100 m of red blood cell suspension (2) and incubate at 4°C for 20 min. Centrifuge at 0x9 to pellet Anaplasma marginale early bodies and red blood cell membranes. The 6-celled pellets were further soaked in phosphate buffered saline at 30.00" OX9. Hemoglobin was removed from the lysed red blood cells by washing three times. Then, the initial To extract DNA from the body 11), use phenol-chloroform to extract proteins. , digestion with RNasa A and protease K and precipitation with ethanol. It was further purified by

Preparation of recombinant RNA library - Anablasma mal using 5au3A Partially digested Ginare DNA to an average size of 5 kilobases (kb) -:, T4 Ban+Hi cleavage and dephosphorylation of the digested DNA using DNA ligase ligated with pBR322 (25). Highly efficient transformation glotocol and 7-su/ Hanahan made E. coli HB 101 cells resistant to ampicillin. sexually transformed (8). R873 serum (rabbit anti-[Am105U+Am10 5L] complex) for a library containing 8,000 recombinants. Plasmids pAM22 and pAM25 were identified by sequencing. This la Other colonies in the library, such as those containing pAM14, are also R8 73, and in addition to the pAM22 sequence, the 13g111 site is extended along side λ. Contained additional DNA of various lengths.

Anaplasma marginale DNA was completely digested with BgllL and pBR322 A second library containing recombinant 3°000 is created by ligating into the BamHI site of A library was prepared. By expression screening with R873, this live Clones containing pAM97 and pAM113 were identified in the library.

Southern plotting method - The protocol used was that disclosed by Wahl et al. (24) It was a modification of Part of Anaplasma marginale genomic DNA (0.5g ) or the plasmid DNA portion (0,369) is digested with an appropriate restriction enzyme; . To compare plasmid and genomic sequences on the same gel, digest the digested genome. Transfer 0.5g of plasmid DNA or 1.8n9 of plasmid DNA to an agarose gel. Run and plot on a nitrocellulose filter. No Iburi Dye The zation is 5 × S S P E (0-18M NaCff, O-01M NaH=P 0-10.001M EDTA [pH 7,4])-10% sulfuric acid Quitran containing 0.25% sarkosyl (SarkosylX sigma) ma)), denatured bovine thymus DNA 1. OO9/aQ and 3! pH [sensitivity] Translage! The test was carried out at 65°C in a probe. Finally, O,1xSSPE− Filters were washed a total of 5 times in 0,0033% Sarkosyl at 65°C. a The probe isolated from the galose gel was the 1.4kbsstI flag of pAM97. It was ment.

Genomic library and AM105 expression by E. coli - Initial experiments Anablasma marginale proteins immobilized on nitrocellulose filters investigated the specificity and sensitivity of the immunoprot assay in detecting (26 ). Previous studies have shown that anaplas possesses specificity for various surface proteins. Monoclonal and polyvalent antisera against Suma marginale were prepared III ( 17-19). Reactions of these antisera with positive and turtle control antigens are shown in Figure IA. . All antibodies detected Anablasma marginale erythrocytes and non-infected erythrocytes. Didn't react. Sensitivity of detection for immunoaffinity isolation A, m105 was highest for rabbit antiserum R873. R873 applies to filters However, a small amount of parasitized red blood, such as 1200 in 1 microliter spot. A ball was also detected. The specificity of each antibody in the immunoplot is determined by the immunoprecipitation reaction experiment. It was the same as that previously observed in . Am105 or surface protein A Multivalent or monochrome antibodies against m36 reacted with appropriate na proteins: There was no cross-reactivity or reaction with the negative control ovalbumin. R873 is the minimum Quantity purification, lng of AM105 was detected. R781 is an isolated Anaplasma ・It was an antiserum prepared against the early form of A. marginer; AmLO5 (A m105tJ and Am105L) and Am36 were immunoprecipitated together. (data not shown), Am1058 and Am36 were recognized in the immunoplot. (Figure IA). This test was performed in recombinant E. coli for Anaplasma malus. be sufficiently sensitive and specific to detect the expression of Ginare protein. it was thought.

The above data demonstrate that Rickettsiae gene regulatory sequences function in E. coli. (10, 13.25). Therefore, Anaplasma margi Parasitic DNA was extracted from bovine red blood cells containing the Florida isolate of Nale7. Ta. The DNA was partially digested with 5au3A and phosphatase-treated pBR322 into the BamHI site of the E. coli HBOI and use it to ampicify the E. coli HBOI. Transformed into phosphorus tolerant. Immunoplot for expression of Am105 antigenic determinant This genomic library was screened with R873 in the assay.

E. coli colonies containing recombinant plasmids of various sizes are It reacted stably with the supernatant (Figure IB). Minimal grasmid pAM22 and expression colonies and restriction of insert DNA from pAM25 (3.75# and 4°15 kb, respectively). The enzyme map is shown in Figure 2. All plasmids from the expressing bacteria are present in pAM22 It contained an inserted sequence. Bgl consumed more than 11 parts There were other insert DNAs of various lengths in the plasmids. Restriction enzyme mapping and The satin blotting method uses a 240 base pair sequence shaded in pAM25. is close to the remaining part of pAM25 DNA in the Anaplasma marginale genome. and that during cloning two 5au3A7 fragments were added to this This suggests that they were linked in glass and mid. Plas from expression colonies Both possible insertion orientations for pBR322 DNA were seen in (Figure 2).

Analysis of each expression plasmid DNA and genomic DNA by satin blotting method Analysis revealed that the inserted sequence in pAM22 was derived from Anaplasma marginale gene. suggesting that it should be contained within a single-BgllI fragment of the mu DNA. Ta. A second library was prepared to confirm this. Anag in Bgllll The Lasma marginale DNA was completely digested and inserted into the BamHI site of pBR322. inserted into.

In this library, expression screening with R873 revealed that the identified pAM97 and pAM113; they are bitropically It contained the expected Bgll+7 fragment (Figure 2).

Protein expressed by recombinant E. coli pAM25 or pBR322 was used to characterize the new protein synthesized. Bacteria containing either [315] methionine are released by metabolic uptake. Labeled. Radiolabeled proteins were determined by immunoprecipitation and SDS gel electrophoresis. analyzed. The protein profile of recombinant E. coli is shown in '@3, lane 7. . This is similar to the similar profile of control bacteria containing only pBR322 (lane 8). Main radiolabel with molecular weight 105,000 in recombinant bacteria All protein bands were present in both lanes except for the identification polypeptide. mark Immunoprecipitation of the recognition protein with R873 revealed that one normal E. coli protein Park was recognized. However, in recombinant bacteria, molecular weight 1 as , ooo proteins were also precipitated (lanes 58 and 10, ratio 112).

Similar results were obtained using a different antiserum R874 against Am, 105 (Ref. 38 and 12). These results were hooded with pAM25 DNA. It was shown that the novel protein was expressed as the main component of the recombinant bacteria. This tongue Pak has the same molecular weight and has the same molecular weight and It shared antigenic determinants with the affinity mutant Am105.

R873 and R874 are likely to be used undiluted in immunoprecipitation reactions. Before exposing the rabbit to the bacteria, ■ or two normal E. coli Reacted with protein. Similarly, antiserum against lysed non-recombinant E. coli Because Am105 was not recognized, the relationship between AM105 and E. coli protein The possibility of cross-reactivity is unlikely (Figures 5 and 6). of the antiserum used Dilution (124.ooo) maintains the activity against Am105 while maintaining anti-E. coli activity. effectively removes the ・No reaction with stiffness was observed.

The molecular weight of the recombinant protein is pAM25, pAM22, pAM97 or pA Identical in bacteria containing the M113 plasmid. These recombinants The level of expression in each was determined by the relative band intensity on the SDS gel. comparison is possible if

The orientation of the insert DNA with respect to pBR322 has a clear effect on expression. (Bitropism was equally represented in the four plasmids). These de The data suggest that: (i) Anablasma marginale genes; functions in E. coli; (ii) Bgl in which the gene has been cloned; [contained within the I7 fragment: and (iii) the expressed molecule is pBR322 encoded amino acids and Anablasma marginae encoded amino acids It must not be a fusion protein consisting of both amino acids.

Recombinant Am105 is structurally homologous to non-recombinant WAm105L. set The variant Am105 is recognized by R873 and therefore Am105Uj5 and/or or was antigenically homologous to the Am105L polypeptide. However, as Recombinant Am105 expressed by any recombinant can be expressed by immunoprecipitation or is monoclonal antibody 22B or 15D in immunoplot assay. 2 (data not shown) or by R781. (Figure 3, lanes 2 and 13). Therefore, recombinant Am105 and There were important antigenic differences between Am105 and Am105.

Each component of the Am105 pair, Am105L and Am105U, in terms of structural homology and Am105. Anazurasma marginale [”with Sl methionine] Radiolabeled, lysed, and neutralizing monoclonal antibody 22B! immunoprecipitate with The proteins were transferred to a 7.5% polyacrylamide-5DS gel containing 4M urea. (Fig. 4A, lane 3). Am105 pair is clear It was broken down into. Control lane (Anaplasma marginale + 24A.

No band was observed with the monoclonal antibody (lane 4). R873 The immunoprecipitated recombinant fiAm105 was analyzed on the same gel. recombinant Am105 migrated as a single band at the same position as Am105L (Fig. 4A, lane l).

The Am105 pair in this gel system is the immunoprecipitated Am105 from the dried gel. It was sufficiently degraded to excise the L and Am105U components. Then each polype Gel 7 fragments containing peptides were rehydrated and analyzed by peptide mapping. (5). Recombinant Am105 immunoprecipitated by R873 was also excised. ,analyzed.

Figure 4B shows partial quenching of eluted polypeptide by N. aureus v8 protease. The peptide map obtained by the analysis is shown. The decomposition peptide of recombinant base Am105 is , very similar to that of Am105L. Recombinant Am 105 and Am 10 Both initial proteolysis products of 5L have molecular weights of 75,000.59,000 and and 51.500 polypeptides. The same low molecular weight component (34,300, 18°600 and 13.000-16.000) also occur. Therefore, the group The recombinant Am105 and Am105L molecules were homologous or identical.

In contrast, the degraded peptides produced from Am105U It is very dissimilar to both mutant Am105. 22,000-27,000 The main digestion products of Am105U in the molecular weight range are Am105L or recombinant It had no relative portion in type Am105. Another molecular weight 16.0 00 peptide is not present in Am105L and recombinant fiAm105. won. Various peptides are converted to Am105L and Am105 by proteolysis. However, the sensitivity of this method is generally between Am105L and Am105U. There was no determination of non-homology. For example, the degraded peptide of 29.500 is A was produced from both m105L and Am105U. These two low molecular weight Whether the putidos share homology will require further structural analysis .

Antibiotics between recombinant Am105, Am105L and Am105U polypeptides primordial relationship. Antigen between Am105L, Am105U and recombinant Am105 The relationship between anti-bacteria expressing recombinant Am105 in four rabbits and were studied by preparing serum; as a control, containing pBR322. Four other rabbits were immunized with E. coli. Non-paper conversion by ELISA Serum was tested for recognition of efiAm105. All rabbits, l: l 00-1: Proliferate antibodies against Am105 within a titer range of 1.000. immunized with recombinant bacteria. Control E. coli grown with antibodies against Am105 None of the rabbits were immunized with li. Anti-recombinant Am105 serum was [3SS] Am105L and Am105 from thionine-labeled Anaplasma marginale (data not shown), therefore 1. R873 and 22B1 antibody.

Two explanations are possible for these results. First, Am105L and A m105U share antigenic determinants and therefore can be immunoprecipitated together. secondly , Am105L and Am105U are antigenically unrelated but can be complexed.

To distinguish between these possibilities, we refine Am105L and Am105U separately. and immunoprecipitated. [3%5] Methionine sm Anagrasma marguicile The washed extract was first subjected to immunoprecipitation reaction with monoclonal antibody 22B1, and the precipitate Am105L and Am105U components were separated by SDS gel electrophoresis. . Am105L and Am105U bands were excised, electroeluted, and then monomerized. Clonal antibody 22B or rabbit anti-Mi recombinant Am105 serum and dog IJ Subjected again to immunoprecipitation reaction (Fig. 5), only Am105U was immunoprecipitated by 22BI. Aml05L was not recognized (lanes 4 and 5). contrast Generally, when the two components are separated before immunoprecipitation, the anti-recombinant Am105 serum is A, immunoprecipitation of Am105L but not m105U (lanes 8 and 9). Therefore, recombinant Am105 is antigenic only to Am105L. They were homologous.

Therefore, Arn105 has two polypeptides, Am105L and Am1 It exists as a complex of 05U. Monoclonal antibody 22B, Am105U binding to this epitope leads to the precipitation of both components of the complex. occurs. The complex present during the immunoprecipitation reaction was precipitated with 1% Nonidet P-40 and Stable in 0.1% SDS, but reduced by boiling in SDS gel sample buffer. It is dissociated. The molecular weight of reduced molecules changes when electrophoresis is performed under non-reducing conditions. Am105L and Arn105U are linked by disulfide bonds. It seems that it has not been done. Recombinant Am105 has structural and anti-inflammatory properties with Am105L. Primarily homologous. Between recombinant Am105 and Am105U polypeptides or evidence of structural or antigenic homology between Am105L and Am105U. No light was obtained. These data demonstrate that rabbit anti-Am105 serum and recombinant fi Explain the forward reaction with Am105 and the member reaction with monoclonal antibody 22B. There is.

The surface radiolabels of Anaplasma marginale early bodies are Am105L and Am1. Label both 05U. Using lactoperoxidase according to (19) above Then, the viable initial body is l! Radiolabeled with J. The labeled extract was then converted to R9 11 (anti-recombinant Am105), R873 (anti-Am 105), monoclonal Immunoprecipitation reactions were performed with antibody 22B1 or appropriate control antibodies. The sediment was collected in 4M urine. (Figure 6). From the results, Both Am105L and Am105U polypeptides contain radiolabels. , R911, R873 and 22B. 22B Increase in band intensity of Am105U when precipitated by 1 and R911 The increase in the band intensity of Am105L when precipitated is due to the increase in the band intensity during this immunoprecipitation reaction. Am105 suggests some dissociation of the Am105L-A complex.

Anaplasma marginale genome cloned L8gll17 fragment Contains numerous copies of . Anagrasma marginale genomic DNA is cut by restriction enzymes; the DNA 7 fragments were separated by gel electrophoresis. and plotted on nitrocellulose to identify bacterial sources expressing recombinant Am105. probed with conventional sipmil-labeled plasmid DNA. in a loop array Multiple hybridization bands were observed by using enzymes that were not cleaved by (Fig. 7A, lanes 7 and 8). These clone sequences or To find out whether the polymorphism represents a partially homologous copy of the polymorphism, Genomic DNA was digested with restriction enzymes that are thought to generate fragments. Hint clI + M1uI digestion to H1nel [-H1ndl H probe A hybridizing 2,8 kb fragment should be obtained. for comparison Plasmid DNA containing the entire 3.9 kb Bgllr fragment was Digested with ncll+MluI and analyzed in the adjacent gel line (Figure 7A, lanes 5 and 6). The expected 2.8kb7 fragment was present in both digests. However, hybridization of genomic DNA 1:t; 4.0 and 67.7 kb Bands were also observed. The 4.0 and 6,7 kb bands are Hinc[l or 3 and 9 kb clones 78g11 that do not have the Mfu1 site or both 17 fragments must represent partially homologous copies.

Hincll + Bgll or 5stI, Bgll or BgHI only A similar digestion with 2-41’i additional hybridization bands, but with Fig. 2 always produced the DNA7 fragment expected from the map in Figures 7A and 7A. B). Which of the Hinc [I - H1ndl II or 5stI gloves will be tested? Multiple hybridizing bands were detected (Figure 7B, 2 and 5). Cloned globe and cow clay 1! Hybrid between DNA5 There was no formation (Fig. 7B, lane 4), indicating the parasitic origin of the cloned sequence.

Therefore, the cloned DNA is based on the Anaplasma marginale genome sequence. accurately represent. However, there is a clone 3.9kBgll17 in the genome. Further partially homologous copies of the segment also exist.

The data presented are based on an anaplymph of approximately 105°000 molecular weight in recombinant E. coli. Expression of Lasma marginale protein is shown. immunoaffinity isolation Antiserum prepared in rabbits against non-recombinant Am105 was Recognizes Am105 and vice versa, which indicates that it shared Ebidog show. Antiserum against recombinant Am105 was used as an anagrath in immunofluorescence assay. reacts with M. marginale and aggregates the purified progenitors, which are assembled into the parasite itself. The presence of the modified WAm105 epitope is shown. Recombinant Am105 is , structurally and antigenically homologous to Am105L; homologous to Am105U No proof could be obtained regarding this.

Non-recycled Aml 05 containing both Am105L and Am105U provides protection to cattle against attack by Anaplasma marginale (17 ). Either Am105L or Am105U was isolated and used as an immunogen. It is not known whether this provides any protection. Both Am105L and Am105U Radiolabeling surfaces can be reached on viable primary bodies. This is immune protection This is one of the important criteria regarding protective proteins (1). Am105U uses this poly The peptide contains the epitope recognized by the neutralizing monoclonal antibody 22B8. It is thought that it induces more protection because of the presence of the protein (Fig. 5). However, among others Japanese-sensitive epitopes may also be present in Am105L. Am by antibody 22B The epitope recognized in 105U was found in eight geographically distinct isolates. (17), which is an important practical concern regarding possible immunization.

Rabbit anti-recombinant Am105 serum was tested in the immunofluorescence assay. isolates, but changes in surface-exposed epitopes may result in polyvalent sera such as It was not shown by. Anaplasma margi by Southern blotting Nale Genome's test is based on the presence of amyl 7 in the Am105L gene and antigenic variation. suggests the possibility of change.

A single Am105L gene was isolated in a recombinant library by expression screening. Child copy detected. Another copy of the gene is found in Neisseria gonorrhoeae (N Similar to the pilin gene of Eisseria gonorrhoeae), the complete is not functional (15,16,22), whereas other Am105L genes (i) a non-functional product in E. coli or Anaplasma marginale; or (ii) not detected in the expression assay. can encode antigenically different forms of the protein. Peptide map is Am10 Without excluding the possibility of limited homology between 5L and Am105U, Am The 105L-related gene could encode Am105U. however , later tests revealed that the proteins Arn105L and Am105U were further may be the product of the isolated Anablasma marginale gene, as detailed in and showed.

Ongoing trials show that recombinant Am105 produces protection in cattle against disease and the same components of the Am105 complex could be tested for protection. We test whether Am105U can be expressed in E. coli. Immunoplot test and those shown in Figure 5 are recognized by the neutralizing monoclonal antibody 22Bl. Ebidog in Am105U was mixed with 2% SDS and 2.5% mercaptoeta. Modified by solvents such as alcohol, 10% vinegar haze and 25% isopropanol. Indicates that the Therefore, this epitope can be used for example in Trypanosoma brucei. relative to the surface-exposed epitope of It is resistant (4a). Other data show that immune affinity separation of Am 105 is This suggests that the epitope recognized by antibody 22B1 is not codylated. The results show that the protein is sensitive to proteases (G.H. Pal v-( G, H, Paf+ar), Niss D Waghe-ra (S, D.

Waghela), Dublin II A. C. Davis (W.C. Davis), A. Z7 Barbet (A, F, Barbet) and T.C. McGuy Yar (T, C, McGuire), International Journal of Paracytology (Int, J+parasitol, ), in press).

Therefore, expression of the Am105U neutralization-sensitive epitope is important for e.g. Direct monoclonalization of fusion protein expression libraries in phage λgtll should be easily obtained by routine antibody screening (26). These la In the library, expression of the Am105U epitope was determined by E. coli. It will not depend on the recognition of Chicha regulatory DNA sequences (21).

The most effective vaccine against Anaplasma marginale is a It may be a recombinant. These are Am86. Am61, Am36j; and Am3 1 as well as Am105, Am105L or Am105U. In this specification The structural and anti-oxidant relationships between the cloned and native surface proteins have been Cloning and development of the Anaplasma marginale gene in primordial interrelationships The current status is listed. Cattle were infected with Am105 purified from infected red blood cells. Because immunization protects against Anaplasma marginale, these The results suggest that recombinant vaccines are suitable, and there is a consensus regarding their development. Give a rational principle.

Bart - cloning of the MSP-1 gene for various geographical isolates; Their DNA and amino acid sequences Characterizing MSP-1a associated with Am105U expression from widely different isolates For this purpose, the cloning and sequence analysis studies and methods described below are chosen. Such methods are similar to those cited or described herein. It should be interpreted in the light of the law. For analysis and sequencing, Anaplus The following geographical isolates of Ma. marginale were selected: Florida (FL) and Ba. The Virginia (VA) isolates represent the largest and smallest polypeptides available, respectively. and previous immunological data and previous immunological data regarding MSP-1. and molecular data were generally obtained by FL as described herein above. be. The Idaho (ID) isolate was found to be the most variable by restriction analysis. It was selected because it is Washington (WA) isolate is FL MSP-1 complex was used in successful cross-challenge experiments in animals immunized by Ru.

For convenience in identifying the specific geographic isolates cited, the antigens will be listed hereafter. rAmJ as used in this article, and with respect to Florida to indicate a geographical isolate. is indicated by an abbreviation such as "F". For example, as generally cited herein above, Am105 opened Am105L column analysis to show association with 70 Lida isolates. To begin, we first isolated the anaplus by partial digestion with the restriction enzyme 5au3A. A pseudo-random genomic library was obtained from DNA isolated from Ma marginale Florida. Ta. The resulting genomic DNA fragment was added with an additional C-tail ((-t ails). Obtained modified DNA 7 fragment was previously cleaved using the restriction enzyme Pstl to G-tailed ) was inserted into the plasmid pUC9. Then, the obtained recombinant grasmid It was used to transform E. coli (strain TB1). The obtained transformed cells Bacteria were tested for expression of the AmF105 epitope by I-enriched s1 protein A and mono Screening was performed using clonal antibody 22Bl. Cloned into plasmid pUCQ In the inserted 2.7 kilobase pair (kbp) insert, the AmF105U subunit We obtained a portion of the MSP-1a gene for the Florida isolate that encodes Knit. . When plasmid pAMT1 is inserted into E. coli, it has approximately 56,000 daltons. Synthesis of an antigen containing subunits of AmF105U with a molecular weight of Jiru. The part of the ArnFLO5U antigen expressed by this recombinant heather bacterial cell is , starting from ■ to about amino acids 220-230 in repeat 8. As shown in the amino acid sequence information given in FIG. 12 for the Da isolate.

MSP-11 Remains in the Chromosome of Florida Isolates of Anaphrasma Marginale To measure the number of gene copies, restriction endonuclease digestion of Anaplasma Southern blot analysis of D. marginale genomic DNA as a DNA hybridization probe. A 2°7 kbp insert of pAMTl was used as a sample. In most cases, Only one band was hybridized with the glove. This represents one gene copy. is suggesting. Therefore, each of the various Anaplasma marginale geographical isolates These isolates for the corresponding MSP-1 protein complexes produced by The size polymorphism that exists between rather than due to allelic variation at a certain location on the chromosome.

Using the same probe, Anaplasma spp. Three other isolates of Marginale were compared to FL. Limitations for all four isolates The map shows the variable length between the internal Kpnl and Hindlrl restriction enzyme cleavage sites. With the exception of the regions, they were substantially identical as shown in FIG. shows the gene Restriction to be more easily seen with respect to the 3' region relative to the hatched region. This similarity in enzymes can be mapped and each M of the same chromosomal location in each isolate Confirm the location of the SP-1a allele.

Plasmid pAMT1 insert region containing partial MSP-1a gene The region was analyzed to determine its location found in the "right" region of the KpnI site. (See Figure 8). [Left j] of the DNA insert of plasmid pAMTI using restriction enzymes. We created progressive deletions in the halves and investigated the effects of these deletions on in vitro synthesis. and monitoring the size of the 56 kDa product encoded by pAMTl. This analysis was conducted by.

To isolate the intact MSP-1a gene from FL isolates, the isolated DNA was Randomly sheared genomic lysing of Anaplasma marginale DNA using sonication Got braley. The sonicated DNA7 fragment was subjected to DNA polymerase I fragments. The Klenov 7 fragment was used to make blunt ends. After that, Nc An oI linker was added to the modified DNA fragment. Obtained DNA fragment The vector was ligated to the expression vector plasmid pKK233-2. The obtained recombinant archetype The lasmid was transplanted into E. coli and the resulting bacterial culture was infected with anti-*AmF105 or monoclonal antibodies with respect to the expression of antigens that produce immunologically similar epitopes Screening was performed by 22Bl. In this screening, plasmid p KAna420' kFI was determined, and the expression product was determined by electrophoresis and immunoprecipitation reaction. Further analysis of the product indicates that fully sized immune reaction products are expressed. showed that.

To perform DNA sequencing, plasmid pGEM4 was inserted into the Sma [pKA The Anaplasma marginale DNA insert contained in na420 was subcloned. plasmid pFLlo was obtained, and pFLlo was used to transform E. coli strain DH5a. was transformed. Then restriction enzyme Kpnl (for VAJIIL isolation) or by the restriction enzymes KpnI and Pstt (for WA8 and ID isolates) By ligation of the cut DNA 7 fragments, Virginia, Washington and Size-selected genome sequence from DNA of Idaho Anaplasma marginale isolates built a library. The DNA7 fragment is then treated with the enzyme Kpnl or K Cloned into plasmid pGEEM4 linearized using pnI and Pstl. and used to transform E. coli strain DH5a. Bacterial transformants are Grunstein and Academy of Sciences (Proceedings of National Academy of S ciencas) (U, S, A,) 72. ．． 3961 (1975) Screening was performed by colony hybridization method according to the method of ). Applicable Method 3: As a hybrid formation probe! Plasmid pAM radioactively recognized by P. A 1 Kbp DNA7 fragment of T1 was used. This is shown in Figure 8. KpnI site of plasmid pAMT1 (corresponding position on the Florida isolate restriction map) (see location) to its right end. The plasmid diagram shown in Figure 8 The boxed or thick lined region is the sequenced DNA of Anaplasma ma. (Fig. 10 for DNA sequences) reference). The abbreviations used in Figure 8 are as follows: Ac=Accl ; A2-Avail: BmBamHI; BclI-BclI (multiple Restriction sites are shown for this enzyme for WA and FL isolates); H=Hindlll; Hi=Hincrl; Hp-Hpal; K= KpnI; N-N5il; Nc=NcoI; PsmPstl; So+ -3maI; 5s=Sstl; and X-Xmall.

By Southern blot comparison of restriction fragments and genomic DNA (not shown) each transformation as shown by Western blot of (!I9) and (!I9). By expression of sufficiently sized immune reaction products by the body, four clonal flags Confirmed the compatibility of related isolates with Anaplasma marginale chromosomes in all Ta. The electrophoretic migration test illustrated by Figure 9 shows that 50 pg/mff ampicillin A of 0.5 to 0.6 in a broth containing. . The recombinant i - Done by Cori. They were collected by centrifugation and collected by Laemmli (Laemmli). IIIlli), Nature (London), 227.680 (1970) in 5DS-PAGE sample buffer. Disintegrated by boiling for minutes. 7.5~IL5% Gradient Polyacrylamide The polypeptide was bisected on a gel and transferred electrophoretically to nitrocellulose. and monoclonal antibody 22B18. ]:Bi”] Glove with J-Protein.

Contains the product of the MSP-1a gene recognized by monoclonal antibody 22B1. The bands that have this are shown with arrowheads. The molecular weight standard shown on the right side of this figure is kilo Shown in Daltons. Lanes 1.3.5 and 7 are recombinant pVAl, pW, respectively. AO1, ptp6 and pFLlo.

Lanes 2.4.6 and 8 are VA, WA, ID and FL Anagramma Mal. It is a polypeptide produced by the early body of Ginare.

As shown in Figure 10, 4111 of Anaplasma marginale [(7) Geographical unit] The DNA sequence for the detachment was obtained. DN in recombinant plasmid GAGEM4 The A insert was compared to Sanger et al. Naru Academy Op Sciences (Proceedings of Na tional Acade+sy of 5 sciences) (U, S, A,) 82.648 (1985), sequenced using the dideoxynucleotide method of Initial sequencing was performed using SF3 and t7 promoter primers in pGEM4. activated the constant reaction. When inserted into each, a new primer is created based on the resulting sequence. Mers are synthesized and used to extend the sequenced region. The array is Represents the 3' end of the Florida isolate clone insert from the 5'Kpn1 site of the clone. It is given up to the same point. Annotations above the sequence indicate the Kpn1 site, promoter Characteristics of regions, initiation of transcription, start and stop fudons of coding sequences, and repeating units. Show rank. Mutated bases are indicated by stars below the sequence, insertions or deletions are marked with a dash. Indicated by The region of homology near the 3' end included in the repeat region is repeated at that point. Draw a double underline in the indicated area. Further discussion of salient points about DNA sequences is shown below.

The above description of suitable methods for gene identification, isolation, cloning and expression includes: It is also applicable to the remaining antigens of the invention. More specifically, the characteristics studied Using these techniques with appropriate modifications for specific antigens, anaplasmoma Purified antigenic protein similar or identical to the natural antigen shown above for Luginare A recombinant plasmid or recombinant nucleic acid, DNA or can generate other recombinant vectors containing RNA. Furthermore, in particular, the above Am220, Am105 from Florida isolates as used in the study ( complex), Arn105U, Aml05L.

Am86, Am61. Am56, Am42, Am36, Am31j; and Am2 5; Especially preferably Am105 (complex) and Am105U.

Am105L, Am86, Am61. Like Am36, Am31 and Am15 Antigenic antibodies from the indicated antibodies, or other isolates of Anaplasma marginale proteins and polypeptides identical to those produced by such recombinant techniques. It can be done. Similarly, such recombinant techniques can be used to achieve desired antigenic and applicable uses. DNA and/or polypeptides of immunological proteins or polypeptides in It can be applied to determine sequences. The amino acid sequence information is then used to construct Given a commercially available recognition of the desired polypeptide sequence to be constructed, It is possible to produce antigenic polypeptides according to the polypeptide synthesis technology known in the art. can. Antigens, vaccines, recombinant vectors, recombinant cells, and methods of the present invention and other aspects apply to the broader class of rich cares in the broader concept. It should be understood that this is possible. This allows the antigens and vaccines of the invention to is used to ensure that at least one member of the Rickettsiae is immunologically treatable and This is because it can be detected. In particular, this is discussed above and in more detail below. Applies to more specific nucleic acid and amino acid sequences. These are parasitic It is known to be effective in inducing an immune response against organisms.

Explanation of MSP-1a gene structure - Four DNA inserts of recombinant base plasmids pFL10, pID6, pWAOl, Each part of pVAl and bluff, mid pAMT 11, shown with thick lines in Figure 8. by sequencing portions of four isolate-derived plasmids and pFL10 Except for pAMTl, which is not shown in Figure 10 because it overlaps with the part shown for The DNA sequence was determined using the DNA sequence shown in FIG.

To define the gene, first the transcription start site was located.

To do this, the Nuku of 7 Vander Ploeg et al. Nucleic Ac1ds Research (Nucleic Ac1ds Re5earc) h) Florida isolation according to the method set forth by 10s3591 (1982) All cellular RNA from Anaplasma marginale early bodies was collected in a single important Primers specific to the region near the 5' end of the open reading frame (ORF) The sequence was determined using National Aca demy of 5 sciencesX [J, S, A,) 82.648 (198 In the method of 5), Hollingsad et al. Molecular Ce1l Gene- tiics) 207.196 (1987). The RNA was directly sequenced using the myeloblastosis virus reverse transcriptase of did. Synthesis of the efflux transcription stop at base IFL (base number 1 of the F'L isolate sequence) , this was identified as the start of transcription (see Figure 11A). The primer is base 147F It was the reverse complement of L~166FL (see Figure 10). Regarding MSP-1a gene A putative promoter is determined by its position with respect to the transcription start site and by the identical by its striking homology with the E. coli promoter consensus sequence. established. Various geographical isolate alleles and promoter regions of E. coli The structure changes slightly as shown in FIG. 11B. -35 and -IO The region as well as the start of transcription are indicated by double underlining in this figure. E・ Cori Consensus Promoter and Anaplasma Marginale Promo Homologous bases between target sequences are indicated by bold letters. Lowercase letters are common between two organisms. Indicates a base that is not present. Differences between ID and three other MSP-1a alleles The two bases shown are indicated by a single underline. In FIG. 11B, "n" It also represents a deoxynucleotide.

The PL, VA and WA alleles extend from the transcription start site to the 5' end of the 135 region. are the same. The ID allele has a single base deletion within the region of -351 at position -30FL. have a loss However, by insertion of T at position -22FL, -35 and The spacing between the 10 regions was maintained (Figures 10 and 11B), and the four alleles were separated. Compatible with that of the E. coli consensus sequence. Then the 5′ to 135 region Until now, the very A+ It is a T-rich extension.

The four alleles include initiation of transcription at base IFL and bases 128FL to 13 FL, WA and as defined by the start methionine codon at 0FL of 127 nucleotides for VA isolates or 71 nucleotides for ID There is an obvious untranslated leader. For FL, the VA and WA alleles are Identical in this region except for the A to G base transition at position 10FL.

On the other hand, the ID allele has a T to G transversion at position 8FL and a 1151 and a deletion of 4 bases.

Despite these differences in the 5′ untranslated region, FL, WA and ID Alleles are expressed at comparable levels in E. coli (DH5a) . VA is not expressed to a comparable extent, but this may be due to plasmid copy number or or the sequence from 3' to the end of the MSP-1a gene, which is not found in other recombinants. This is due to the differences in the encoded products. This question was not followed up.

Translation occurs at the methionine codon of bases 128FL-130FL for the following reasons. 81) The only long open coding frame in this gene that appears to be initiated The system starts just 5' to this encoding codon; 2) upstream bases 45FL to 4 There are other methionine codon coding sequences in 7FL, but this long open decoding frame 3) mono Clonal antibody 22B1 is a synthetic oligonucleotide encoded by this coding frame. (see Figure 14); and 4) a fragment of the polypeptide. to a point beyond that contained in the plasmid pAMTl expressing the There are no other methionine codons in the reading frame. in each allele , extending to the same obvious stop codon sequence at bases 2429FL to 2431FL. There is one long open decryption frame (see Figure 10).

From bases 1686FL to 2324FL, which are completely conserved between alleles A very high overall There is homology. However, there are three regions with a high degree of variability in the coding sequence. be. The first 30 bases of the DNA coding sequence consist of 4 FL, 4 VA, and 4 WA each. ID has only 27 bases in the same region, 7 of which are from other isolates. Consists of hypervariable regions that deviate from the body. This result shows that there are four substitutions between the isolates. has been shortened from 10 to 9 amino acids, three of which are non-conservative. Related N-terminal amino acids. A similar clustering of substitutions at the 3′ end results in Five amino acids differ in the last 35 residues. Finally, base 1184FL? A 120 bp extension from A highly variable region with 11 base substitutions (see Figures 10 and 12). , 8 of these substitutions are non-conservative and 7 of 11 are short spirals (c It is in a region expected to be a oi 1-turn) structure. This antigen is important for the host response to

The salient features of the MSP-1a gene for all four isolates are 84 or D containing a series of similar tandem repeat DNA sequences containing 87 bp. This is the presence of the NA tandem repeat region.

These DNA tandem repeat sequences have 28 or 29 amino acids, respectively. encodes the expression of a polypeptide sequence. Tandem repeat sequences are isolated from VA isolates. twice in the WA isolate, four times in the jD isolate and six times in the FL isolate. Repeated 8 times in detachment. Each allele has 2 in number of repeats. At this point, this observation does not seem to be of significant significance. You can't get sex. Tandem repeats of 28 or 29 amino acid units are N - immediately follows the terminal 10 (or 9) amino acids.

Figure 13 shows Anablasma marginale isolates with four repeating amino acid sequences. With respect to all repeat sequences contained in the tandem repeat region, herein The 5 gene, referred to as repeat form A-E, contains two repeat forms. . The primary structure of the various repeat forms is the entire tandem repeat sequence of these isolates. 28 or 29 that is completely conserved in all five repeating forms that define Ugly e' is completely conserved by 25 amino acids in the sequence. In each allele If the tandem repeat domain starts with one copy of one repeating form or However, the second repeat form is present in one of the seven copies. each pair Changes in the number of tandem repeats present in the erected genes may be used to explain the differences. These four geographical units of Anaplasma marginale can be easily combined without the need to include other mechanisms. The size polymorphism of Am105U protein can be completely explained in terms of dissociation. Ru.

The 28- and 29-mer amino acid sequences shown in Figure 13 are one conserved saleta amino acid sequence. acid sequence DSSSA, GQQQESSVSSQS, EASTSS or QASTS Contains S and QLG. one or more of these sequences or their subs The unit may be important in defining the antigen of the invention. Antibody 22B1 is The arrays EASTSS and QASTSS are selected, as explained in more detail below. Combine selectively. Florida isolate 29+m shown in Figure 13 as repeating form B Bovine antibody titers against the ar polypeptide increased. Additional polypeptide sequences Combining one or more of these repeating arrays into a column can be is the 29mar amino acid sequence or the conserved subnucleotides shown herein. Stimulating an immune response characterized by their subunits like units It can also be important when These highly conserved tandem repeat units regions of homology may be conserved in other Rickettsia microorganisms, and Therefore, products containing these amino acid sequences, subunits or their recombinant The use of antigens and immunogens to detect further Rickettsia infections It can be administered as a treatment or as a prophylactic.

Analysis of amino acid sequence information encoded by the MSP-1a gene of various isolates. The true molecular weight of all antigenic proteins tested was determined using the standards used in the test. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility comparison It clearly shows an anomaly with respect to the expected molecular weight. Each antigen has a code It migrates in electrophoresis by a means that appears to be significantly larger than the size given.

This discrepancy between electrophoretic mobility and true molecular weight is due to the It is a recognized property of proteins containing domains.

Significant homology with tandem repeats in addition to various tandem repeat units There are five other known sequence regions in the FL alleles that share . this Four of the homologous regions (exemplified by bases 236FL to 277FL) and) form a series that overlaps the same region within a repeating structure. First homology region , bases 2240FL to 2254FL are the DNA sequence GGTGGcCAGCAGC Contains Ag (lowercase letters are mismatched; see FIG. 10).

This sequence shares 13 out of 15 bases with the homology region of the tandem repeat.

This region is in a long open decoding frame, is in frame synchronization, and is The radical differences are silent (encoding the same amino acid) and therefore It encodes the amino acid sequence GGQQQ. The second homology region (see Figure 10) (not shown) is the DNA sequence TTAtGcGCaGATgcCaCcTC It is encoded by bases -1188FL to -1208FL containing A. this The regions share 14 out of 21 bases. Third region of homology, base −129 2FL~-1308FL (XTCAGOGGGGTcC, TCAG not shown CA) shares 16 out of 17. Fourth homology region, base -145Q FL~-146IFL (CgGCAGgAAGcG) has 9 out of 12 bases are sharing. The four homology regions are bases 260FL to 274FL, 23 By 6FL~256FL, 254~270FL and 266FL~277FL It overlaps with the region of the repeating array illustrated in . Fifth homology region, base -49 6FL~-516PL (CAGGaCcGcAaATGgGcCTCAA) is 15 of the 21 bases are shared with the extension exemplified by bases 302FL to 322FL. have These regions of homology may indicate the origin of the repeats as described below. .

Mapping of neutralization-sensitive epitopes - In particular, the in vitro neutralization of anti-11 [Am105 (complex)] The potency and efficacy and binding of monoclonal antibody 22Bl to this immunological depth is The monoclonal 22 The epitope recognized by B1 was mapped. Plasmid pAMTl is mono AmF105U subunit poly recognized by clonal antibody 22B1 encodes a peptide with an N-terminal 10 amino acid extension and 7 complete repeats and contains only one partial repeat. For this, we use a repeating structure. The focus of the research was narrowed. The study demonstrated that various synthetic compounds containing overlapping extensions of B repeats By assaying the binding ability of monoclonal 22B1 to oligopeptides. It was. The minimum structure required to bind this antibody is the six amino acid sequence QAS It turned out to be TSS. This arrangement consists of two homologous forms in each repeat. The other sequence is RASTSS. 5+ner polypeptide, A Whereas STSS and QASTS did not bind antibodies, synthetic peptides , both bound isometrically to monoclonal antibody 22B1. Results of these experiments is schematically described in FIG.

Different lengths and different A8 and B repeats of the anti-jKAmF I O5U polypeptide The oligopeptide containing the region containing the binding region of monoclonal antibody 22B1 was assayed for inity. (+) reaction is monochrome in these assays. Shows ronal 22B1 binding.

A (-) reaction indicates no detectable monoclonal 22B1 binding. Result is , obtained by solution-phase inhibition radioimmunoassay using 161-labeled AmF105. This is an enzyme-linked immunosorbent assay and an immunoprot assay. confirmed by. Enzyme-linked assays were performed using Balmer (P almar) et al.'s International Journal of Parasitology -(Inter-nationaL Journal for Paras Itology), 17, 1279 (1987). I went to see it. First, apply bovine serum albumin to the microtiter plate. To this was added 0.25% (W/V) glutaraldehyde, 10mM sodium sulfate. , oligopeptide in 94mM ethylenediaminetetraacetic acid, pH 6,8. did. Perform blocking and washing steps using veronal buffer with the following composition. = 145 M sodium chloride, 1.8mM 5.5'-diethylbarbic acid Sodium, 4.5mM barbic acid, 0.5□MMgOffl. visualization system The program was horseradish peroxidase coupled to recombinant protein G. stomach For the Munoplot assay, serial dilutions of oligopeptides were Spotted onto nitrocellulose precoated with serum albumin. This plot The samples were fixed in 2.5% (V/V) glutaraldehyde. Ballmer et al., Saye Tris as described in Science 231.1299 (1986). After extensive washing with buffered saline, the plot was incubated with monoclonal 22Bl. incubate, then incubate with rabbit anti-mouse IgG and finally 1 26! Incubated with protein A.

A synthetic 29 mar ori containing the sequence shown in Figure 13 as MMB The gopeptide was tested in cattle to determine the antigenic potential of this sequence. , two cormorants Four doses of 400 μ9 were administered to the patient. The first injection was 70 indian full aj. The bunt was about 1-2 taQ. Then, the same amount of antigen oligopeptide was added. Three boosters were administered approximately two weeks apart. In another three cows, the molecular weight with an approximate electrophoretic mobility corresponding to 20,000 to 200,000 Daltons Polymerized protein 29m with the cross-linking agent carbodiimide to produce an antigen Other vaccines containing the same amount of antigen containing the er oligopeptide were similarly administered. Seed, Science 144.1344 (1964). 5 All cattle were vaccinated approximately 2 months after the first vaccination and immediately after the last booster injection. Serum antibody titers were tested. The titer is applied in the wells of the titration plate. Immunoaffinity purified AmF 105 and 29mer synthetic oligopeptide were analyzed for both. The results of these tests are shown in Table 6 below. all the cows , tested for pre-inoculation titer and found to have negligible response. .

29-r oligopeptide vaccine Bovine 1 and 2 1:100 1:10100O29 oligopeptide and Polymerized carbodiimide vaccine Cow 3 1:10,000 1:10. ooO cow 4 and 5 1:1.000 t:to, oo.

Immunoaffinity purification AmF105 vaccine Kush 6 1:10,000 1:1.000 completely repeating domain is a structural algorithm Coils (coi 1/t) that match short linear epitopes by the algorithm urn) segment. However, the complete 2911er The binding affinity of monoclonal antibody 22Bl for repeat B is approximately two orders of magnitude greater than for the pitope, with some structural effects It suggests power.

These results demonstrate that the MSP-1a gene and recombinant its encoded polypeptide product containing Am105U; revealed some characteristics of the subunit antigen derived from et al. Tande The M repeat domain is found in streptococcal M proteins, which are taxonomically distant, and in some cases. their presence in the surface antigens of Rickettsia spp. It had not been previously reported in Pakistan. repeatability in this domain The variables account for the extreme size polymorphism of this polypeptide. monoclonal anti The epitope bound by body 22B1 is important even in neutralizing parasite infectivity. Functionality was precisely conserved in each replicate of each isolate.

In addition to the variable number of repeats, three non-terminus polypeptides containing the N-terminus There is always an area of variability. The gene is based on the E. coli promoter consensus sequence. using a promoter structure and spacing that is very similar to . MSP -1a Promoter Oyohii coli Similarity between promoter consensus sequences Regardless of sex, one significant difference emerged: clear ribbon-like coupling. site, even if this gene is expressed in appreciable amounts in E. coli. However, it was not detected at all in the untranslated leader region. -11 to -5 The sequence GTGTGTG found in position (relative to the ATG codon) is lower It is a base pair with liposomal RNA that has a low affinity.

The exceptional structural features of the AmF105 polypeptide are that it is a surface protein and body, but to facilitate its translocation to the outer membrane bilayer. No obvious single sequence was detected. Predicted polypeptide The bite lobularity plot shows amino acids 255FL~270FL, 541FL~ 557FL.

567FL~585FL, 631FL~650FL and 662FL~678F We reveal five major hydrophobic extensions from L. The last four of them are Sufficient length and hydrophobicity to act as a permeable membrane domain. These canals One of the aqueous domains may act as a non-degradable internal signal sequence.

The hypervariable nature of the N-terminus of AmF105 suggests that this domain may This suggests that the design function is not satisfied. On the other hand, this sequence and amino acids The highly variable region sequences of 353FL to 392FL satisfy immunological functions. and will provide the necessary epitope for T-cell recognition. If so , T-cells and host subpopulations capable of responding to AmF105. tion can be regulated by these regions. Therefore, in these areas mutations in the host population that are immune to heterologous isolates. can provide a level of antigenic variability that allows the parasite to survive.

Tandem repeat structures are susceptible to unequal homologous recombination and/or slipped strands. Numerous instances of slipped-strand mispairing It was hypothesized that it increases with fruit. Tandem repeat in AmF l 05 The origin of is unknown. Sequences that share significant homology with repeats are MSP It is also found at other sites within and outside the -1a gene coding sequence. tandem repeat Given the length of the sequence (84 or 87 bp), the longer sequence is In order to reduce the probability of Rand mispairing, unequal homologous recombination is a more promising mechanism. Ru.

A number of the MSP-1a gene alleles of the present invention are characterized by We have revealed one molecular principle regarding is polymorphism.

By defining several features of the MSP-1a gene, the T of AmF105 −Cellular evitopes can be mapped to Anaplasma marginale. The potential for antigenic variation based on T-cell epitopes can be assessed. Ru. Adaptability to other rickettsial organisms can be further studied. preserved neutralization sensitivity The definition of sexual epitopes allows further evaluation of synthetic immunoprotection, and this This includes vaccines based on recombinantly produced vegutides.

In addition to the four alleles of MSP-1a, the same or cloned the Florida isolate MSP-1b gene using a very similar method. Ta. Anaplasma margi + -le (7) MSP-1 complex in 70 Lida isolates MSP-1b for Florida isolates, the second surface protein subunit of the body The gene encodes the production of recombinant AmF105L. Figure 15 shows various Restriction enzyme map for MSP-1b Florida showing cleavage sites for the enzyme. . Figure 16 shows the DNA encoded by the AmF105L gene and related amino acids. The amino acid sequence is shown. ! ! The last part of 116 is the amino acid of the expressed recombinant antigen. The acid composition and theoretical molecular weight of 80,359.85 Daltons are shown, and the theoretical molecular weight is 80,359.85 Daltons. The molecular mass is consistent with electrophoretic mobility measurements of approximately 100-105 kilodaltons.

Therefore, the production of recombinant MSF'-1 complexes is similar to that of MSP-1 in a heterologous system. 1a and M'3F-1b genes by co-expression.

Amino acid sequence abbreviations used herein are shown in appendix A. .

20 types of amino acids Amino acid type 3 letter symbol 1 letter symbol Hydrophobic (aliphatic side chain) Glutamic acid Glu E Aspartic acid Asp D Asparagine Asn N Contains human σxyl Methionine Met M Ea to 9J Figure 1. Antibody and I+Z% labeled protein A on nitrocellulose ・Detection of marginale (A, marginale) protein.

(A) Known positive and negative control antibodies were applied to all filters in duplicate. Added in serial 10-fold dilutions: red blood cells infected with A. marginale (60% parasitic) 2X10' to 2X10'' total cells) and uninfected red blood cells (same concentration), Am105 protein (10-0.1 ng), Am36 protein (10-0.1 ng). 1 ng), and ovalbumin (IO ~ 0.1 ng). each with a different antibody Tested on filters: R873 (1:4,000 dilution), R781 (1:4 00 dilution), F34Cl (2pg/m1), and Fl9 El (2s g/m+). (B) There is a possibility that the previous screening was positive. Recombinant E. coli (E. coli) selected as colonies were transferred to R873 Rescreened on a dual filter for reaction with. Right of each filter The two spots at the top are 2XlO' whole blood with 60% parasitemia. Dual signal derived from positive control anti-W containing spheres = 1 μm. uninfected red blood cells (1p+) also has a compound equation, so that in addition to each filter, t; gives no signal at all. won.

”Edit ==12 Anabrasma marginale gene L R AM22 R.L. AM25 L R AM97 R.L. AM113 kilohes χ・1 Figure 2. Plasmid insertion from an E. coli colony expressing the Am105 determinant DNA restriction enzyme map. L (left) and R (right) are insert DNA pBR322 Represents the orientation to the array. L is close to the EcoRV site of pBR322, R is close to the sph1 site. A, AvaI; Ap, ApI; Ban, B anI[;Bg, Bgu　;BllBgl　II　;Ec, EcoRV　;H, HpaI;Hc, HincI[;Hd, H4ndlI[;Ml, MluI:N d, Ndel　; Pu, Pbull　; SmSSm5S　; Sp%5phl　; Ss.

5stI; Tt, TthIII; Xa, Xmalff; XblXbaI; s　X　h　o　I　.

”5==wo-1 Figure 31. E. marginale protein synthesized by recombinant E. coli.

E. coli containing pBR322 or pAM25 plasmid DNA was incubated in During VijrO incubation, radiolabel with ["Sl" methionine and surfactant extract] It was subjected to immunoprecipitation reactions with various antisera. Immunoprecipitates were diluted with 7.5-17.5% poly It was analyzed by acrylamide-5DS gel electrophoresis and fluorescence analysis.

Lane 1. +4 (: labeled molecular weight standard protein; lanes 2 to 7, E. coli and pAM25; lanes 8-13, E-Furi and pBR322; lane 78 and 8. Total 3s3 protein profile. Antibodies to be immunoprecipitated were obtained from normal rabbit serum. (lanes 6 and 9), R873 (lanes 5 and 10), R612 (lane 4) 8 and 11), R874 (lanes 38 and 12) and R781 (lane 2 8 and 13).

Ku Figure 41 Recombinant Am105 (rAm105), Am105L and Am105 Comparison with U. (A) pAM25 (lane 1) or pBR:322 (lane 2) ) containing E. coli cells during ir + viLro culture. After labeling with onine, the surfactant extract was subjected to immunoprecipitation reaction with R873. . A. marginale was also labeled with [35s]methionine and a neutralizing monoclonal anti- antibody 22B, (lane 3) or control monoclonal antibody 24A+ (lane 4). The t-0 immunoprecipitate was subjected to immunoprecipitation reaction with 7.5% polyacrylate containing 4Mi〉. Analyzed on Rylamide Gel-5DS gel: lane 5.14c labeled molecular weight standard tag. Npaku. CB) 0.025 μg of Nis aureus (S, aureus) V8 Recombinant Aml0 produced by degradation in a stacking gel with proteases 5. Partial proteolysis products of Am1.051-1 and Am105U were dissolved at 15% Comparisons were made on polyacrylamide-3DS gels.

=5 Figure 58 Antigen comparison of recombinant Am105, Am105U, and Am105L. 3 53-labeled Am105U and Am105L with various antibodies as indicated, either independently or Both were subjected to immunoprecipitation reaction. All sediment was washed with 7.5% polyamide containing 4M urea. Analyzed on acrylamide gel-3DS gel: Am 1 05 L, lane 1, 4.8, and 11; Am105U, lanes 2.5.9, and 12: and both Am105U and Am105L (“SIN Re (7) 22B, sediment), lane 3.6.7, and 10° large x5z7/! 5 Figure 6. Surface radiation perception and immunity of early A. marginale bodies? Descending reaction. initial body , using lactoperoxidase, +4! Radiolabeled with r and extracted with surfactant The solution was mixed with R873 (lane 1), seven-null antibody 22B+ (lane 2), and R9. 11 (lane 3), monoclonal antibody IE (lane 4), and R907 ( Lane 5) was subjected to immunoprecipitation reaction. The immunoprecipitate was transferred to a 5% bore containing 4M urea. Analyzed on acrylamide gel-5DS gel.

Figure 7. Paired with A. marginale genomic DNA by Southern plotting method comparison with plasmid DNA. (A) pAM14(p) or A. margi Nale genomic DNA (g) was digested with restriction enzymes, subjected to electrophoresis, and pAM1 Nick translation of 4, t7ed 1, 4kb HincU-) (indl l! Probed with insert DNA. (B) Genomic DNA of A. marginale ( g) t or bovine leukocyte DNA (wbc) is digested with restriction enzymes and subjected to electrophoresis, 1.4 kb HinclI-Hindl [I fragment of pAM14 (lanes 1 to 3) Alternatively, probe with the 2-Okb 5stI fragment of pAM97 (lanes 4, 4, and 5). I was disappointed. Compared to that produced from the cloned 3.9kb Bglll fragment. The corresponding genomic bands are indicated by thin arrows on the gel.

ノ===丞EIIE 1/'2 DNA sequences of the FLSV'A+W and ID alleles of the m5pla gene.

2B 422=teEJ71e ! =3=7JJIE;! of the promoter regions of the FL, VA, WA and ID alleles of the m5pla gene. structure.

-35-,. Starting allele Middle B encoded by the FL, VA, WA and ID alleles of the mspail gene. Polypeptide sequence of AmF105 mutant.

! =3=column L7 The FLSVA+WA and ID alleles of the mspla gene are encoded by A repeating structure.

number in allele Mapping of mAb22B1 binding epitopes sensitive to neutralization reactions.

reactivity I minister Shisan AMF105L SYN restriction site from base 1 to base 2746.

Positions are numbered starting with base number 1.

All enzymes listed in this figure are commercially available.

The mark is controll! Shown below the base located just before the enzymatic cleavage site.

If the cut site is unknown, a mark is placed in the center of the site. The first letter of enzyme core is Located below the ° mark.

- Cleavage by the asymmetric recognition sequence of many enzymes occurs at a location distant from the sequence. Please note that

MboII Hlnfl Rial BgLL2J3 2J3 'AgaX　-XbaI HpaKX NLaH engineering Alu, 1 ^1uK N1aXlN Fnu48Z BstXX HxLX N5LX SEa [NsLXHgaZ BbvXAlu I　KboI! RiaISea Mountain ITaql Psr, I1 (boII Mn1I Fnu4HI BbVJ scl =JE 4 Giant West Thea &amp; CACAGCG Ding G TA (i ATA TCT CAT (TA Ckk) (:A CCT iG CCCCAA TCA (:CA b to A AGT C CT　τC＾ 2530 251tO25502560257025aOaMccc CTCc Ac　TTCC(CT(:CATT　CACCAA　Cf1ACGCACT　A TT CCT T1’CCCCCCCCbCCτCC cTCAGT CCCTAG A? (:　CcT　TCCTTCCCA　(IT G TTCATCATCTCA A clove (:TA (:C` Clove CCGCC ＾Ding Noomi Hyakuman 2& Table of codon usage for AMF105L r koj translated as above.

Number of identified codons - 756 Number of unspecified codons - 〇 Calculated molecular weight - 80359,85 Translation begins at base 170.

Translation stops at the stop codon (base 2438).

The sequence is printed from base 1'B to base 2746.

Sequences are numbered starting with base number ].

-Gu---^-"""-PCT/U!On/(1167R++11##lml- 1--Mm, Mausoleum/lls90101678

Claims (1)

[Claims] 1. The following amino acid sequences are selected from the group consisting of: glutamic acid-alanine-serine-threonine-serine-serine, and glutamine-alanine-serine-threonine-serine-serine. A purified antigenic peptide comprising at least one amino acid sequence selected. 2. The purified antigenic peptide according to claim 1, wherein the antigenic peptide is produced by a cell containing recombinant DNA encoding the production of the antigenic peptide. 3. The essence according to claim 1, wherein the antigenic peptide is produced by artificial peptide synthesis. manufactured antigenic peptides. 4. The purified antigenic peptide according to claim 1, wherein the antigenic peptide has immunogenicity that causes resistance to infection with at least one Rickettsial microorganism. 5. The purified antigenic peptide according to claim 1, wherein the antigenic peptide has immunogenicity to cause resistance to infection with at least one rickettsial symptom of the genus Anaplasma. Petite. 6. The antigenic peptide has immunogenicity that causes resistance to at least Anaplasma marginale infection. The purified antigenic peptide according to claim 1. 7. The purified antigenic peptide of claim 1, wherein the antigenic peptide has immunogenicity in mammals that causes resistance to at least Anaplasma marginale infection. Do. 8. Claim 1, wherein the antigenic peptide has a molecular weight of at least 15 kilodaltons. Purified antigenic peptides. 9. The antigenic peptide corresponds to an approximate molecular weight of at least 15 kilodaltons. The purified antigenic peptide according to claim 1, which has a relative mobility in electrophoresis. 10. A purified antigenic peptide as claimed in claim 1 and further identified as comprising an amino acid sequence having the sequence described as type A in FIG. 11. A purified antigenic peptide as claimed in claim 1 and further identified as comprising an amino acid sequence having the sequence described as type B in FIG. 12. 3. A purified antigenic peptidic acid identified as comprising an amino acid sequence as claimed in claim 1 and further described as type C in FIG. Specified as containing the amino acid sequence described in claim ⊥ and further described as type D in Figure 13. Steel antigen good peptide. 14. A purified antigenic peptide as claimed in claim 1 and further identified as comprising an amino acid sequence having the sequence described as type E in FIG. 15. The amino acid according to claim 1 further comprising glutamine-leucine-glycine. Purified antigenic peptides identified as containing amino acid sequences. 16. A purified antigenic peptide as claimed in claim 1 and further specified as comprising an amino acid sequence having aspartic acid-serine-serine-serine-alanine. 17. Claim 1 further comprising glycine-glycine-glutamine-glutami A purified antigenic peptide identified as containing an amino acid sequence having glutamine. 18. A purified antigenic peptide as claimed in claim 1 and further specified as comprising an amino acid sequence having the following: serine-glycine-glutamine-glutamine-glutamine. 19. Claim 1 further comprising glycine-glutamine-glutamine-gluta. A purified antigenic peptide identified as comprising an amino acid sequence having the following: min-glutamic acid-serine-serine-parine-serine-serine-glutamine-serine. 20. The amino acid sequence according to claim 1 further includes: glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine-glycine. Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri - valine - serine - serine - glutamine - serine manufactured antigenic peptides. 21. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. at least two tandem lipids comprising an amino acid sequence having -serine-serine A purified antigenic peptide that is identified as containing a native amino acid sequence. 22. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. a first amino acid sequence having 1-serine-serine and glutamine-leucine; at least two tandem repeats comprising a second amino acid sequence having a lysine; a purified antigenic peptide identified as comprising an amino acid sequence; 23. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. at least two amino acid sequences comprising a first amino acid sequence having -serine-serine and a second amino acid sequence having aspartic acid-serine-serine-serine-alanine. A purified antigenic peptide identified as containing a tandem repeat amino acid sequence. 24. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. a first amino acid sequence having Glycine-Glycine-Serine and Glycine-Glycine-Glue. and a second amino acid sequence having tamine-glutamine-glutamine. A purified antigenic peptide identified as containing two tandem repeat amino acid sequences. Do. 25. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. a first amino acid sequence having 9-serine-serine and a serine-glycine-glutamic acid sequence; A purified antigenic peptide identified as comprising at least two tandem repeat amino acid sequences comprising: - glutamine - a second amino acid sequence having glutamine. 26. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. a first amino acid sequence having -serine-serine and glycine-glutamin Rutamine-Glutamine-Glutamic acid-Serine-Serine-valine-Serine-Seri A purified antigenic peptide that is identified as comprising at least two tandem repeat amino acid sequences comprising - glutamine - a second amino acid sequence having serine. 27. as claimed in claim 1, further comprising glutamic acid-alanine-serine-threonine. at least two tandem repeat amino acid sequences comprising a first amino acid sequence having 1-serine-serine and at least a second and a third amino acid sequence. the second and third amino acid sequences are the following amino acid sequences: glutamic acid. -leucine-glycine, aspartic acid-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide selected from the group consisting of: -valine-serine-serine-glutamine-serine. 28. The amino acid sequence as claimed in claim 1 and further includes: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, and glycine-gluta. Min - Glutamine - Glutamine - Glutamic acid - Serine - Serine - Palin - Seri A spermatozoon identified as containing at least two tandem repeat amino acid sequences containing 1-serine-glutamine-serine. manufactured antigenic peptides. 29. having immunogenicity conferring resistance to infection with at least one rickettsial microorganism and consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine, and glutamine-alanine-serine-threonine-serine-serine. Select from the group A purified antigenic peptide comprising at least one amino acid sequence selected. 30. 30. A purified antigenic peptide as claimed in claim 29 and further specified as comprising an amino acid sequence having: glutamine-leucine-glycine. 31. 30. A purified antigenic peptide as claimed in claim 29 and further specified as comprising an amino acid sequence having aspartic acid-serine-serine-serine-alanine. 32. Claim 29, further comprising glycine-glycine-glutamine-gluta A purified antigenic peptide identified as comprising an amino acid sequence having min-glutamine. 33. Claim 29, further comprising serine-glycine-glutamine-glutami A purified antigenic peptide identified as containing an amino acid sequence having glutamine. 34. Claim 29, further comprising glycine-glutamine-glutamine-glue. A purified antigenic peptide identified as comprising an amino acid sequence having the following: tamine-glutamic acid-serine-serine-parine-serine-serine-glutamine-serine. 35. Claim 29 further includes the following amino acid sequence: glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine-glycine. Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri - valine - serine - serine - glutamine - serine manufactured antigenic peptides. 36. at least one atom as claimed in claim 29 and further selected from said group. Contains at least two tandem repeat amino acid sequences containing a amino acid sequence Purified antigenic peptide. 37. 29, further comprising glutamic acid-alanine-serine-threo at least two tandem repeats comprising a first amino acid sequence having nin-serine-serine and a second amino acid sequence having glutamine-leucine-glycine. A purified antigenic peptide that is identified as containing an amino acid sequence. 38. 29, further comprising glutamic acid-alanine-serine-threo A purified antigen identified as comprising at least two tandem repeat amino acid sequences comprising a first amino acid sequence having nin-serine-serine and a second amino acid sequence having aspartic acid-serine-serine-serine-alanine. sex peptide. 39. 29, further comprising glutamic acid-alanine-serine-threo A first amino acid sequence having nin-serine-serine and glycine-glycine a second amino acid sequence having glutamine-glutamine-glutamine. A purified antigenic peptide identified as containing two tandem repeat amino acid sequences. Chido. 40. 29, further comprising glutamic acid-alanine-serine-threo A first amino acid sequence containing nin-serine-serine and serine-glycine-glue. and a second amino acid sequence having tamine-glutamine-glutamine. A purified antigenic peptide identified as containing two tandem repeat amino acid sequences. Do. 41. 29, further comprising glutamic acid-alanine-serine-threo a first amino acid sequence having nin-serine-serine and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine-serine-serine; and a second amino acid sequence having phosphorus-glutamine-serine. A purified antigenic peptide identified as containing a tandem repeat amino acid sequence. 42. 29, further comprising glutamic acid-alanine-serine-threo identified as comprising at least two tandem repeat amino acid sequences comprising a first amino acid sequence having nin-serine-serine and at least a second and third amino acid sequence, the second and third amino acid sequences The following amino acid sequence: Gluta Mine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide selected from the group consisting of: -valine-serine-serine-glutamine-serine. 43. as claimed in claim 29, further comprising the following amino acid sequence: glutamic acid-arani Serine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A spermatozoon identified as containing at least two tandem repeat amino acid sequences containing 9-valine-serine-serine-glutamine-serine. manufactured antigenic peptides. 44. an amino acid sequence having immunogenicity conferring resistance to infection with at least one rickettsial microorganism and comprising an amino acid sequence having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences comprising a first repeat amino acid sequence; sequence and a second repeating amino acid sequence, the first repeating amino acid sequence consisting of the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, and glutamine-alanine-serine-threonine-serine-serine. the second repeating amino acid sequence is selected from the first group, and the second repeating amino acid sequence has the following amino acid sequence: glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine-glycine. Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide selected from the second group consisting of: -valine-serine-serine-glutamine-serine. 45. An amino acid sequence having at least two tandem repeat amino acid sequences and having immunogenicity conferring resistance to infection with Rickettsia symptoms of at least one species. sequence, and the two tandem repeat amino acid sequences each contain the following amino acid sequences. Column: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising at least one repeating amino acid sequence selected from the group consisting of: -parin-serine-serine-glutamine-serine. 46. an amino acid sequence having immunogenicity conferring resistance to infection with at least one rickettsial symptom, and comprising an amino acid sequence having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences having the following amino acid sequence: : Glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising at least two repeating amino acid sequences selected from the group consisting of: -valine-serine-serine-glutamine-serine. 47. having immunogenicity conferring resistance to infection with at least one rickettsial microorganism and comprising an amino acid sequence having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences having the following amino acid sequences: Glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising at least three repeating amino acid sequences selected from the group consisting of: -valine-serine-serine-glutamine-serine. 48. having immunogenicity conferring resistance to infection with at least one rickettsial microorganism and comprising an amino acid sequence having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences having the following amino acid sequences: Glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising at least four flanking amino acid sequences selected from the group consisting of: -valine-serine-serine-glutamine-serine. 49. An amino acid that has immunogenic properties that confers resistance to infection with at least one Rickettsial microorganism and that is substantially similar to that described in Figure 12 for the Florida isolate. Purified antigenic peptides containing amino acid sequences. 50. 12 with immunogenic properties conferring resistance to infection with at least one Rickettsial microorganism, and which is similar to that described in Figure 12 for the Virginia isolate. Purified antigenic peptides containing amino acid sequences. 51. An immunogenic compound conferring resistance to infection with at least one rickettsial microorganism and is generally similar to that described in Figure 12 for the Washington isolate. Purified antigenic peptides containing amino acid sequences. 52. An amino acid that has immunogenicity that confers resistance to infection with at least one Rickettsial microorganism and that is substantially similar to that described in Figure 12 for the Idaho isolate. Purified antigen injection peptide containing amino acid sequence. 53. have immunogenicity conferring resistance to infection with at least one rickettsial symptom and have the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine -Serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising an amino acid sequence having at least two amino acid sequences selected from the group consisting of: -valine-serine-serine-glutamine-serine. 54. 54, further comprising an immunogenic resistance to infection with Rickettsial microorganisms. A purified antigenic peptide characterized by its ability to bind to the immune serum of an animal fed the rickettsial microorganism for which resistance is desired. 55. It has immunogenicity conferring resistance to infection with at least one Rickettsial microorganism and has the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine- Serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising an amino acid sequence having at least three amino acid sequences selected from the group consisting of: -parin-serine-serine-glutamine-serine. 56. 56, further comprising an immunogenic resistance to infection with Rickettsial microorganisms. A purified antigenic peptide characterized by its ability to bind to the immune serum of an animal fed the rickettsial microorganism for which resistance is desired. 57. Immunogen conferring resistance to infection with at least one rickettsial microorganism Note, the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine-glycine. Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri A purified antigenic peptide comprising an amino acid sequence having at least four amino acid sequences selected from the group consisting of: -valine-serine-serine-glutamine-serine. 58. 58, further comprising an immunogenic resistance to infection with Rickettsial microorganisms. A purified antigenic peptide characterized by its ability to bind to the immune serum of an animal fed the rickettsial microorganism for which resistance is desired. 59. 40, comprising at least one purified antigenic peptide as claimed in claim 24, 30, 31, 37, 38, 39 or 40, which protects against Rickettsia and reduces the severity of or prevents infection by Rickettsia. A vaccine that induces an immune response in susceptible animals. 60. The following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri at least one amino acid sequence selected from the group consisting of -valine-serine-serine-glutamine-serine Contains purified antigenic peptides to protect against and reduce the severity of or prevent infection with Anaplasma rickettsiae. Vaccines that induce an immune response in pancreatic animals. 61. Claim 60, further comprising at least one of Anaplasma marginale sp. are also characterized by their suitability for eliciting an immune response against one rickettsiae. Vaccine that can be killed. 62. The following amino acid sequences are selected from the group consisting of: glutamic acid-alanine-serine-threonine-serine-serine, and glutamine-alanine-serine-threonine-serine-serine. at least one purified antigenic peptide comprising at least one amino acid sequence selected from Administrative agents that contain and protect against and reduce the severity of infection or prevent infection. A vaccine that induces an immune response in animals susceptible to infection with Naplasma marginale. 63. 63. The vaccine of claim 62, wherein the antigenic peptide is produced by a cell comprising a recombinant nucleic acid encoding the production of the antigenic peptide. 64. 63. The vaccine according to claim 62, wherein the antigenic peptide is produced by artificial peptide synthesis. 65. The antigenic peptide further contains the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri 63. The vaccine of claim 62, having at least one additional amino acid sequence selected from the group consisting of: -valine-serine-serine-glutamine-serine. 66. 66. The vaccine of claim 65, wherein the vaccine is adapted to immunize a mammal. 67. The antigenic peptide further comprises at least one amino acid selected from the above group. identified as containing at least two tandem repeat amino acid sequences containing an acid sequence. 63. The vaccine of claim 62. 68. The antigenic peptide further contains the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri At least two amino acid sequences comprising at least one amino acid sequence selected from the group consisting of 63. The vaccine according to claim 62, which is specified as comprising a tandem repeat amino acid sequence of hmm. 69. The antigen injection peptide is further identified as comprising at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences comprising a first repeating amino acid sequence and a second repeating amino acid sequence; Repetitive the second repeating amino acid sequence is selected from the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine, and a first group consisting of glutamine-alanine-serine-threonine-serine-serine; has the following amino acid sequence: glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri 63. The vaccine of claim 62, wherein the vaccine is selected from the second group consisting of: -valine-serine-serine-glutamine-serine. 70. It contains at least one purified antigenic peptide having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences having the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine. Line-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine - Glutamine + - Glutamine, Serine - Glycine - Glutamine - Gluta amine-glutamine, and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-seri Anablasma margina comprising at least two repeating amino acid sequences selected from the group consisting of: A vaccine that induces an immune response in animals susceptible to infection. 71. Contains at least one purified antigenic peptide having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences having the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine. Line-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri Anaplasma margina comprising at least three repeating amino acid sequences selected from the group consisting of: A vaccine that induces an immune response in animals susceptible to infection. 72. Contains at least one purified antigenic peptide having at least two tandem repeat amino acid sequences, each of the two tandem repeat amino acid sequences having the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine. Line-serine, glutamine-leucine-glycine, aspartic acid-serine-serine-serine-alanine, glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri Anaplasma margina containing at least four repeating amino acid sequences selected from the group consisting of: A vaccine that induces an immune response in animals susceptible to infection. 73. Is it inoculating an animal with an immunogenic antigen in sufficient quantity to induce an immune response? If the immunogenic antigen has the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri at least one amino acid sequence selected from the group consisting of -valine-serine-serine-glutamine-serine inducing an immune response in an animal susceptible to infection with at least one Rickettsial microorganism and reducing or preventing infection with the at least one Rickettsial microorganism. How to give a degree of protection. 74, at least one purified antigen in which the immunogenic antigen comprises at least one amino acid sequence having glutamate-alanine-serine-threonine-serine-serine; 74. The method of claim 73, comprising peptide. 75. at least one purified peptide wherein the immunogenic antigen comprises at least one amino acid sequence having glutamine-alanine-serine-threonine-serine-serine; 74. The method of claim 73, comprising tide. 76. The immunogenic antigen comprises at least two tandem repeat amino acid sequences. at least one purified peptide comprising at least two tandem repeats having the following amino acid sequence: glutamic acid-alanine-serine-threo Nin-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri 74. The method of claim 73, comprising at least one amino acid sequence selected from the group consisting of: -valine-serine-serine-glutamine-serine. 77. The following amino acid sequences are selected from the group consisting of: glutamic acid-alanine-serine-threonine-serine-serine, and glutamine-alanine-serine-threonine-serine-serine. at least one purified antigenic peptide comprising at least one amino acid sequence selected from A diagnostic test that detects antibodies against rickettsial parasites, including Tide. 78. 78. The method of claim 77, wherein at least one amino acid sequence selected from said group is produced from a cell containing a recombinant nucleic acid encoding the production of said amino acid sequence. Break test. 79. 78. The diagnostic test of claim 77, wherein at least one amino acid sequence selected from said group is produced by artificial peptide synthesis. 80. 78. The diagnostic test of claim 77, wherein the test is suitable for detecting antibodies raised against rickettsial parasites of the genus Anaplasma. 81. The test was successful against the rickettsial parasite Anaplasma marginale species. 78. A diagnostic test according to claim 77, which is suitable for detecting antibodies of different types. 82. A recombinant nucleic acid encoding the expression of at least one antigenic peptide having the ability to elicit an immune response against a Rickettsial parasite that reduces the severity of or prevents and protects against infection by the Rickettsial parasite. 83. 83. The recombinant nucleic acid of claim 82, further specified as deoxyliponucleic acid. 84. Furthermore, the ability to induce an immune response against rickettsial parasites of Anaplasma spp. 83. A recombinant nucleic acid according to claim 82, characterized as a nucleic acid encoding the expression of at least one antigenic peptide having potent recombinant properties. 85. 85. The recombinant nucleic acid of claim 84, further specified as deoxyribonucleic acid. 86. In addition, the immune response to rickettsial parasites of Anaplasma marginale spp. 83. The recombinant nucleic acid of claim 82, specified as a nucleic acid encoding the expression of at least one antigenic peptide having the ability to induce a response. 87. 97. The recombinant nucleic acid according to claim 86, 94, 95, 96, further specified as deoxyribonucleic acid. 88. 83. The recombinant nucleic acid of claim 82, further identified as a nucleic acid encoding the expression of at least one antigenic peptide comprising an amino acid sequence having the following: glutamic acid-alanine-serine-threonine-serine-serine. 89. Furthermore, it has glutamine-alanine-serine-threonine-serine-serine. a nucleus encoding the expression of at least one antigenic peptide comprising an amino acid sequence 83. The recombinant nucleic acid of claim 82, specified as an acid. 90. Furthermore, the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri at least one amino acid sequence selected from the group consisting of -valine-serine-serine-glutamine-serine 83. The recombinant nucleic acid of claim 82, identified as a nucleic acid encoding expression of an antigenic peptide. 91. Furthermore, the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri At least one amino acid sequence comprising at least two amino acid sequences selected from the group consisting of: -valine-serine-serine-glutamine-serine 83. The recombinant nucleic acid of claim 82, identified as a nucleic acid encoding expression of an antigenic peptide. 92. Furthermore, the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri At least one amino acid sequence comprising at least three amino acids selected from the group consisting of: -valine-serine-serine-glutamine-serine 83. The recombinant nucleic acid of claim 82, identified as a nucleic acid encoding expression of an antigenic peptide. 93. Furthermore, the following amino acid sequence: glutamic acid-alanine-serine-threonine-serine-serine, glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri At least one amino acid sequence containing at least four amino acids selected from the group consisting of: -valine-serine-serine-glutamine-serine 83. The recombinant nucleic acid of claim 82, identified as a nucleic acid encoding expression of an antigenic peptide. 94. In addition, the immune response to rickettsial parasites of Anaplasma marginale spp. glutamic acid-alanine-serine-threonine-serine-serine; glutamine-alanine-serine-threonine-serine; -Serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri The antigenic peptide comprises at least one amino acid sequence selected from the group consisting of: -valine-serine-serine-glutamine-serine 83. The recombinant nucleic acid according to claim 82, characterized by: 95. In addition, the immune response to rickettsial parasites of Anaplasma marginale spp. glutamic acid-alanine-serine-threonine-serine-serine; glutamine-alanine-serine-threonine-serine; -Serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri At least two amino acid sequences comprising at least one amino acid sequence selected from the group consisting of -parin-serine-serine-glutamine-serine characterized by the antigenic peptide containing the tandem repeat amino acid sequence of 83. The recombinant nucleic acid of claim 82. 96. In addition, the immune response to rickettsial parasites of Anaplasma marginale spp. a nucleic acid encoding the expression of at least one antigenic peptide capable of inducing a response, and having the amino acid sequence glutamic acid-alanine-serine-thread. onine-serine-serine and the following amino acid sequence: glutamine-alanine-serine-threonine-serine-serine, glutamine-ro Isine-glycine, Aspartic acid-serine-serine-serine-alanine, Glycine-glycine Lutamine-Glutamine-Glutamine, Serine-Glycine-Glutamine-Glutamine Glycine-Glutamine-Glutamine-Glutamine-Glutamic acid-Serine-Seri The antigenic peptide comprises at least two tandem repeat amino acid sequences comprising at least one amino acid sequence selected from the group consisting of: 83. The recombinant nucleic acid according to claim 82. 97. Antibiotics against Anaplasma marginale when inoculated into resistant cattle. A substantially pure antigenic surface protein of Anaplasma marginale, an active fragment thereof, or an immunologically similar protein produced by polypeptide synthesis or genetic engineering, which is capable of eliciting an immune response in a human organism. 98. A nucleic acid probe for detecting natural DNA of a Rickettsia parasite, comprising at least one nucleic acid sequence selected from the group consisting of: AGTGGTCAGCAGCAA and GGTGGTCAGCAGCAA.