KR20120113581A - Monoclonal antibody specific to vibrio vulnificus rtxa1, hybridoma producing the monoclonal antibody and diagnostic kit comprising the monoclonal antibody - Google Patents

Abstract

PURPOSE: A monoclonal antibody produced from a hydridoma clone is provided to quickly and accurately diagnose, prevent and treat vibrio vulnificus sepsis. CONSTITUTION: A monoclonal antibody binds to vibrio vulnificus RtxA1 antigen prepared by hybridoma cells(deposit number KCLRF-BP-00261) and thermal-treated vibrio vulnificus RtxA1 antigen. The monoclonal antibody binds to 3981-4380th amino acid sites of vibrio vulnificus RtxA1. A composition for detecting vibrio vulnificus contains the monoclonal antibody. A diagnosing kit for detecting vibrio vulnificus contains the monoclonal antibody. A therapeutic agent for treating vibrio vulnificus contains the antibody.

Description

Monoclonal antibody specific for Vibrio septic bacterium AltiAX, hybridoma secreting it, and diagnostic kit comprising same {Monoclonal antibody specific to Vibrio vulnificus RtxA1, hybridoma producing the monoclonal antibody and diagnostic kit including the monoclonal antibody}

The present invention relates to a monoclonal antibody specific for Vibrio septic bacterium RtxA1, a hybridoma secreting the same, and a diagnostic kit comprising the same. More specifically, the present invention relates to a vibrio septic bacterium RtxA1 and a thermodenatured RtxA1. The present invention relates to a monoclonal antibody specific for the Vibrio sepsis fungus RtxA1, a hybridoma secreting the same, and a diagnostic kit including the same.

Vibrio vulnificus is a basophilic pathogenic bacterium that lives primarily in the estuary close to the sea and is associated with most seafood-related deaths. Although vibrio sepsis infection is relatively short-lived, it is one of the emerging hot spots in which clinical cases continue to increase worldwide due to global warming. In particular, absolute cases worldwide are less serious than cholera and salmonella food poisoning, but cause serious social problems due to high mortality and tragic clinical symptoms.

Vibrio sepsis was first reported in 1976 by the US Center for Disease Control (Hollis), who reported the bacteriological properties of basophilic, pathogenic Vibrio bacteria isolated from humans for 11 years. It has been named lactose-fermenting Vibrio or Lac (+) because of its ability to degrade lactose. In 1979, CDC Blake et al. Analyzed the data of 39 patients reported in the CDC and classified them into primary septicemia and wound infections according to clinical symptoms (Blake, PA). , Merson, MH, Weaver, RE, Hollis, DG, Heublein, PC, N. Engl. J. Med. 300: 1-6, 1979). In the same year, Farmer was named Vibrio vulnificus (vulnus = wound, ficus = forming) as a new species, and is coming today (Farmer, J.J. III, Lancet 2: 903, 1979).

V. vulnificus sepsis has a short incubation period and, once developed, progresses rapidly and misses the timing of effective antimicrobial treatment. V. vulnificus sepsis mostly occurs in men over 40 years old (about 90-95%) (more than 90%), rarely seen in normal people, and mainly in patients with underlying diseases. In case of primary sepsis, most patients have chronic diseases such as hepatic disease and drinking wall, and hepatic cirrhosis, chronic hepatitis, liver cancer, etc. Underlying diseases such as osteomyelitis and rheumatoid arthritis have been identified. However, in some cases, below 5%, no special underlying disease can be found. In the United States, diabetes, malignancy, hemochromatosis and thalassemia are relatively high. Recently, V. vulnificus sepsis has been reported in AIDS patients, and the US CDC recommends AIDS patients not to eat oysters or other seafood during the summer. Therefore, in order to rapidly determine the infection of Vibrio sepsis, to prevent the rapid spread of the disease, and to maximize the efficiency of the initial treatment, the development of an effective and rapid diagnosis method for early diagnosis of the infection is urgently needed.

Several virulence factors have been reported through the study of the mechanism of the development of vibrio sepsis, which began in the mid-1980s. That is, hemolytic toxin (Kreger, A, Lockwood, D, Infect. Immun. 33: 583-590, 1981), metalloproteinases (Kothary, MH, Kreger, AS, J. Gen. Microbiol. 133: 1783-1791 , 1987), phospholipase A2 (Testa, J., Daniel, LW, Kreger, AS, Infect. Immun. 45: 458-463, 1984), polysaccharide capsular (Wright, AC, Simpson, LM, Oliver JD, Morris , JG, Jr., Infect.Immun. 58: 1769-1773, 1990), Siderophore (Simpson, LM, Oliver, JD, Infect.Immun. 41: 644-649, 1983), RtxA1 (Kim YR, Lee) SE, Kook H, Yeom JA, Na HS, Kim SY, Chung SS, Choy HE, Rhee JH. Cell Microbiol 10: 848-862, 2008). Among these, RtxA1 is a major virulence factor secreted in the early stages of vibrio sepsis infection, and plays an important role in the transmission of vibrio sepsis bacteria and death of cells into the blood through the intestine. This shows that RtxA1 is a useful target for the development of new diagnostics and therapeutics for early infections.

For early diagnosis of vibrio sepsis, bacterial nucleic acid detection using polymerase chain reaction (PCR) has been used. However, nucleic acid detection using polymerase chain reaction can effectively diagnose early infection, but requires relatively expensive and skilled professionals and expensive experimental facilities. Moreover, there is a limit to diagnosis of various Vibrio sepsis strains due to the high specificity of the nucleic acid diagnostic method using a polymerase reaction. Therefore, for the early diagnosis of infection and the above-mentioned problems, there has been a continuing need for the production of monoclonal antibodies against specific antigens and the development of diagnostic agents using the same.

Furthermore, when a sample is taken and analyzed from a patient for diagnosing Vibrio sepsis, there is a problem in which the inspector is infected with Vibrio sepsis during the examination and processing.

The present invention has been made to solve the above-mentioned problems, the first problem to be solved of the present invention specifically binds to Vibrio sepsis RtxA1 and monoclonal antibodies capable of binding to high cross-reactivity and thermodenatured RtxA1 and production thereof It is to provide hybridoma cells.

A second object of the present invention is to provide a diagnostic kit comprising the monoclonal antibody of the present invention.

Vibrio sepsis RtxA1 antigen and heat-treated vibrio sepsis RtxA1 produced by hybridoma cells of Accession No. KCLRF-BP-00261 and hybridoma cells of Accession No. KCLRF-BP-00262 for solving the first problem of the present invention. Provided are monoclonal antibodies that bind to the antigen.

According to a preferred embodiment of the present invention, amino acid sites 3981 to 4380 of the Vibrio sepsis RtxA1 antigen may bind.

According to another preferred embodiment of the present invention, there is provided a composition for detecting vibrio septic bacterium comprising the monoclonal antibody described above.

In order to achieve the second object of the present invention, a diagnostic kit for detecting vibrio sepsis containing the monoclonal antibody described above is provided.

According to one preferred embodiment of the present invention, there is provided a method for detecting vibrio sepsis by contacting the monoclonal antibody with a biological sample to detect antigen-antibody complex formation.

According to another preferred embodiment of the present invention, there is provided a method for detecting vibrio sepsis by contacting the monoclonal antibody with a heat treated biological sample to detect antigen-antibody complex formation.

According to another preferred embodiment of the present invention, the detection may be an ELISA or Western blot.

According to another preferred embodiment of the present invention, it may be a Vibrio sepsis treatment comprising the antibody.

According to another preferred embodiment of the present invention, (A) immunizing a mouse using a recombinant protein of SEQ ID NO: 6 derived from Vibrio sepsis RtxA1 antigen or a fusion protein thereof; (B) fusion and culture of splenocytes of the immunized mice with myeloma cells; (C) selecting the hybridoma cells of Accession No. KCLRF-BP-00261 that produce a monoclonal antibody that binds to the Vibrio sepsis RtxA1 antigen in the fused hybridoma cells; Provided are methods for preparing bridoma cells.

Hereinafter, terms used in this specification will be briefly described.

Unless stated otherwise, the term 'nucleic acid' refers to deoxyribonucleotides or ribonucleotides in single- or double-helix form, and inherently nucleotides that bind to nucleic acids in a manner similar to naturally occurring nucleotides.

Known analogs of tide, and unless otherwise indicated, certain nucleic acid sequences include their complementary sequences.

'Operatively linked to' means a functional binding between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, thereby The regulatory sequence will control the transcription and / or translation process of said other nucleic acid sequence.

The term 'recombinant' as used in connection with a cell refers to the cell's replicating a heterologous nucleic acid or expressing a peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can also express genes found in the cell's original form, but modified genes can be reintroduced into cells by artificial methods.

'Primer' means a synthetic or natural oligonucleotide. The primer serves as an initiation point for the synthesis under conditions in which the synthesis of the primer extension product complementary to the template is induced, i.e. the presence of polymers such as nucleotides and DNA polymerase, and conditions of suitable temperature and pH. For maximum efficiency of amplification, preferably the primer is single stranded. Preferably, the primer is a deoxyribonucleotide. The primers of the present invention may comprise naturally occurring dNMPs (i.e. dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides. For example, oligonucleotides of the present invention may be selected from the group consisting of backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohex Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Thityl-, propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro- , Ah Iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazoryl-, pyridyl-), inosine and diaminopurine.

'Vector' refers to a DNA molecule as a carrier that can stably transport foreign genes into a host cell. To be a useful vector, it must be replicable, have a way to enter the host cell, and have a means to detect its presence. The foreign gene herein refers to a sequence encoding the 3491-4701 amino acid region of the Vibrio septic bacterium RtxA1 antigen or a sequence in which a His-tag sequence is added to the end of the sequence to facilitate purification.

'Plasmid' generally refers to a circular DNA molecule formed by operably linked to a vector so that a foreign gene can be expressed in a host cell. However, the plasmid can be used as a vector which is degraded by specific restriction enzymes and introduces a new gene by gene recombination to construct a plasmid containing the gene of interest. Thus, plasmids and vectors are used interchangeably herein, and those skilled in the art of genetic engineering will fully understand their meanings even if they do not distinguish their names.

The "fusion protein" refers to the Vibrio pulmonary fungus RtxA1, whose His-tag is attached to the C-terminus for the convenience of protein purification. it means. Therefore, although expressed as "fusion protein" in the present invention, it does not necessarily mean that His-tag is attached, and may not use a fusion protein if you take the inconvenience of protein purification. Therefore, in the present invention, the term "recombinant Vibrio pneumococcal RtxA1" is defined as "Vibrio pneumococcal RtxA1" or "Vibrio pneumococcal RtxA1 with a chain added for N-terminal or C-terminal for convenience of purification".

The term 'monoclonal antibody' of the present invention means a highly specific antibody directed against a single antigenic site (epitope) as it is known in the art. Typically, unlike polyclonal antibodies that include different antibodies directed against different epitopes, monoclonal antibodies are directed against a single epitope on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage that is not contaminated by other immunoglobulins because it is produced by the culture of hybridomas. .

As used herein, the term "biological sample" includes tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, urine, secretions, and the like. These biological samples can be reacted with the antibodies of the invention without heat treatment or manipulation.

As used herein, the term "antigen-antibody complex" refers to a combination of the monoclonal antibody with the Vibrio pneumoniae RtxA1 protein reacted to confirm the expression of Vibrio pneumoniae RtxA1 in a biological sample.

Recombinant RtxA1 (3491-4701) according to the present invention provides a recombinant RtxA1 protein antigen which is very useful for the production of monoclonal antibodies of Vibrio pneumococcal RtxA1. It also provides a recombinant RtxA1 (3491-4701) protein antigen which is very useful for diagnosis with the Vibrio pneumococcal RtxA1, and facilitates the mass expression and purification of the recombinant RtxA1 (3491-4701) protein antigen.

Monoclonal antibodies produced in the hybridoma clone according to the present invention is excellent in sensitivity and specificity, can be very useful for rapid and accurate diagnosis and prevention of vibrio sepsis infection. In addition, the recombinant protein RtxA1 antigen expressed in Escherichia coli is excellent in sensitivity and specificity, and can be very useful for diagnosing and preventing infection of Vibrio sepsis.

Meanwhile, since the monoclonal antibody of the present invention is excellent in cross-reactivity to other types of vibrio sepsis RtxA1 and can also bind to heat-treated vibrio sepsis, the examiner heat-processes the biological sample infected with Vibrio sepsis and vibrio sepsis. It is very safe because it can kill the bacteria and eliminate the risk of infection and then diagnose the infection.

1 is a cleavage map of a recombinant RtxA1 (3491-4701) expression vector introduced with recombinant RtxA1 (3491-4701) according to a preferred embodiment of the present invention.
2A shows the results of confirming the expression and purification of recombinant RtxA1 (3491-4701) of Vibrio sepsis by 10% SDS-PAEG.
2B shows the results of analyzing the specificity of purified recombinant RtxA1 (3491-4701) protein by immunoblot.
Figure 3 shows the results of analysis of purified 5RA monoclonal antibody with 10% SDS-PAEG.
4A schematically shows a recombinant RtxA1 (3491-4701) fragment protein expressed in transgenic E. coli.
4B shows the results of confirming the expression of recombinant RtxA1 (3491-4701) fragment protein with 10% SDS-PAEG.
4C shows the results of analyzing the specificity of the expressed recombinant RtxA1 (3491-4701) fragment protein by immunoblot.
FIG. 5 shows the results of immunoblot analysis of reactivity of monoclonal antibodies (1RA, 5RA, 11RA) against wild species Vibrio sepsis.
FIG. 6 shows the results of immunoblot analysis of cross-reactions against various strains of vibrio sepsis of monoclonal antibodies (5RA and 11RA).
7 shows the results of analysis of the reactivity of monoclonal antibodies (1RA, 4RA, 5RA, 11RA) in the cells infected with Vibrio sepsis by immunofluorescence antibody method.

Hereinafter, the present invention will be described in more detail.

The present inventors immunized mice using recombinant RtxA1 antigen expressed in E. coli, which was determined as a specific toxin antigen secreted early in Vibrio infection, and splenocytes and B-lymphoblasts of immunized mice. The present invention was completed by preparing a hybridoma clone fused with phosphorus P3X63Ag8.653 and producing monoclonal antibody against the Vibrio sepsis RtxA1 antigen therefrom. The monoclonal antibody according to the present invention showed excellent reactivity with Vibrio sepsis RtxA1 antigen. In particular, some monoclonal antibodies showed cross-reactivity to thermodenatured RtxA1 antigen and several Vibrio sepsis strains. This shows that the monoclonal antibody of the present invention is a diagnostic antibody that can be used for various diagnostic methods, such as indirect immunofluorescent antibody, immunoblot blot, and enzyme immunosorbent antibody method. In addition, since the monoclonal antibody of the present invention can be used as a neutralizing antibody of the virulence factor RtxA1, it can be used as a therapeutic agent.

Meanwhile, each monoclonal antibody produced in 10 hybridoma cells fused using recombinant RtxA1 protein as an immunogen was bound to amino acids 3491-3980 according to the amino acid binding site of RtxA1 of Vibrio sepsis. For example, monoclonal antibodies that bind to amino acids 3981-4380 and monoclonal antibodies that bind to amino acids 4381-4701 may be classified. Among these, 5RA (binding to the 3981-4380 amino acids), 11RA are summarized in terms of mass production potential according to isotype, reactivity to heat-denatured antigens, affinity with RtxA1 antigen, and cross-possibilities for other types of vibrio sepsis. (Binding to amino acids 3491-3980) showed the best effect. Therefore, 5RA was deposited with the Korean Cell Line Research Foundation under the accession number KCLRF-BP-00261, and 11RA was deposited under the accession number KCLRF-BP-00262 (see Table 1).

Monoclonal antibodies can be made by fusion methods well known in the art (Kohler et al., European Journal of Immunology 6; 511-519). In general, hybridoma cells secreting monoclonal antibodies are made by fusing cancer cell lines with immune cells from immunologically suitable host animals such as mice injected with antigenic proteins. These two populations of cell fusion can be fused using methods known in the art, such as polyethylene glycol, and the antibody producing cells can be propagated by standard culture methods. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing antibodies specific for the antigen can be cultured in large quantities in vitro or in vivo.

The monoclonal antibodies of the present invention can be used without purification, and also in high purity using various conventional methods such as dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like. It can be used by purification.

Specifically, according to a preferred embodiment of the present invention, the sequence represented by SEQ ID NO: 3 encoding the Vibrio septic bacterium RtxA1 protein of SEQ ID NO: 4 is used as a template and is located at positions 3491 to 4701 in the amino acid sequence of Vibrio septic bacterium RtxA1. PCR is performed with the front and rear primers of the novel primer sequences SEQ ID NOs: 1 and 2 to selectively amplify the amino acid sites. Then, the amino acid sequence of the amplified Vibrio septic bacterium RtxA1, which encodes an amino acid at positions 3491 to 4701 (SEQ ID NO: 5) or a conventional His-tag sequence is added to the end of the sequence to facilitate purification. One sequence is inserted into a plasmid vector and transformed into a host cell. Thereafter, the expressed protein is purified, injected into the abdominal cavity of the mouse, and splenocytes are extracted, fused with myeloma cells, and cultured to obtain a plurality of hybridoma cells. Since then, the monoclonal antibodies produced from the hybridoma cells were evaluated comprehensively for mass production potential, cross-reactivity, and binding ability of the heat-treated Vibrio sepsis RtxA1 protein, and the best 5RA and 11RA were respectively assigned to the Korea Cell Line Research Foundation. Deposited as -BP-00261 and KCLRF-BP-00262. The 5RA monoclonal antibody thus produced can specifically bind to the amino acid positions 3981-4380 of the amino acid sequence of Vibrio sepsis RtxA1.

Hybridomas secreting monoclonal antibodies can be cultured in large quantities in vitro or in vivo. The monoclonal antibodies produced by the hybridomas may be used without purification, but for best results, the monoclonal antibodies may be purified with high purity (eg, 95% or more) according to methods well known in the art. It is preferable to use. Such purification techniques can be separated from the culture medium or ascites fluid using, for example, purification methods such as dialysis, salt precipitation, chromatography and the like.

The monoclonal antibodies of the present invention bind to antigens to inhibit or neutralize their actions, and further kill pathogens and cells infected with pathogens. For example, a monoclonal antibody that specifically recognizes RtxA1, a toxin portion of Vibrio pneumococcus, can bind to and inhibit or neutralize its action, and the antibody to which the antigen is bound can activate complement. Complement may allow the antigen to be removed. Monoclonal antibodies can also bind to antigens, making them more readily consumed by phagocytic cells. In addition, antigen cells to which monoclonal antibodies are bound are more easily killed by natural killer cells (NK cells), so that the monoclonal antibodies can be used to remove antigens through an immune response and vibrio sepsis that produce these antigens. Can be. Therefore, the antibody alone can be expected to result in the removal and reduction of antigens and vibrio pneumococci through the immune response, and thus the antibody of the present invention can be used for diagnosis or treatment of vibrio pneumococci.

Those skilled in the art will readily appreciate that the monoclonal antibodies according to the present invention can be converted to chimeric antibodies, humanized antibodies and human monoclonal antibodies having reduced immunogenicity for application in the human body. Such chimeric antibody is a recombinant region of the monoclonal antibody of the present invention with the constant region of the human antibody, the humanized antibody complementary determining region (complementarity determining) that directly binds to the antigen in the variable region of the monoclonal antibody of the present invention Regions (CDRs) or only specificity determining residues (SDRs), which are involved in antigen binding specificity, are implanted in human antibodies.

The human antibody replaces the heavy or light chain variable region of the variable region of the monoclonal antibody of the present invention instead of the heavy or light chain variable region of the human antibody, and as a result, a hybrid (mouse heavy / human light chain) showing antigen binding ability Alternatively, the mouse light chain / human heavy chain) antibody is replaced with a human heavy or light chain variable region, and then a human human variable region showing antigen-binding ability is selected and linked to the human antibody constant region. It can be easily prepared from the monoclonal antibody of the present invention using a known method such as such, it is natural that such a variant is within the scope of the present invention. Chimeric antibodies, humanized antibodies and human monoclonal antibodies prepared as described above can be produced in animal cells using known methods.

In addition, the monoclonal antibody of the present invention, as long as it has the characteristics of binding as described above, is not only a complete form having the full length of two heavy chains and two light chains, but also a functional fragment of an antibody molecule, for the treatment and diagnosis of Vibrio pneumonia. Can be used. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and can include Fab, F (ab '), F (ab') 2, Fv, etc.

According to a preferred embodiment of the present invention, the present invention provides a vibrio sepsis diagnostic kit comprising the monoclonal antibody described above. As the monoclonal antibody used in the diagnostic kit for the detection of vibrio sepsis of the present invention, a fragment of the monoclonal antibody can be used as long as the antibody can selectively recognize the RtxA1 antigen of vibrio sepsis. Such antibody fragments may include F (ab ') 2, Fab, Fab', Fv fragments, and the like.

Vibrio sepsis diagnostic kits of the invention may include monoclonal antibodies or fragments thereof that selectively recognize the RtxA1 antigen and tools / reagents used for immunological analysis.

Tools / reagents for use in immunological assays can include suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, Fluorine

Resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

Assay systems for use in detection methods and diagnostic kits for antigen-antibody complex formation of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography test strips and radiation split immunoassay devices, and Flow-through devices and the like. Preferably, antigen-antibody complex formation is performed by tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, Complement Fixation Assays, FACS, protein chips, and the like.

The pharmaceutical composition comprising the monoclonal antibody of the present invention may be formulated or used in combination with drugs such as antihistamines, anti-inflammatory analgesics and antibiotics that are already in use.

The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Oligonucleotide Synthesis for Recombinant RtxA1 Synthesis of Vibrio Sepsis

 The following two oligonucleotides with NheI and XhoI restriction enzyme recognition sites were synthesized. In addition, RA-4701R oligonucleotide contains six histidines for further purification by chromatographic chromatography using histidine-binding resin in the purification of recombinant RtxA1.

RA-3491F: 5'-ACATGAATTCATGCAGAGAAGTTTGGCGACTAC-3 '(SEQ ID NO: 1, anterior primer)

RA-4701R: 5'-CTCCTCGAGCTAATGATGATGATGATGATGCACCGTTATACCCTTTTTATG-3 '(SEQ ID NO .: 2, rear primer)

The nucleotides were used for the subsequent amplification of the Vibrio sepsis RtxA1 gene (Example 2 below) and the production of recombinant vectors for recombinant RtxA1 protein production.

Example 2 Polymerase Chain Reaction (PCR)

Vibrio septic bacterium RtxA1 full-length DNA (SEQ ID NOs: 3, 14106 bp) was used as a template in a reaction solution mixed with a polymerase and a front primer (SEQ ID NO: 1) and a rear primer (SEQ ID NO: 2) synthesized in Example 1 above. The polymerase chain reaction was performed by mixing to amplify the amino acid sequence 3491 to 4701 of vibrio sepsis RtxA1 (named RtxA1 (3491-4701)). SEQ ID NO: 4 is the amino acid sequence of the full length Vibrio sepsis RtxA1. Thermal cycler (PTC-100), MJ Research. Inc., USA was used for neutralization chain reaction. After 3 minutes of predenaturation at 94 ° C, denaturation at 94 ° C for 1 minute, Annealing at 55 ° C. for 1 minute, and extension at 4 ° C. for 4 minutes at 30 ° C. were repeated 30 times, and finally further reacted at 72 ° C. for 10 minutes. Next, to remove the polymerase and non-specific amplification products, the desired amplification product band region after electrophoresis on the agarose gel was fragmented and purified using a gel extraction kit (Geneall, Korea).

Example 3 Preparation of Recombinant RtxA1 Preparation Vector

A recombinant RtxA1 plasmid vector having the cleavage map of FIG. 1 was constructed. Specifically, the amplified product of RtxA1 (3491-4701) inserted with his-tag sequence CATCATCATCATCATCAT at the C-terminal purified in Example 2 was digested with EcoRI (Fermentas, Canada) and then E. coli polymerase Klenow (New England) Biolab, USA) was used to fill the cuts (fill in) and cut with XhoI (Fermentas, Canada). In addition, the pET-21 (a) (Novagen, USA) vector was digested with NheI (Fermentas, Canada), and then filled in with the cut surface using E. coli polymerase Klenow (New England Biolab, USA), and XhoI (Fermentas, Canada). The cleaved fragment and the vector were purified using the gel extraction kit (Geneall, Korea) used in Example 2, and then mixed with 1 μg of the purified RtxA1 gene fragment and 30 ng of the pET-21 (a) fragment, and 10-fold. 1 μl of conjugation concentrate (10 mM DTT, 100 mM MgCl 2, 10 mM ATP, 600 mM Tris-HCl, pH7.5) and 10 units of T4 DNA ligase (Takara) were added to the total volume. Adjusted to μl and reacted at room temperature for 16 hours, a circular plasmid (pET-) having a cleavage map of 9,027bp in which the recombinant RtxA1 gene (SEQ ID NO: 5 + His-tag sequence) was inserted into the pET-21 (a) vector. RtxA1 (named 3491-4701). The plasmid was then amplified and inserted into E. coli DH5α (Invitrogen, USA) for pure separation of only pET-RtxA1 (3491-4701) into which the RtxA1 (3491-4701) gene (SEQ ID NO: 5) was inserted. Re-extracted recombinant pET-RtxA1 (3491-4701) from the transformed E. coli, DNA sequencing, restriction enzyme treatment and agarose gel electrophoresis to pET-RtxA1 (3491-4701) for Vibrio sepsis RtxA1 (3491) -4701) The presence of the gene was confirmed.

Example 4 Expression of Recombinant RtxA1 (3491-4701) Protein

For expression of the recombinant RtxA1 vector pET-RtxA1 (3491-4701) prepared in Example 3 was inserted into E. coli BL21 (DE3) / pLy S (Invitrogen, USA). The transformed Escherichia coli was incubated for 4 hours in a 37 ℃ shaking incubator, followed by further incubation for another 3 hours by adding 1 mM IPTG. The cultured transformed E. coli was centrifuged at 8,000 rpm for 30 minutes, and then the E. coli precipitates were eluted with elution solution (300 mM NaCl, 25 mM Tris-HCl (pH 7.5), 10% glycerol, 0.1% Tween-20). Suspended. E. coli sediment suspended for protein extraction was disrupted by using a sonicator, and then the superior protein extracts were collected by centrifugation, and 10% SDS-PAGE was performed on the cell disruptions (FIG. 2A). The molecular weight in FIG. 2A is shown today on FIG. 2A, and lanes 1 and 2 are proteins extracted before and after expressing the recombinant RtxA1 protein in E. coli transformed with the recombinant RtxA1 vector pET-RtxA1 (3491-4701), respectively.

 Arrows indicate the location of the expressed recombinant RtxA1 (3491-4701) protein. As can be seen in Figure 2A it can be seen that the recombinant RtxA1 (3491-4701) protein is expressed in E. coli transformed with pET-RtxA1 (3491-4701).

Example 5 Purification of Recombinant RtxA1 (3491-4701) Protein

After extracting the protein by the method of Example 4 in Escherichia coli transformed with the vector pET-RtxA1 (3491-4701) for the production of recombinant RtxA1 (3491-4701), Ni-NTA chromatography (Qiagen, USA) was subjected to manufacturer's instructions. Recombinant RtxA1 (3491-4701) protein was first purified from the protein extract. Recombinant RtxA1 (3491-4701) protein first purified using Ni-NTA chromatography was purified secondarily using gel filtration liquid chromatography (size exclusion-HPLC) (GE Healthcare, USA). 3491-4701) was produced (SEQ ID NO: 6 + Hi-tag). Purification modalities and purification efficiencies were confirmed by performing SDS-PAGE (FIG. 2A). Lanes 3 and 4 in FIG. 2A are purified proteins in Ni-NTA chromatography and gel filtration liquid chromatography, respectively, and arrows indicate positions of purified recombinant RtxA1 (3491-4701) proteins. Purified recombinant RtxA1 (3491-4701) protein, as identified in lane 4 of FIG. 2A, showed a purity of 98%.

Example 6 Specificity Analysis of Recombinant RtxA1 (3491-4701) Protein

SDS-PAGE was performed on the recombinant RtxA1 (3491-4701) protein and the control bovine serum albumin (BSA) protein of 130 kDa identified in Example 5, and the gel phase protein of Tobin (Towbin) after electrophoresis was terminated. Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA 1979; 76: 4350-4354) were transferred to Nitrocellulose membrane (Bio-Rad). The protein-transferred membrane was blocked with a nonspecific reaction with a phosphate buffer solution containing 5% skim milk. Herein, the recombinant RtxA1 antigen protein of Example 7 was mixed with Sigma adjuvant in the same amount (1: 1 by volume), and then injected into the abdominal cavity of BALB / C (female, 8-week old) mice four times at three-week intervals. One month later, the polyclonal antibody against Vibrio pneumococcal RtxA1 protein obtained from mouse serum was diluted 1: 1000 and reacted at room temperature for 1 hour, and peroxidase-labeled IgG (Jackson labatory, USA) Secondary antibodies were used diluted in accordance with the manufacturer's instructions. After reacting with the primary and secondary antibodies, it was washed three times with phosphate buffer solution. After the secondary antibody reaction and washing, the membrane was treated with ECL western blot substrate solution (Amersham, USA), and the results were analyzed using a LAS-1000 luminescent image analyzer (Fujifilm, Japan). (FIG. 2B). Lane 1 in FIG. 2B is the control bovine serum albumin (BSA) protein and lane 2 is the recombinant RtxA1 (3491-4701) protein. As shown in FIG. 2B, the recombinant RtxA1 (3491-4701) protein showed a positive response to the antibody against the Vibrio sepsis RtxA1 protein, but did not respond to the control right iron albumin. Therefore, it was confirmed that the 130 kDa purified protein purified in Example 5 was RtxA1 (3491-4701) protein.

In conclusion, the recombinant RtxA1 (3491-4701) according to the present invention provides a recombinant RtxA1 protein antigen which is very useful for the production of monoclonal antibodies of the Vibrio pneumococcal RtxA1. It also provides a recombinant RtxA1 (3491-4701) protein antigen which is very useful for diagnosis with the Vibrio pneumococcal RtxA1, and facilitates the mass expression and purification of the recombinant RtxA1 (3491-4701) protein antigen. Therefore, the recombinant RtxA1 (3491-4701) protein expressed in the transformed Escherichia coli may be used for the production of a diagnostic or diagnostic kit for Vibrio sepsis, a vaccine for preventing or treating Vibrio sepsis, and the production of antibodies to Vibrio sepsis. Can be.

Example 7 Mouse Immunity for Monoclonal Antibody Production Cell Line Construction

RtxA1 (3491-4701) recombinant antigen protein (SEQ ID NO: 6 + His tag) prepared in Example 5 was mixed with Sigma adjuvant (Sigma, USA) in the same amount (1: 1 by volume) and then 4 times at 3 week intervals. 200 μL was injected into the abdominal cavity of BALB / C (female, 8 week old) mice. One month after the fourth immunization, three days after administration of the purified recombinant RtxA1 (3491-4701) protein into the tail vein, the spleen was extracted and used for cell fusion.

Example 8 Culture of P3X63Ag8.653 as B-lymphoblast

Four to five days prior to cell fusion, myeloma cells P3X63Ag8.653 (ATCC accession number CRL-1580) were removed from the nitrogen bottle and suspended in IMDM (Invitrogen, USA) medium supplemented with 10% fetal bovine serum and 5 minutes at 1,000 rmp. Centrifuged. The supernatant was discarded and the precipitated cells were carefully resuspended in IMDM with 10% fetal calf serum and incubated in a 37 ° C. incubator fed with 5% carbon dioxide gas.

Example 9 Cell Fusion

Cell fusion was performed according to the general method using polyethylene glycol (Ed Harlow, David Lane: Antibodis, A laboratory manual. Cold Springs Harbor press, 1988 P139-244). Splenocytes were isolated from the spleen extracted in Example 7 using a cell strainer (Falcon, USA) and diluted to a concentration of 1 × ^{10 8} cells / ml. 1x10 ⁷ cells / ml of myeloma cells and 1ml of PEG1500 (Sigma, USA) were mixed and fused in equal amounts. After completion of the fusion, the cells were diluted in 200 ml of HAT (Sigma, USA) medium, and then, 100 µl was dispensed into 96-well microplates, and cultured in a 37 ° C. incubator supplied with 5% carbon dioxide.

<Example 10> RtxA1 (3491-4701) antibody production investigation in cell fusion hybridoma

Antibody production of the fused cells was examined by ELISA using a cell culture fused with recombinant RtxA1 (3491-4701) antigen expressed in E. coli. Recombinant RtxA1 (3491-4701) antigen protein was diluted to 10 μg / ml concentration in carbonate buffer (pH 9.4) and then 100 μl per well of Maxisorp ELISA plate (Nunc, USA) added at 4 ° C. The reaction was coated for 16 hours at. Each antigen-coated well was treated with blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) to block non-specific reactions at 37 ° C. for 1 hour. It was. 50 μl of the cell culture solution fused to each well was added and reacted at 4 ° C. for 1 hour, and then washed three times with a washing buffer solution (PBS, 0.05% Tween-20, 0.05% BSA). After washing, 100 μl of Biotin-condensed anti-mouse IgG + IgA + IgM antibody (1: 2,000; Sigma, USA) diluted 1: 1000 times was added to each well and reacted at 37 ° C. for 1 hour, followed by washing with buffer. Wash three times. After washing, 100 μl of HRP (Horseradish peroxidase) -condensed streptavidin (Invitrogen, USA) was added to each well and reacted at 37 ° C. for 1 hour, followed by washing three times with washing buffer solution, followed by TMB (3,3 ′, 100 μl of 5,5′-tetramethyl-benzidine) (Sigma, USA) substrate solution was added to each well and allowed to react for 30 minutes in the dark for color development, followed by 2N H ₂ SO ₄ treatment to stop the enzymatic reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. As a result, 10 hybridomas were reacted with the Vibrio sepsis RtxA1 (3491-4701) antigen (Table 1).

Example 11 Cloning of Hybridoma Cells

For cloning of 10 hybridoma cells fused, hybridomas reacting with the Vibrio sepsis RtxA1 (3491-4701) antigen obtained in Example 5 were allowed to enter about 10 cells in the first well of a 96 well plate. After the suspension was added, two-fold dilution was performed in rows, and the resultant was cloned twice by diluting it twice in a column. Selected hybridoma clones were stored in liquid nitrogen suspended in IMDM medium containing 30% fetal calf serum and 7.5% dimethyl sulfoxide (DMSO).

Example 12 Purification of Monoclonal Antibodies

The 10 cloned hybridoma cells producing the antibody were cultured in a 37 ° C., 5% carbon dioxide gas incubator, and the antibodies were purified from the supernatant. Purification of the antibody was performed using protein A or G chromatography (Peptron, Korea), the experiment was carried out according to the manufacturer's method. After purification, SDS-PAGE was performed, and the degree of purification was found to be 98% or higher (FIG. 3). Lane 1 of FIG. 3 is labeled with protein standard molecular weight, and lane 2 shows the purified antibody in Protein A chromatography.

Example 13 Screening for Monoclonal Antibodies by Isotyping

Determination of isotypes of monoclonal antibodies from selected 10 hybridoma clones was examined by ELISA. For isotype determination, purified antibodies to each mouse isotype fished in rabbits were diluted to a concentration of 10 μg / ml in carbonate buffer (pH 9.4), followed by Maxisorp ELISA plate (Nunc, USA). 100 μl of each well was added and the reaction was carried out at 4 ° C. for 16 hours to coat the antibody. Each well coated with each isotype antibody was treated with blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) for 1 hour at 37 ° C. Specific reactions were blocked. 50 μl of the cell culture solution fused to each well was added and reacted at 4 ° C. for 1 hour, and then washed three times with a washing buffer solution (PBS, 0.05% Tween-20, 0.05% BSA). HRP (Horseradish peroxidase) -condensed anti-mouse IgG + IgA + IgM antibody (1: 2000; Sigma, USA) diluted 1: 1000 after actual washing was added to each well and reacted for 1 hour at 37 ° C. It was then washed three times with a wash buffer solution. After washing, 100 µl of TMB (3,3 ', 5,5'-tetramethylbenzidine) (Sigma, USA) substrate solution was added to each well, and reacted for 30 minutes in the dark, followed by color development, followed by 2N H2SO4. I was. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. As a result of determining the isotype, as shown in Table 1 below, when classified according to the heavy class subclass, six were IgG1 subclass, three were IgG2a subclass, and the other was IgG2b subclass. In general, IgM antibodies have the characteristic of nonspecific adsorption with components other than solid surface or target antigen used for the development of diagnostics. Therefore, all ten monoclonal antibodies obtained through the present invention were selected as antibodies that can be used as diagnostic monoclonal antibodies. In the following experiments, the antibody was characterized using only this antibody.

Example 14 Expression of RtxA1 (3491-4701) Fragments for Affinity Analysis and Epitope Analysis of Monoclonal Antibodies

The recombinant RtxA1 (3491-4701) protein including amino acid sequences 3491 to 4701 of vibrio sepsis RtxA1 was used as expressed in Example 4. The gene regions of RtxA1 (3491-4380) comprising amino acid sequences 3491 to 4380 of Vibrio sepsis RtxA1 are oligonucleotides and polymerases of two of the following (RA-3491F2 and RA-4308R) having EcoRI and XhoI restriction enzyme recognition sites; Amplification was carried out in the same manner as in Example 2 using the chain reaction.

RA-3491F2: 5´-ACATGAATTCATACCATGGCAGAGAAGTTTGGCGACTAC-3´ (SEQ ID NO: 7, anterior primer)

RA-4380R: 5´-CCATTCTCGAGCTAATGATGATGATGATGATGCGTGCCTGTTGCGTAGAACAC-3´ (SEQ ID NO: 8, rear primer)

In addition, the gene regions of RtxA1 (3491-3980) including amino acid sequences 3491 to 3980 of RtxA1 are oligonucleotides and polymerase chain reaction of two of the following (RA-3491F2 and RA-3980R) having EcoRI and XhoI restriction enzyme recognition sites. Was amplified in the same manner as in Example 2.

RA-3491F2: 5′-ACATGAATTCATACCATGGCAGAGAAGTTTGGCGACTAC-3´´ (SEQ ID NO: 9, anterior primer)

RA-3980R: 5´-CCATTCTCGAGCTAATGATGATGATGATGATGCTCACCCGAGGTGGCAATGC-3´ (SEQ ID NO: 10, Rear Primer)

The amplified RtxA1 (3491-4380) and RtxA1 (3491-3980) gene regions were inserted into the pET-21 (a) vector in the same manner as in Example 3, and transformed E. coli was transformed using the same method as in Example 4. Recombinant RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins were expressed and extracted (FIG. 4B). Specificity analysis of the extracted recombinant protein was confirmed using the same method as in Example 6 (Fig. 4C). 4A schematically shows a recombinant RtxA1 (3491-4701) fragment protein expressed in hemolytically converting Escherichia coli. In FIG. 4B, the molecular weights are indicated on the left side of the diagram, and lane 1, lane 2, lane 3 and lane 4 are control recombinant proteins, recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491- 3980) is a protein extracted from transgenic E. coli. Arrows indicate the positions of the expressed recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), RtxA1 (3491-3980) proteins. In FIG. 4C, specificity of recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins was analyzed using antibodies against the Vibrio sepsis RtxA1 protein. The molecular weight size is shown on the left side of the diagram, and lane 1, lane 2, lane 3 and lane 4 represent control recombinant proteins, recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980), respectively. It is a protein extracted from the transforming Escherichia coli. The arrows indicate the positions of the RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins that the antibody to the RtxA1 protein is positive. 4B and 4C, recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) were identified.

Example 15 Affinity Analysis and Determinant Analysis of Monoclonal Antibodies

Affinity analysis and epitope analysis of monoclonal antibodies used as useful information for the selection of diagnostic and therapeutic monoclonal antibodies were performed by fragments of recombinant RtxA1 (3491-4701) identified in Example 14 and ELISA. Transgenic Escherichia coli extracts expressing recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins were each diluted with carbonate buffer (pH 9.4) and then Maxisorp ELISA plate (Nunc). 100 μl per well of US), and reacted at 4 ° C. for 16 hours to coat each recombinant protein. Each well coated with each recombinant protein was treated with blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) and non-specific at 37 ° C. for 1 hour. The reaction was blocked. 50 μl of the cell culture solution containing monoclonal antibody was added to each well, followed by reaction at 4 ° C. for 1 hour, and then washed three times with washing buffer (PBS, 0.05% Tween-20, 0.05% BSA). After washing, 100 μl of Biotin-condensed anti-mouse IgG + IgA + IgM antibody (1: 2,000; Sigma, USA) diluted 1: 1000 times was added to each well, and reacted at 37 ° C. for 1 hour, followed by washing with buffer. Wash three times. After washing, 100 μl of HRP (Horseradish peroxidase) -condensed streptavidin was added to each well, and reacted at 37 ° C. for 1 hour, followed by washing three times with washing buffer solution, followed by TMB (3,3 ', 5,5'-). 100 μl of tetramethyl-benzidine) (Sigma, USA) substrate solution was added to each well, followed by reaction for 30 minutes in the dark, followed by 2N H ₂ SO ₄ treatment to stop the enzymatic reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader (Table 1). As a result of affinity analysis and epitope analysis of monoclonal antibodies, five (5RA, 7RA, 8RA, 9RA, 11RA) strongly recognized the recombinant protein RtxA1 (3491-4701) antigen as summarized in Table 1. In contrast, the 1RA and 10RA monoclonal antibodies recognized moderate levels of RtxA1 (3491-4701) antigens, while the 2RA, 3RA and 4RA monoclonal antibodies weakly recognized RtxA1 (3491-4701) antigens. The 2RA, 3RA, 8RA and 11RA monoclonal antibodies recognized amino acid sequences 3491 to 3980 parts of RtxA1, and the 1RA, 5RA, 7RA and 9RA monoclonal antibodies bound to amino acid sequences 3981 to 4380 parts of RtxA1. Monoclonal antibodies at 4RA and 10RA recognized amino acid sequences 4381 to 4701 of RtxA1. Therefore, the present invention was able to secure three groups of monoclonal antibodies with different epitopes, and the monoclonal antibodies of each group could be usefully used for basic research on vibrio sepsis as well as the development of antigenic diagnostic agents for vibrio sepsis. have.

Example 16 Reactivity Analysis of Monoclonal Antibodies to Heat Denatured RtxA1 (3491-4701) Antigens.

Monoclonal antibodies specific for specific antigens can be used to develop diagnostic agents that detect antigens under denaturing conditions, such as high temperatures in infected specimens. In the present invention, the recombinant RtxA1 (3491-4071) antigen was heat-treated at 100 ° C. for 3 minutes to obtain a thermodenatured RtxA1 (3491-4701) antigen, and then subjected to ELISA to investigate the reactivity of each monoclonal antibody. Heat-denatured recombinant RtxA1 (3491-4701) antigen was diluted with carbonate buffer (pH 9.4), then 100 μl of each well of Maxisorp ELISA plate (Nunc, USA) was added and reacted at 4 ° C. for 16 hours. Each recombinant protein was coated. Each well coated with each recombinant protein was treated with blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) and non-specific at 37 ° C. for 1 hour. The reaction was blocked. 50 μl of the cell culture solution containing monoclonal antibody was added to each well, followed by reaction at 4 ° C. for 1 hour, and then washed three times with washing buffer (PBS, 0.05% Tween-20, 0.05% BSA). After washing, 100 μl of the Biotin-condensed anti-mouse IgG + IgA + IgM antibody (1: 2,000, Sigma, USA) diluted 1: 1000 times was added to each well and reacted at 37 ° C. for 1 hour, followed by washing with buffer. Wash three times. After washing, 100 μl of HRP (Horseradish peroxidase) -condensed streptavidin was added to each well, and then reacted at 37 ° C. for 1 hour, followed by washing three times with a washing buffer solution, followed by TMB (3,3 ', 5,5'-). 100 μl of tetramethyl-benzidine) (Sigma, USA) substrate solution was added to each well and allowed to react for 30 minutes in the dark for color development, followed by 2N H 2 SO 4 treatment to stop the enzymatic reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. Response analysis of monoclonal antibodies against the heat-denatured RtxA1 (3491-4701) antigens of each monoclonal antibody revealed that 1RA, 2RA, 3RA, 5RA, 7RA, 8RA, 9RA and 11RA were opened as shown in Table 1. Denatured RtxA1 (3491-4701) antigens were recognized, but 4RA and 10RA did not recognize heat denatured RtxA1 (3491-4701) antigens. Therefore, the monoclonal antibody obtained in the present invention may be usefully used for the development of a diagnostic agent for detection of denatured antigen after high-temperature treatment of a test sample.

Example 17 Reactivity Analysis of Monoclonal Antibodies to Wild Species Vibrio Sepsis.

Immunoblot was performed to analyze the reactivity of the monoclonal antibody against the RtxA1 toxin protein produced in wild vibrio sepsis. The wild type Vibrio sepsis M06-24 / O strain (Reddy GP, Hayat U, Abeygunawardana C, Fox C, Wright AC, Maneval DR, Jr., Bush CA, Morris JG, Jr. J Bacteriol 1992; 174: 2620-2630 ) Inoculated in HI medium containing 2.5% NaCl and incubated for 16 hours in a 37 ℃ shaking incubator. The culture solution was inoculated at 1/200 in HI medium containing 2.5% NaCl, and then cultured for 2 hours in a 37 ° C. shaking incubator. The culture medium thus obtained was centrifuged at 12,000 rpm for 10 minutes, and then 300 μl of the supernatant and 1,200 μl of cold acetone (Sigma, USA) were mixed and treated at −20 ° C. for 30 minutes. The culture supernatant and acetone mixture treated for 30 minutes were centrifuged at 12,000 rpm for 15 minutes, and then precipitates were collected. The precipitate was eluted in 20 μl 2 × sample buffer and subjected to 10% SDS-PAGE. After completion of SDS-PAGE, the gel phase protein was transferred to Nitrocellulose membrane (Bio-Rad, USA) according to Tobin's method (Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA 1979; 76: 4350-4354). Moved to. The protein-transferred membrane was blocked with a nonspecific reaction with a phosphate buffer solution containing 5% skim milk. It was reacted for 1 hour at room temperature using a Vibrio septic bacterium RtxA1 monoclonal antibody diluted to 50 μg / ml, and the peroxidase-labeled IgG (Jackson labatory, USA) secondary antibody was diluted according to the manufacturer's instructions. Was used. After reacting with the primary and secondary antibodies, it was washed three times with phosphate buffer solution. After the secondary antibody reaction and washing, the membrane was treated with ECL western blot substrate solution (Amersham, USA), and the results were analyzed using a LAS-1000 luminescent image analyzer (Fujifilm, Japan). (FIG. 5). In FIG. 5, the first lane shows the mutant Vibrio sepsis strain CMM744 (Kim YR, Lee SE, Kook H, Yeom JA, Na HS, Kim SY, Chung SS, Choy HE, Rhee JH. Cell Microbiol 2008; 10: 848-862), and lane 2 is culture of wild species Vibrio sepsis. As confirmed in FIG. 5, the 1RA, 5RA, and 11RA monoclonal antibodies showed a positive response at 130 kDa with respect to the cultivar Vibrio sepsis culture. Thus, 1RA, 5RA, 11RA monoclonal antibodies would be useful for the development of diagnostics for detection of Vibrio sepsis RtxA1 antigen.

Example 18 Cross Reaction of Several Vibrio Septic Bacterial Strains of Monoclonal Antibodies.

In order to develop an efficient and useful diagnostic agent for detecting vibrio sepsis, the cross-reactivity of monoclonal antibodies obtained from the present invention and several vibrio sepsis strains should be investigated because monoclonal antibodies should show cross-reaction with several vibrio sepsis bacteria strains. It was. For this purpose, Vibrio sepsis strain isolated from patients (ATCC 29307 (ATCC, USA), CMCP6 (Kim YR, Lee SE, Kim CM, Kim SY, Shin EK, Shin DH, Chung SS, Choy HE, Progulske-Fox A, Hillman JD, Handfield M, Rhee JH.Infect Immun 2003; 71: 5461-5471), YJ016 (Chen CY, Wu KM, Chang YC, Chang CH, Tsai HC, Liao TL, Liu YM, Chen HJ, Shen AB, Li JC, Su TL, Shao CP, Lee CT, Hor LI, Tsai SF.Genome Res 2003; 13: 2577-2587) and environmental strains (Env1 (Rosche ™, Smith B, Oliver JD. Appl Environ Microbiol 2006; 72: 4356-4359.) Were inoculated in HI medium containing 2.5% NaCl, respectively, and incubated for 16 hours in a 37 ° C shaking incubator. The culture solution was inoculated at 1/200 in HI medium containing 2.5% NaCl, and then cultured for 2 hours in a 37 ° C. shaking incubator. The culture medium thus obtained was centrifuged at 12,000 rpm for 10 minutes, and then 300 μl of the supernatant and 1,200 μl of cold acetone (Sigma, USA) were mixed and treated at −20 ° C. for 30 minutes. The culture supernatant and acetone mixture treated for 30 minutes were centrifuged at 12,000 rpm for 15 minutes, and then precipitates were collected. The precipitate was eluted in 20 μl 2 × sample buffer and subjected to 10% SDS-PAGE. After completion of SDS-PAGE, the gel phase protein was transferred to Nitrocellulose membrane (Bio-Rad, USA) according to Tobin's method (Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA 1979; 76: 4350-4354). Moved to. The protein-transferred membrane was blocked with a nonspecific reaction with a phosphate buffer solution containing 5% skim milk. It was reacted for 1 hour at room temperature using a Vibrio septic bacterium RtxA1 monoclonal antibody diluted to 50 μg / ml, and the peroxidase-labeled IgG (Jackson labatory, USA) secondary antibody was diluted according to the manufacturer's instructions. Was used. After reacting with the primary and secondary antibodies, it was washed three times with phosphate buffer solution. After the secondary antibody reaction and washing, the membrane was treated with ECL western blot substrate solution (Amersham, USA), and the results were analyzed using a LAS-1000 luminescent image analyzer (Fujifilm, Japan). (FIG. 6). In FIGS. 6A and 6B, lanes 1 were cultures of mutant Vibrio sepsis strain CMM744 in which the RtxA1 gene was deleted as a control, lanes 2 were cultures of ATCC 29307 Vibrio sepsis strain, lanes 3 were cultures of CMCP6 Vibrio sepsis strain, and lanes 4 were YJ016 Vibrio sepsis. Culture of the strain, lane 5 is the culture of the Env1 Vibrio sepsis strain. As shown in FIG. 6A, the 5RA monoclonal antibody showed a positive response at 130 kDa with respect to ATCC 29307, CMCP6 and YJ016 vibrio sepsis cultures, and in FIG. 6B, the 11RA monoclonal antibodies were ATCC 29307, CMCP6, YJ016 and Env1 Vibrio. The cultures of the sepsis strains were positive at 130 kDa. Thus, 5RA and 11RA monoclonal antibodies would be useful for the development of diagnostics for the detection of the RtxA1 antigen in several vibrio sepsis strains.

Example 19 Reactivity Analysis of Monoclonal Antibodies in Vibrio Sepsis Infected Cells

Until recently, the immunofluorescence assay has been widely used as a standard diagnostic method for the diagnosis of bacterial tissue infection. Vibrio sepsis was inoculated to investigate the reactivity of the monoclonal antibody and Vibrio sepsis bacterium obtained in the present invention. Indirect immunofluorescent antibody was performed using HeLa cells (ATCC, USA). Vibrio septic bacteria were inoculated into HeLa cells monolayered on a slide for FA (fluorescence assay) (Nunc, USA) for 1 hour at 100 MOI, and cells infected with 3.7% formaldehyde (formaldehyde) were fixed. Immobilized Vibrio sepsis infected cells were treated with 0.2% Triton X-100 and each monoclonal antibody was added at a concentration of 10-50 μg / ml using a phosphate buffer solution containing 1% BSA. The solution was diluted, reacted at 37 ° C. for 30 minutes, and washed three times using a phosphate buffer solution. After washing, Alexa Flour 488 condensed anti-mouse IgG antibody (20 μg / ml; Invitrogen, USA) was treated and reacted at 37 ° C. for 30 minutes. After the reaction, the solution was washed three times with phosphate buffer, mounted with ProLong Gold anti-fade (Invitrgen, USA) solution containing DAPI, and observed under a fluorescence microscope. As shown in FIG. 7, monoclonal antibodies showed a negative response in the RtxA1 mutant Vibrio sepsis strain CMM744, but a positive response was apparent in cells infected with the wild type Vibrio sepsis. Therefore, 1RA, 4RA, 5RA, 11RA monoclonal antibodies will be useful for the development of diagnostics for the detection of RtxA1 antigen in cells infected with Vibrio sepsis.

Monoclonal antibody
Isotype

Reactivity to Heat Denatured Antigens
Binding activity RtxA1 (3491-4701) RtxA1 (3491-4380) RtxA1 (3491-3980) 1RA IgG1 positivity medium about voice 2RA IgG2a positivity about about medium 3RA IgG1 positivity about about medium 4RA IgG1 voice about voice voice 5RA IgG2a positivity River about voice 7RA IgG1 positivity River about voice 8RA IgG2b positivity River River River 9RA IgG1 positivity River about voice 10RA IgG1 voice medium voice voice 11RA IgG2a positivity River medium River

As can be seen from Table 1, mass production potential according to isotype among each monoclonal antibody produced in 10 hybridoma cells fused using recombinant RtxA1 protein as an immunogen, responsiveness to heat-denatured antigen, and RtxA1 The combination of the affinity with the antigen and the possibility of crossover to other types of vibrio sepsis showed the best effects of 5RA and 11RA. Accordingly, the 5RA was deposited with the Korean Cell Line Research Foundation under accession number KCLRF-BP-00261, and 11RA was deposited with the accession number KCLRF-BP-00262.

Diagnosis, prevention and treatment of Vibrio sepsis can be easily performed through the present invention, and thus it can be widely used in the medical industry.

Korea Cell Line Research Foundation KCLRFBP00261 20110328

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Monoclonal antibody specific to Vibrio vulnificus RtxA1,          hybridoma producing the monoclonal antibody and diagnostic kit          comprising the monoclonal antibody <130> P-1 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 acatgaattc atgcagagaa gtttggcgac ta 32 <210> 2 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 2 ctcctcgagc taatgatgat gatgatgatg caccgttata cccttttta 49 <210> 3 <211> 14106 <212> DNA <213> Vibrio vulnificus <400> 3 atgggaaaac cattttggag aagtgttgaa tacttcttca cagggaacta ttccgccgac 60 gatggaaaca acagtattgt tgctatcggt tttggtggag aaatccatgc ctacggtggt 120 gatgatcatg tcactgtcgg atcgattggt gcaacggttt ataccggcag cggcaatgat 180 acggtcgtag gcggttcggc atatctaaga gtggaagaca ccacagggca cctttctgta 240 aaaggcgcag ctgggtatgc agacattaat aaaagtagcg atggcaatgt gtcattcgct 300 ggtgccgctg gcggtgtgtc gattgaccac ttaggtaatc atggtgatgt cagttacggc 360 ggtgccgcgg cttataatgg cattacccgc aaaggtttga gcggcaacgt cacctttaaa 420 ggcgctggcg gttacaatgc cctatggcat gaaaccaacc aaggtaatct ctcttttgct 480 ggcgcaggag cgggcaataa actagaccgt acttggttca accgttatca aggctcgcgt 540 ggtgatgtga cgtttgatgg cgctggcgcg gcaaacagca tcagttcacg tgttgacaac 600 attactttcc gtggggctgg tgctgacaac catttggtcc gtaaaggcaa agtgggcgac 660 attaccttgc aaggtgcggg cgcatcaaac cgcattgagc gtacacgcca agcggaagat 720 gtttacgcgc aaacacgcgg taacattcgc tttgaaggtg tcggtggtta caacagcctt 780 tactccgatg tggcacacgg tgacatccat ttctccggtg gcggtgctta taacaccatt 840 acacgaaaag gcagtggtag tagcttcgat gctcaaggta tggaatacgc gaaagcggaa 900 gacattgttc tcaccgcggc gcagatgcat ggtttatcga ttgataacgg caacaagttc 960 catgcggtta ccgctgtaaa atcagagcga gagccaaata cttatctgtt tgctatcgca 1020 gatggtactt acaccaaaat caacaaagtt cgtctctata acgatccaga aacgggcaaa 1080 ctcaaatact actctgaagc ttggttcaag cgcggtaatc atttagcgga gcttgctcgt 1140 tctgatgtct cttctgctgg tggttttgaa gtcaatccaa tcaatggtgg ttacaccctt 1200 tctaatattg ccgttgagca tcagcaatcg ttaaccgttc atgctgtcga gaaggacctg 1260 accgaatatg agtgggtgac ctacgccaat ggcgcactga ttgatgcgaa agatgtggca 1320 ttatcagatg cgaaaatggg cggtcacgct atctcgaccg atgggacgac agtcgatgtt 1380 caagcggtga aatcaaaccg aaaaccaaac acttatgttt acgcaaaagt gcttgggcct 1440 tacacaaaaa ttgtggtggt tgaacttgct aatgatccca aaacaggggc gcttaagtat 1500 caagctcgtt cttggtacaa agagggtaat cacaccgcaa acctagccaa tgaagatatt 1560 tcgtctgcaa acggatatca ctcgatgggc aaaggcggct actcactcag cgatctgcat 1620 tacagtgtga acgcggtaag aagcaccagc gaaaccgtgg ccgacatcga cgagtacacc 1680 gatcaaacct tgtttaagcc agcgaccgat agcggtgaaa gctctggtga tgtgcgcttc 1740 aatggtgcgg gtggcggtaa cgtcatcaag tccaatgtga ctcgcggcaa tgtctacttt 1800 aatggtggcg gtatcgctaa cgtcatcctc cacagttcgc aatttggtca taccgagttc 1860 aacggtggtg gcgcggcaaa cgtgattgtc aaaagtggcg aagaagggga tctcaccttc 1920 cgcggcgcgg gtttggcgaa cgtgttggta caccaaagca agcaaggcaa gatggatgtt 1980 tacgcgggcg gtgcggtaaa cgtactggtt cgtattggcg atggtcaata ccttgcgcat 2040 ttgctggctt acggcaacat ttccgtgcac aaaggcaatg gcaacagccg tgtcgttatg 2100 ctcggtggtt acaacaccca tacccaaatt ggctccggca acggcttgtg gttggcggcg 2160 ggcggcttca acgtgatgac gcaagtgggt aaaggtgatg tagcatcggt actggctggc 2220 ggcgccaacg tgctcaccaa agtgggtgac ggcgatctaa ccgcaggtat gttgggcggc 2280 gcaaacgtga ttacccgcat tagcggcgac aatgaaacct cgaataccac agccgtagcc 2340 ttaggcggtg ctaacattct caccaaaaaa ggcaaaggga atacccttgc ggtgatgggc 2400 ggtggcgcaa acgtgctgac gcatgtgggt gatggtacaa caactggcgt aatggtcggt 2460 ggtgcgaaca ttctcaccaa agtaggtaac ggcgacacga ccggcattat gcttggtgtt 2520 ggcaacgtgc taacgcatgt tggtgatggc caaactcttg gtgtgatggg cgcggcgggt 2580 aacatcttca ccaaagtggg tgatggaacc tctattgcgg tcatgattgg tgctggcaac 2640 atcttcaccc atgtgggaga agggaatgct tgggcgctga tgggcggctt gggtaacgtc 2700 tttaccaaag tcggcaacgg tgatgctctc gcgttaatgg tggcagaagc caacgtcttc 2760 actcacattg gtgatggcat gtcggtcgca ctaatgttag ccaaaggcaa cgttgcgaca 2820 aaagtgggta atggaaccac actcgctgca atggttggta acgccaatat cttcactcac 2880 gtaggcagtg gcagcacgtt tgctgcaatg atcggccaag ccaacatcat gaccaaagtg 2940 ggcaatgatc tcaccgcggc actgatggtc ggtaaagcca acatctacac ccatgtcggt 3000 gatgggacca gcctaggaat attcgcgggc gaagtgaacg tgatgactaa ggtcggtaac 3060 ggcacaacct tggcggcgat gttcggtaaa gccaacatca tgacgcatgt cggcgatggt 3120 ctaacgggcg tgttagcact gggcgaagcc aatattgtca ccaaagtggg tgatgatttc 3180 atgggtgttg tggcggcagc aaaagccaac gtcgtgactc atgttggtga tgcaacaacc 3240 gcggctgtgt tggctggtaa aggcaatttc ctaaccaaag tgggtgaagg caccacggta 3300 gggttgttga tttccgatat cggcaacgta atgacccacg tcggagatgg cacgaccatt 3360 ggtattgcca aaggcaaggc aaacatcatt accaaagtcg gtgatggttt gggcgtcaat 3420 gtcgcttggg ggcaagcaaa cgtgtttacc caagtgggcg acggtgatcg ctataacttt 3480 gccaaaggtg aagccaacat catcaccaaa gtaggtgatg gccaagaagt ctccgtggtg 3540 caaggtaaag ctaacatcat tactcatgtg ggcaatggcg acgactacac cggagcttgg 3600 ggtaaagcga acgtcattac taaagtgggt aatggccgta acgtggtact ggcaaaaggt 3660 gaagccaaca tcgtgaccca agtgggggat ggcgacagct ttaacgcact ttggagcaaa 3720 ggcaacatcg tcaccaaagt gggtgatggt atgcaggtga cggcagcaaa aggcaaagcc 3780 aatatcacta ccaccgttgg taatggcttg agtgtgacaa ccgcctatgg tgatgccaac 3840 atcaatacca aagtcggtga tggcgtatcc gtcaacgtcg cttggggcaa atataacatc 3900 aacacgaaag tgggtgatgg cctgaatgtt gccgtgatga aaggcaaagc taacgccaat 3960 attcacgtgg gcgatggcct caacatcaat gcctcttacg cacaaaacaa cgttgcaatc 4020 aaagttggta acggcgattt ttacagtttg gctgttgcgt caagcaacac cagcagcaac 4080 aagctctcag cactgtttga taatatcaag caaaccgtac ttggtgtcgg tggtagccaa 4140 gccatcaact acttggttca aggtgatgaa gcatcgtcat ccggtacgca aaaaggccgt 4200 ggtgcgattg caacaccgga aatcaccaag ctggatggtt tccaaatgga agccattgaa 4260 gaagtgggct cggacttggg cgatagccta accggcagtg ttactaaggt agatacccca 4320 gatctcaaca agatgcaaaa tgcactcgac gttgacggat cttctgatca aactcaagct 4380 ccgaatctca ttgtgaatgg tgattttgag caaggtgatc gtggttggaa atcgacacac 4440 ggtgttgaag cttcctattc gggcaatgtc tatggcgtga acggcgaagg tcacggcgca 4500 cgtgtgacgg aactggatac ctacaccaac accagtctct atcaagatct gaccgatctt 4560 acggaaggtg aagtcattgc tgtgagcttt gattttgcga aacgtgcagg tctatcgaat 4620 aacgaaggca ttgaagttct ttggaatggc gaagtcgtct tctcatcgtc tggtgatgcc 4680 tctgcttggc agcaaaaaac cttaaaactg accgcgcatg ctggcagtaa ccgaatcgaa 4740 ttcaaagcca caggccacaa tgatggactg ggctatatcc tagataacgt cgtggcgaaa 4800 tcggaatcgt cgcaacaagc caacgcagtg agtgagcacg cgacacaaaa tcaagcgtcg 4860 cagaatgtcc tatcagataa agagcgcgca gaagcggatc gccaacgtct tgaacaagaa 4920 aagcagaaac agcttgatgc tgtcgcaggg tcacaaagtc agttggagtc gaccgaccaa 4980 caagcgctag gaaataacgg tcaagctcaa cgtgatgcgg tgaaagaaga gtcggaagcc 5040 gtgaccgccg agttgacaaa actggcgcaa ggtctcgatg tgcttgatgg ccaagcaacg 5100 catactggcg agtcaggcga ccaatggcgc aatgattttg ccggtggtct gctcgatggc 5160 gttcaaagcc agctcgacga tgccaagcaa ctcgcgaatg acaagattgc tgcagcgaag 5220 cagacgcagt ttgacaataa cagcaaagtc aaagaatccg ttgcgaaatc tgaagcgggt 5280 gtggcacaag gtgagcaaaa tcgcgcaggt gcagagcaag acattgccga agctaaagcc 5340 gatgcagaaa cacgcaaagc cgatgctgtg gcgaaaagta acgatgcgaa acaggccgaa 5400 tctgacgcac acagcgccgc aaacgacgcg caatcgcgtg gtgatcgcga tgcgatgaat 5460 gccgaaaaca aagtcaacca agcgcagaat gatgccaaag ggaccaagca aaacgaaggc 5520 gatcgccctg accgtgaagg cgtggctggc agcggtcttt caggtaacgc tcatcgcgtg 5580 gaaggcgccg gcgaaacggg cagtcatgtc aacaccgaca gtccaaccaa cgccgatggt 5640 cgattcagtg aaggtctgag cgaacaagag caagaagcgc tggaaggtgc gacaaacgca 5700 gtgaaccgtc tgcaaattaa cgcaggtatt cgcggaaaga acagcggtag cacaatcacc 5760 tcaatgttca ccgaaacaaa ctccgacagc attgtggtgc caaccactgc gtctcaggat 5820 gtggttcgaa aagagattcg catttctggg gtgaatttag aaggacttgg tgaagcttcg 5880 catgactctg ctgaatcgct tgtggctgct cgcgcggaaa aagtggcgaa tctctatcgc 5940 tggctagaca ctgataatga cgtggcgacg gacaagtatg tacctgtccc tggctttgaa 6000 cgcgtcgatg ttgatgtctc tgatgaagtc aagcagcgca tgattcaatc tatgagcggt 6060 tacatcgaac acaccgacaa tcaagtaccg aaagatcaag cggaagcatt agccactttg 6120 tttgttgaat cgacgctcga ttatgactgg gacaaacgcg ttgagtttct cactaagctt 6180 gagagctatg gttatagttt tgaagcgcca cacgcagaga aaagcattgt ttccttctgg 6240 tctggtaaaa acttcaagca ataccgagac attctcgaca atgcccagac tgatggcaaa 6300 aaggtcgtgt acgacatcga tgttaaaggc aacgcatttg caatcgacct gaataagcat 6360 ttaatgcgtt ggggcggcct gttccttgac ccagataacg ccgagcaaaa tcagttgaaa 6420 tcatcaatcg atgcagcaac gttcagcaat acaggttttt ggagttcggt ctatgcgacg 6480 ggggcacaaa atgatgtgta tgtcatcgca gaaggtggtg tgcgtcttgg caactatttc 6540 tggaatgttg agctacctgc attacgccaa ttgcagcgtg aagggttagt cggtgaaatt 6600 cgactgcttg ataagccagt atccgagtac aaagatctac cagcagacca gattggacgc 6660 cgacttaccg atgcaggtgt tgccgttaaa gtacgctttg atgcgttgag tcacgagaga 6720 caagctgagc ttttggccga caatccagat ggttacaaag ccgatacgct ggttgagttg 6780 gatgtgaagc taagtgcgat cgatagcatg ttgcgtgaat ctctaccatt ctattcactt 6840 cgtactgagc gcaacctctt agtgcaagaa ggcgaagaag gatttgaagt tcgctcatgg 6900 ccgggaatcg atgaaaagag caagactatt ctgcttgata acccagaaga tgcagctcaa 6960 caaaaatcca ttgagcgctt tatcttggca aacttcgaca acttcgagca gatgccagat 7020 gaacttttct tagtggataa caaggtgctc tcgcatcacg atggccgcac gcgaattata 7080 gcgcaaaaag aagacggtgc ttggacgtac aacaccaatg ttgaactgat gtctgtcact 7140 gagctactcg atgctgctca tgtgaacggt aaagttcgcg gtgatagtta ccaacaagtg 7200 atcgacgccc tgacggagta tcatgcgagt acggttgaac acgcagatta cgaactagaa 7260 tctgtggaaa aactgctcaa cctgcgtaaa cagattgaag gttatgtact cgggcatccg 7320 gattctggcc gtgtggaagc aatgaactca ctgttaaatc aggtgaattc tcgtctggaa 7380 gaagtctctg ttcttgcggt ctctgagcaa agcatcaaag cgcacgatag ctttagccgc 7440 ctatatgacc agcttgataa tgccaactta aaagaaagta aacatctgta tctggatgga 7500 aacggcgatt tcgtcactaa gggcaaaggc aacctagcca ctatcgatca gctaggtggt 7560 agcgatgctg tacttgaaaa agtaaaagca gccgtaactc atgagtatgg tcaagtcgtt 7620 gccgatacga tcttcgcaag gctttctgca aacgatctgg caaaagatgg taaaggcatt 7680 gatattgctg gtttaaacaa agtacaccaa gcgattgaac agcacatgtc gccagtgagc 7740 gcgaccatgt acatctggaa gccgagtgat cacagcacac taggtcatgc cgcattgcaa 7800 attggccaag gtcgcacgca gcttgaaggt caagccgctg ctgacttcaa taagcagaat 7860 tacgtgagtt ggtggccact cggtagcaag tcatccaata tccgcaatat cttcaacgtt 7920 gctacagaag atcagccgga tcttaaacta cgttggagcg atttcagcca acctgctcat 7980 caaaatgaca cacttgagca cgatatggca tctgaagaaa acgacggctt tggtttgaaa 8040 gatggtgaaa ctaagctgaa gcgctttatc gaaaaactca acgccgcgaa aggtattgat 8100 gcttcataca aagatgcttc tgaaggctat gcgagtgtac tactgggcaa tccggatatg 8160 cttgcatcga caggcattcc agcgcatgtt ttccagccat tcgtcgacca atggaatgac 8220 accagctacg acatgatgga cgtagcaaat cgttttgctg aagagctaca gaaacaagct 8280 caggcaagcg gtgacccagc ccttgtggaa aaacgtatcg acaacgtggt tcgtctgttt 8340 gctgaaagag cgctagaaga aatcgaagcc tttaaggcca gccaagcaga tgaaggccga 8400 gtgttccgca ttaacctaga agggttggat gtggcggcga tgcaagctga atggaaacgt 8460 ctaagcaacg atccggatgc tcgatatcag ttattgacca aaaactgctc aagtaccgtc 8520 gcaaaagtgt tgaaagcggg tggtgctgac aaacttatcg gccacacatg gcgacctaag 8580 tttggtgttt ggacgccgac agagcttttc aactttggtc aggcgctgca ggaagctcag 8640 cttgaaattg ccgcgaagaa gcaaagtcat caagtcactg atgtccttga tgccttgtca 8700 ggcaatgaga agcacaaaga aaacgtggcg attgaaaatg atggcacgcc accgcgcgat 8760 aaagaatctc ttagcccact gactcgattc ctcaacaatg agttatatgg tgagaaagac 8820 gcacgtcgta agattggcga aatcacgcaa accttacttg accatgcggt agaaaatgga 8880 gaatcgcaga aagttaccct caaaggggaa gtgggccgtc taacggggta ttaccatcag 8940 ggagcggctt caagcgaagg tgaaacaagc gcgacaagcg gcaaagtcgt gttgttcctg 9000 catggttctg gttcttctgc tgaagagcaa gcgagcgaga ttcgcaacca ctaccaaaaa 9060 caaggtatcg acatgctcgc agtcaacctg cgtggctatg gtgaaagcga cggcggacca 9120 agcgaaaagg gcttgtacca agacgctcgc accatgttca actacctagt gaacgacaag 9180 ggtattgatc caagcaatat catcattcac ggctactcaa tgggtggtcc aattgccgca 9240 gatttagcac gttatgccgc gcaaaatggc caagcggtgt ctggcttatt gcttgaccgt 9300 cctatgccaa gcatgaccaa agccatcacc gctcatgaag tggcgaatcc agcgggcatt 9360 gtaggggcta tcgcgaaagc ggttaacggt cagttctcag tggagaagaa cctaaaaggc 9420 ttgccaaaag agacgccgat tctgctgcta acggacaacg aaggtttagg cgaagaaggc 9480 gagaagctac gagctaaact cgcgattgct ggctacaacg tcacgggtga acaaaccttc 9540 tatggccacg aagcgagcaa ccgcttgatg ggtcagtacg cggatcaaat tgtctccggt 9600 ctgttcaatg cagagcaagc agcggtagaa gcgggcgaag tgctgaaagg actagagaaa 9660 gactttaaac gctatggcga cgcgctgaaa ccagatacga gcgtaccggg taaatcgaaa 9720 gacattcgca ccactaaaga tttcctaaat ggttacaaaa atgaccatgc aaaagaaatc 9780 gttgacggct tccgctcaga tatgagtatc aagcaactgg tggatctgtt cgttaaaggt 9840 agctggagtg cacagcaaaa aggtgcgctt gcttgggaaa ttgaaagtcg tgcactgaaa 9900 gtgacgttcc agaacaagtc tgagaagtac aaccgattgt tccgtgagat tgcttctgct 9960 ggcgtggtgg atgcgaaagc gactgaacag cttgcgccac agttaatgct gttgaaccta 10020 tcgaatgacg gttttggtgg acgttgtgat ccactttcta aactcgtttt ggttgctaaa 10080 cagcttgaaa acgatggtca agttggcgtg gcaagacaac tgctagagaa gatgtactct 10140 gcggcagcgg tgctgagcaa tccaaccctt tactcagaca gtgaaaatgc caatgcaagc 10200 aagttgctca gcagcttggc ggccattcat gcgaagaacc caatgcatga tacgtcgatg 10260 aaagtgtggc aggaaaagct gaaaggaaag caagcactga ccgtaaacgg tgtggttgag 10320 aaaatcactg atgcatcggc taacggcaaa cctgtgttgt tggaacttga tgctccgggg 10380 catgcgatgg cagcttgggc aaaaggctca ggcgacgatc gtgtttacgg cttctacgat 10440 ccaaatgctg gcattgttga gttctcgtca gcagagaagt ttggcgacta cctaacgcgt 10500 ttcttcggca agtccgatct gaacatggct caaagctata agctgggtaa aaatgacgca 10560 ggtgaagcaa tcttcaaccg cgtggtggta atggatggca acacgttagc aagctacaag 10620 ccgaccttcg gtgacaagac caccatgcag gggatcctag atctacctgt gtttgacgct 10680 acaccgatga aaaagcccgg tacttcagat gtcgatggca atgcaaaagc cgtagatgat 10740 acgaaagaag cattggctgg tggaaagata cttcacaacc aaaatgtgaa tgactgggaa 10800 cgtgttgttg tgactccgac agcggacggc ggtgaaagcc gttttgatgg tcaaatcatc 10860 gtgcaaatgg agaacgatga tgtcgttgca aaagccgctg cgaaccttgc gggtaagcac 10920 ccagaaagca gtgtggtggt gcagatcgat tcagacggca actatcgcgt ggtgtatggc 10980 gatccgtcaa agctggatgg aaagctacgt tggcagttag taggtcatgg tcgagatgac 11040 tcagaaagta acaacacgcg tttaagtggc tacagtgccg acgagctggc agtgaaattg 11100 gccaagttcc aacagtcgtt taatcaagcg gaaaacatca acaataagcc tgatcatatc 11160 agtattgttg gttgttcttt ggtgagtgac gataagcaaa aaggctttgg tcatcagttt 11220 attaacgcga tggatgcgaa tggtcttcgt gtcgatgtct ctgtacgcag ttctgaactg 11280 gccgtagacg aggcagggcg taaacatacc aaggacgcga atggtgattg ggtccaaaaa 11340 gccgaaaaca acaaagtttc gctaagctgg gacgagcaag gtgaagttgt tgccaaggat 11400 gaacgtattc gcaacggtat tgcggaaggc gacatcgatc tctctcgtat tggtgtcagc 11460 gacgttgacg agccagctcg tggtgcaatc ggtgacaaca atgatgtgtt tgatgcgcca 11520 gaaaaacgca aagcggagac agaaacctca tcttcttctg caaacaataa actcagctac 11580 tcaggtaaca ttcaagtcaa tgtgggtgat ggtgagttta cggcagtgaa ctggggcaca 11640 tcgaatgtgg gcattaaagt cggcacgggt ggctttaagt cgctggcttt tggtgacaat 11700 aacgtcatgg ttcacatcgg caatggtgag agcaagcaca gcttcgatat tggtggttat 11760 caggcactgg aaggtgcgca aatgttcatc ggtaatcgta atgtgagctt caacttaggt 11820 cgaagtaatg atctgattgt gatgatggac aagtcgattc cgactccgcc attggttaat 11880 ccgttcgatg gtgccgctcg tatttcgggc gtactgcaaa gcattgccac ctcgggtgag 11940 ggccaagatt ggctagcggc tcaagagcag caatggacat tgtctggcgc caagaagttc 12000 gtcaaagata tgtctggttt ggatcagagc agcagtgttg attacaccag tttggttgaa 12060 ctggactcgc agaacgagcg aagcagccgt ggcttgaagc acgatgcaga agcggctctg 12120 aacaagcagt acaatcaatg gttaagtggc aatagcgatt ctgacaccag caagctcagc 12180 cgcgcagata agcttcgtca agccaatgaa aagcttgcgt ttaactttgc tgtgggtggt 12240 caaggtgcgg atatccaagt cacgacaggt aactggaact tcatgtttgg tgacaacatc 12300 cagtctattt tggataccaa cctaggttca ctgtttggcc tcatgacaca gcagttctct 12360 gctacgggtc aggccaagac aaccttcacc tacacgccag aagatttgcc tcgtcagctt 12420 aagaacaagc tacttgggca gttagcgggt gtaggagccg agaccacgct agcggatatt 12480 tttggtgtgg attacaccgc gtcaggtcaa attgtttcgc gtaatggtga agctgtcgat 12540 ggtgtagcga ttctcaaaga gatgttggag gtcattggtg agttcagtgg tgatcaactg 12600 caagcttttg tcgacccagc taagttactg gatagcttga aggcgggtat caacatgggt 12660 gcggatggca ttaagtcttt tgctgaaact catggactga aagagaaggc gccagaagag 12720 gaagaggaca actcttcggt ttctgttaat ggtgcgagcg taaacagtgc gcaaggcgcg 12780 acggtggctg atggcagcac tgaaacagca gaaacaccag atcgtgcctt tggctttaac 12840 tcgcttaacc tgccgaactt gttcgccact atctttagtc aagacaagca gaaagagatg 12900 aaatcgctgg tggaaaatct caaagagaat ctcaccgccg atctgctgaa tatgaaagag 12960 aaaacgtttg atttccttcg taacagtggt catctccaag gtgatggtga tatcaacatc 13020 tccctaggaa actacaactt caactggggt ggtgatggta aagatctcgg agcgtatcta 13080 ggagacaaca acaacttctg gggcggacga ggcgatgatg tgttctacgc aacaggcacg 13140 tcaaacatct tcacgggcgg cgaaggcaac gacatgggcg ttctgatggg acgtgaaaac 13200 atgatgtttg gcggtgatgg caacgacaca gcagtggttg caggacgcat taaccatgtc 13260 ttccttggtg ccggtgatga ccagtcgttt gtctttggtg agggcggtga aattgacacc 13320 ggttcaggcc gcgactacgt ggtgacgtct ggcaacttca accgtgtgga tacgggggac 13380 gatcaagact actccgtgac gattggcaac aacaaccaag tagagctagg cgctggcaat 13440 gactttgcta atgtcttcgg taactacaac cgtatcaatg caagcgctgg caacgatgtt 13500 gtgaagctaa tgggctatca cgccgtgttg aatggtggtg agggcgagga ccatctcatc 13560 gcagcggcca tctctaagtt cagtcaattc aacggtggcg aagggcgcga tctgatggtg 13620 ttgggtggtt atcaaaacac gttcaaaggt ggcacggatg tggacagctt tgtggtgagc 13680 ggtgatgtta tcgacaacct tgttgaagac attcgcagcg aagataacat tgtcttcaat 13740 ggcatcgatt ggcagaaact gtggttcgaa cgcagcggat atgacctgaa gttgtctatt 13800 cttcgtgacc cggcaagcga cagtgaccaa gcgaagtttg agcatattgg ttcggtgacg 13860 tttagtgatt actttaacgg taatcgagcg caggtgatca tcgcaatggg tgagaaagac 13920 gcgacgggtg aacgtgagta caccaccttg tctgagagtg caattgatgc gctggtacaa 13980 gccatgagtg gctttgaccc tcaggcgggt gacaatggat tcatcgataa cctagacagc 14040 aaatctcgcg tggcgattac cactgcgtgg gcagacgttg ttcataaaaa gggtataacg 14100 gtgtaa 14106 <210> 4 <211> 4701 <212> PRT <213> Vibrio vulnificus <400> 4 Met Gly Lys Pro Phe Trp Arg Ser Val Glu Tyr Phe Phe Thr Gly Asn   1 5 10 15 Tyr Ser Ala Asp Asp Gly Asn Asn Ser Ile Val Ala Ile Gly Phe Gly              20 25 30 Gly Glu Ile His Ala Tyr Gly Gly Asp Asp His Val Thr Val Gly Ser          35 40 45 Ile Gly Ala Thr Val Tyr Thr Gly Ser Gly Asn Asp Thr Val Val Gly      50 55 60 Gly Ser Ala Tyr Leu Arg Val Glu Asp Thr Thr Gly His Leu Ser Val  65 70 75 80 Lys Gly Ala Ala Gly Tyr Ala Asp Ile Asn Lys Ser Ser Asp Gly Asn                  85 90 95 Val Ser Phe Ala Gly Ala Ala Gly Gly Val Ser Ile Asp His Leu Gly             100 105 110 Asn His Gly Asp Val Ser Tyr Gly Gly Ala Ala Ala Tyr Asn Gly Ile         115 120 125 Thr Arg Lys Gly Leu Ser Gly Asn Val Thr Phe Lys Gly Ala Gly Gly     130 135 140 Tyr Asn Ala Leu Trp His Glu Thr Asn Gln Gly Asn Leu Ser Phe Ala 145 150 155 160 Gly Ala Gly Ala Gly Asn Lys Leu Asp Arg Thr Trp Phe Asn Arg Tyr                 165 170 175 Gln Gly Ser Arg Gly Asp Val Thr Phe Asp Gly Ala Gly Ala Ala Asn             180 185 190 Ser Ile Ser Ser Arg Val Asp Asn Ile Thr Phe Arg Gly Ala Gly Ala         195 200 205 Asp Asn His Leu Val Arg Lys Gly Lys Val Gly Asp Ile Thr Leu Gln     210 215 220 Gly Ala Gly Ala Ser Asn Arg Ile Glu Arg Thr Arg Gln Ala Glu Asp 225 230 235 240 Val Tyr Ala Gln Thr Arg Gly Asn Ile Arg Phe Glu Gly Val Gly Gly                 245 250 255 Tyr Asn Ser Leu Tyr Ser Asp Val Ala His Gly Asp Ile His Phe Ser             260 265 270 Gly Gly Gly Ala Tyr Asn Thr Ile Thr Arg Lys Gly Ser Gly Ser Ser         275 280 285 Phe Asp Ala Gln Gly Met Glu Tyr Ala Lys Ala Glu Asp Ile Val Leu     290 295 300 Thr Ala Ala Gln Met His Gly Leu Ser Ile Asp Asn Gly Asn Lys Phe 305 310 315 320 His Ala Val Thr Ala Val Lys Ser Glu Arg Glu Pro Asn Thr Tyr Leu                 325 330 335 Phe Ala Ile Ala Asp Gly Thr Tyr Thr Lys Ile Asn Lys Val Arg Leu             340 345 350 Tyr Asn Asp Pro Glu Thr Gly Lys Leu Lys Tyr Tyr Ser Glu Ala Trp         355 360 365 Phe Lys Arg Gly Asn His Leu Ala Glu Leu Ala Arg Ser Asp Val Ser     370 375 380 Ser Ala Gly Gly Phe Glu Val Asn Pro Ile Asn Gly Gly Tyr Thr Leu 385 390 395 400 Ser Asn Ile Ala Val Glu His Gln Gln Ser Leu Thr Val His Ala Val                 405 410 415 Glu Lys Asp Leu Thr Glu Tyr Glu Trp Val Thr Tyr Ala Asn Gly Ala             420 425 430 Leu Ile Asp Ala Lys Asp Val Ala Leu Ser Asp Ala Lys Met Gly Gly         435 440 445 His Ala Ile Ser Thr Asp Gly Thr Thr Val Asp Val Gln Ala Val Lys     450 455 460 Ser Asn Arg Lys Pro Asn Thr Tyr Val Tyr Ala Lys Val Leu Gly Pro 465 470 475 480 Tyr Thr Lys Ile Val Val Val Glu Leu Ala Asn Asp Pro Lys Thr Gly                 485 490 495 Ala Leu Lys Tyr Gln Ala Arg Ser Trp Tyr Lys Glu Gly Asn His Thr             500 505 510 Ala Asn Leu Ala Asn Glu Asp Ile Ser Ser Ala Asn Gly Tyr His Ser         515 520 525 Met Gly Lys Gly Gly Tyr Ser Leu Ser Asp Leu His Tyr Ser Val Asn     530 535 540 Ala Val Arg Ser Thr Ser Glu Thr Val Ala Asp Ile Asp Glu Tyr Thr 545 550 555 560 Asp Gln Thr Leu Phe Lys Pro Ala Thr Asp Ser Gly Glu Ser Ser Gly                 565 570 575 Asp Val Arg Phe Asn Gly Ala Gly Gly Gly Asn Val Ile Lys Ser Asn             580 585 590 Val Thr Arg Gly Asn Val Tyr Phe Asn Gly Gly Gly Ile Ala Asn Val         595 600 605 Ile Leu His Ser Ser Gln Phe Gly His Thr Glu Phe Asn Gly Gly Gly     610 615 620 Ala Ala Asn Val Ile Val Lys Ser Gly Glu Glu Gly Asp Leu Thr Phe 625 630 635 640 Arg Gly Ala Gly Leu Ala Asn Val Leu Val His Gln Ser Lys Gln Gly                 645 650 655 Lys Met Asp Val Tyr Ala Gly Gly Ala Val Asn Val Leu Val Arg Ile             660 665 670 Gly Asp Gly Gln Tyr Leu Ala His Leu Leu Ala Tyr Gly Asn Ile Ser         675 680 685 Val His Lys Gly Asn Gly Asn Ser Arg Val Val Met Leu Gly Gly Tyr     690 695 700 Asn Thr His Thr Gln Ile Gly Ser Gly Asn Gly Leu Trp Leu Ala Ala 705 710 715 720 Gly Gly Phe Asn Val Met Thr Gln Val Gly Lys Gly Asp Val Ala Ser                 725 730 735 Val Leu Ala Gly Gly Ala Asn Val Leu Thr Lys Val Gly Asp Gly Asp             740 745 750 Leu Thr Ala Gly Met Leu Gly Gly Ala Asn Val Ile Thr Arg Ile Ser         755 760 765 Gly Asp Asn Glu Thr Ser Asn Thr Thr Ala Val Ala Leu Gly Gly Ala     770 775 780 Asn Ile Leu Thr Lys Lys Gly Lys Gly Asn Thr Leu Ala Val Met Gly 785 790 795 800 Gly Gly Ala Asn Val Leu Thr His Val Gly Asp Gly Thr Thr Thr Gly                 805 810 815 Val Met Val Gly Gly Ala Asn Ile Leu Thr Lys Val Gly Asn Gly Asp             820 825 830 Thr Thr Gly Ile Met Leu Gly Val Gly Asn Val Leu Thr His Val Gly         835 840 845 Asp Gly Gln Thr Leu Gly Val Met Gly Ala Ala Gly Asn Ile Phe Thr     850 855 860 Lys Val Gly Asp Gly Thr Ser Ile Ala Val Met Ile Gly Ala Gly Asn 865 870 875 880 Ile Phe Thr His Val Gly Glu Gly Asn Ala Trp Ala Leu Met Gly Gly                 885 890 895 Leu Gly Asn Val Phe Thr Lys Val Gly Asn Gly Asp Ala Leu Ala Leu             900 905 910 Met Val Ala Glu Ala Asn Val Phe Thr His Ile Gly Asp Gly Met Ser         915 920 925 Val Ala Leu Met Leu Ala Lys Gly Asn Val Ala Thr Lys Val Gly Asn     930 935 940 Gly Thr Thr Leu Ala Ala Met Val Gly Asn Ala Asn Ile Phe Thr His 945 950 955 960 Val Gly Ser Gly Ser Thr Phe Ala Ala Met Ile Gly Gln Ala Asn Ile                 965 970 975 Met Thr Lys Val Gly Asn Asp Leu Thr Ala Ala Leu Met Val Lys Val             980 985 990 Ala Asn Ile Tyr Thr His Val Gly Asp Gly Thr Ser Leu Gly Ile Phe         995 1000 1005 Ala Gly Glu Val Asn Val Met Thr Lys Val Gly Asn Gly Thr Thr Leu    1010 1015 1020 Ala Ala Met Phe Gly Lys Ala Asn Ile Met Thr His Val Gly Asp Gly 1025 1030 1035 1040 Leu Thr Gly Val Leu Ala Leu Gly Glu Ala Asn Ile Val Thr Lys Val                1045 1050 1055 Gly Asp Asp Phe Met Gly Val Val Ala Ala Ala Lys Ala Asn Val Val            1060 1065 1070 Thr His Val Gly Asp Ala Thr Thr Ala Ala Val Leu Ala Gly Lys Gly        1075 1080 1085 Asn Phe Leu Thr Lys Val Gly Glu Gly Thr Thr Val Gly Leu Leu Ile    1090 1095 1100 Ser Asp Ile Gly Asn Val Met Thr His Val Gly Asp Gly Thr Thr Ile 1105 1110 1115 1120 Gly Ile Ala Lys Gly Lys Ala Asn Ile Ile Thr Lys Val Gly Asp Gly                1125 1130 1135 Leu Gly Val Asn Val Ala Trp Gly Gln Ala Asn Val Phe Thr Gln Val            1140 1145 1150 Gly Asp Gly Asp Arg Tyr Asn Phe Ala Lys Gly Glu Ala Asn Ile Ile        1155 1160 1165 Thr Lys Val Gly Asp Gly Gln Glu Val Ser Val Val Gln Gly Lys Ala    1170 1175 1180 Asn Ile Ile Thr His Val Gly Asn Gly Asp Asp Tyr Thr Gly Ala Trp 1185 1190 1195 1200 Gly Lys Ala Asn Val Ile Thr Lys Val Gly Asn Gly Arg Asn Val Val                1205 1210 1215 Leu Ala Lys Gly Glu Ala Asn Ile Val Thr Gln Val Gly Asp Gly Asp            1220 1225 1230 Ser Phe Asn Ala Leu Trp Ser Lys Gly Asn Ile Val Thr Lys Val Gly        1235 1240 1245 Asp Gly Met Gln Val Thr Ala Ala Lys Gly Lys Ala Asn Ile Thr Thr    1250 1255 1260 Thr Val Gly Asn Gly Leu Ser Val Thr Thr Ala Tyr Gly Asp Ala Asn 1265 1270 1275 1280 Ile Asn Thr Lys Val Gly Asp Gly Val Ser Val Asn Val Ala Trp Gly                1285 1290 1295 Lys Tyr Asn Ile Asn Thr Lys Val Gly Asp Gly Leu Asn Val Ala Val            1300 1305 1310 Met Lys Gly Lys Ala Asn Ala Asn Ile His Val Gly Asp Gly Leu Asn        1315 1320 1325 Ile Asn Ala Ser Tyr Ala Gln Asn Asn Val Ala Ile Lys Val Gly Asn    1330 1335 1340 Gly Asp Phe Tyr Ser Leu Ala Val Ala Ser Ser Asn Thr Ser Ser Asn 1345 1350 1355 1360 Lys Leu Ser Ala Leu Phe Asp Asn Ile Lys Gln Thr Val Leu Gly Val                1365 1370 1375 Gly Gly Ser Gln Ala Ile Asn Tyr Leu Val Gln Gly Asp Glu Ala Ser            1380 1385 1390 Ser Ser Gly Thr Gln Lys Gly Arg Gly Ala Ile Ala Thr Pro Glu Ile        1395 1400 1405 Thr Lys Leu Asp Gly Phe Gln Met Glu Ala Ile Glu Glu Val Gly Ser    1410 1415 1420 Asp Leu Gly Asp Ser Leu Thr Gly Ser Val Thr Lys Val Asp Thr Pro 1425 1430 1435 1440 Asp Leu Asn Lys Met Gln Asn Ala Leu Asp Val Asp Gly Ser Ser Asp                1445 1450 1455 Gln Thr Gln Ala Pro Asn Leu Ile Val Asn Gly Asp Phe Glu Gln Gly            1460 1465 1470 Asp Arg Gly Trp Lys Ser Thr His Gly Val Glu Ala Ser Tyr Ser Gly        1475 1480 1485 Asn Val Tyr Gly Val Asn Gly Glu Gly His Gly Ala Arg Val Thr Glu    1490 1495 1500 Leu Asp Thr Tyr Thr Asn Thr Ser Leu Tyr Gln Asp Leu Thr Asp Leu 1505 1510 1515 1520 Thr Glu Gly Glu Val Ile Ala Val Ser Phe Asp Phe Ala Lys Arg Ala                1525 1530 1535 Gly Leu Ser Asn Asn Glu Gly Ile Glu Val Leu Trp Asn Gly Glu Val            1540 1545 1550 Val Phe Ser Ser Ser Gly Asp Ala Ser Ala Trp Gln Gln Lys Thr Leu        1555 1560 1565 Lys Leu Thr Ala His Ala Gly Ser Asn Arg Ile Glu Phe Lys Ala Thr    1570 1575 1580 Gly His Asn Asp Gly Leu Gly Tyr Ile Leu Asp Asn Val Val Ala Lys 1585 1590 1595 1600 Ser Glu Ser Ser Gln Gln Ala Asn Ala Val Ser Glu His Ala Thr Gln                1605 1610 1615 Asn Gln Ala Ser Gln Asn Val Leu Ser Asp Lys Glu Arg Ala Glu Ala            1620 1625 1630 Asp Arg Gln Arg Leu Glu Gln Glu Lys Gln Lys Gln Leu Asp Ala Val        1635 1640 1645 Ala Gly Ser Gln Ser Gln Leu Glu Ser Thr Asp Gln Gln Ala Leu Gly    1650 1655 1660 Asn Asn Gly Gln Ala Gln Arg Asp Ala Val Lys Glu Glu Ser Glu Ala 1665 1670 1675 1680 Val Thr Ala Glu Leu Thr Lys Leu Ala Gln Gly Leu Asp Val Leu Asp                1685 1690 1695 Gly Gln Ala Thr His Thr Gly Glu Ser Gly Asp Gln Trp Arg Asn Asp            1700 1705 1710 Phe Ala Gly Gly Leu Leu Asp Gly Val Gln Ser Gln Leu Asp Asp Ala        1715 1720 1725 Lys Gln Leu Ala Asn Asp Lys Ile Ala Ala Ala Lys Gln Thr Gln Phe    1730 1735 1740 Asp Asn Asn Ser Lys Val Lys Glu Ser Val Ala Lys Ser Glu Ala Gly 1745 1750 1755 1760 Val Ala Gln Gly Glu Gln Asn Arg Ala Gly Ala Glu Gln Asp Ile Ala                1765 1770 1775 Glu Ala Lys Ala Asp Ala Glu Thr Arg Lys Ala Asp Ala Val Ala Lys            1780 1785 1790 Ser Asn Asp Ala Lys Gln Ala Glu Ser Asp Ala His Ser Ala Ala Asn        1795 1800 1805 Asp Ala Gln Ser Arg Gly Asp Arg Asp Ala Met Asn Ala Glu Asn Lys    1810 1815 1820 Val Asn Gln Ala Gln Asn Asp Ala Lys Gly Thr Lys Gln Asn Glu Gly 1825 1830 1835 1840 Asp Arg Pro Asp Arg Glu Gly Val Ala Gly Ser Gly Leu Ser Gly Asn                1845 1850 1855 Ala His Arg Val Glu Gly Ala Gly Glu Thr Gly Ser His Val Asn Thr            1860 1865 1870 Asp Ser Pro Thr Asn Ala Asp Gly Arg Phe Ser Glu Gly Leu Ser Glu        1875 1880 1885 Gln Glu Gln Glu Ala Leu Glu Gly Ala Thr Asn Ala Val Asn Arg Leu    1890 1895 1900 Gln Ile Asn Ala Gly Ile Arg Gly Lys Asn Ser Gly Ser Thr Ile Thr 1905 1910 1915 1920 Ser Met Phe Thr Glu Thr Asn Ser Asp Ser Ile Val Val Pro Thr Thr                1925 1930 1935 Ala Ser Gln Asp Val Val Arg Lys Glu Ile Arg Ile Ser Gly Val Asn            1940 1945 1950 Leu Glu Gly Leu Gly Glu Ala Ser His Asp Ser Ala Glu Ser Leu Val        1955 1960 1965 Ala Ala Arg Ala Glu Lys Val Ala Asn Leu Tyr Arg Trp Leu Asp Thr    1970 1975 1980 Asp Asn Asp Val Ala Thr Asp Lys Tyr Val Pro Val Pro Gly Phe Glu 1985 1990 1995 2000 Arg Val Asp Val Asp Val Ser Asp Glu Val Lys Gln Arg Met Ile Gln                2005 2010 2015 Ser Met Ser Gly Tyr Ile Glu His Thr Asp Asn Gln Val Pro Lys Asp            2020 2025 2030 Gln Ala Glu Ala Leu Ala Thr Leu Phe Val Glu Ser Thr Leu Asp Tyr        2035 2040 2045 Asp Trp Asp Lys Arg Val Glu Phe Leu Thr Lys Leu Glu Ser Tyr Gly    2050 2055 2060 Tyr Ser Phe Glu Ala Pro His Ala Glu Lys Ser Ile Val Ser Phe Trp 2065 2070 2075 2080 Ser Gly Lys Asn Phe Lys Gln Tyr Arg Asp Ile Leu Asp Asn Ala Gln                2085 2090 2095 Thr Asp Gly Lys Lys Val Val Tyr Asp Ile Asp Val Lys Gly Asn Ala            2100 2105 2110 Phe Ala Ile Asp Leu Asn Lys His Leu Met Arg Trp Gly Gly Leu Phe        2115 2120 2125 Leu Asp Pro Asp Asn Ala Glu Gln Asn Gln Leu Lys Ser Ser Ile Asp    2130 2135 2140 Ala Ala Thr Phe Ser Asn Thr Gly Phe Trp Ser Ser Val Tyr Ala Thr 2145 2150 2155 2160 Gly Ala Gln Asn Asp Val Tyr Val Ile Ala Glu Gly Gly Val Arg Leu                2165 2170 2175 Gly Asn Tyr Phe Trp Asn Val Glu Leu Pro Ala Leu Arg Gln Leu Gln            2180 2185 2190 Arg Glu Gly Leu Val Gly Glu Ile Arg Leu Leu Asp Lys Pro Val Ser        2195 2200 2205 Glu Tyr Lys Asp Leu Pro Ala Asp Gln Ile Gly Arg Arg Leu Thr Asp    2210 2215 2220 Ala Gly Val Ala Val Lys Val Arg Phe Asp Ala Leu Ser His Glu Arg 2225 2230 2235 2240 Gln Ala Glu Leu Leu Ala Asp Asn Pro Asp Gly Tyr Lys Ala Asp Thr                2245 2250 2255 Leu Val Glu Leu Asp Val Lys Leu Ser Ala Ile Asp Ser Met Leu Arg            2260 2265 2270 Glu Ser Leu Pro Phe Tyr Ser Leu Arg Thr Glu Arg Asn Leu Leu Val        2275 2280 2285 Gln Glu Gly Glu Glu Gly Phe Glu Val Arg Ser Trp Pro Gly Ile Asp    2290 2295 2300 Glu Lys Ser Lys Thr Ile Leu Leu Asp Asn Pro Glu Asp Ala Ala Gln 2305 2310 2315 2320 Gln Lys Ser Ile Glu Arg Phe Ile Leu Ala Asn Phe Asp Asn Phe Glu                2325 2330 2335 Gln Met Pro Asp Glu Leu Phe Leu Val Asp Asn Lys Val Leu Ser His            2340 2345 2350 His Asp Gly Arg Thr Arg Ile Ile Ala Gln Lys Glu Asp Gly Ala Trp        2355 2360 2365 Thr Tyr Asn Thr Asn Val Glu Leu Met Ser Val Thr Glu Leu Leu Asp    2370 2375 2380 Ala Ala His Val Asn Gly Lys Val Arg Gly Asp Ser Tyr Gln Gln Val 2385 2390 2395 2400 Ile Asp Ala Leu Thr Glu Tyr His Ala Ser Thr Val Glu His Ala Asp                2405 2410 2415 Tyr Glu Leu Glu Ser Val Glu Lys Leu Leu Asn Leu Arg Lys Gln Ile            2420 2425 2430 Glu Gly Tyr Val Leu Gly His Pro Asp Ser Gly Arg Val Glu Ala Met        2435 2440 2445 Asn Ser Leu Leu Asn Gln Val Asn Ser Arg Leu Glu Glu Val Ser Val    2450 2455 2460 Leu Ala Val Ser Glu Gln Ser Ile Lys Ala His Asp Ser Phe Ser Arg 2465 2470 2475 2480 Leu Tyr Asp Gln Leu Asp Asn Ala Asn Leu Lys Glu Ser Lys His Leu                2485 2490 2495 Tyr Leu Asp Gly Asn Gly Asp Phe Val Thr Lys Gly Lys Gly Asn Leu            2500 2505 2510 Ala Thr Ile Asp Gln Leu Gly Gly Ser Asp Ala Val Leu Glu Lys Val        2515 2520 2525 Lys Ala Ala Val Thr His Glu Tyr Gly Gln Val Val Ala Asp Thr Ile    2530 2535 2540 Phe Ala Arg Leu Ser Ala Asn Asp Leu Ala Lys Asp Gly Lys Gly Ile 2545 2550 2555 2560 Asp Ile Ala Gly Leu Asn Lys Val His Gln Ala Ile Glu Gln His Met                2565 2570 2575 Ser Pro Val Ser Ala Thr Met Tyr Ile Trp Lys Pro Ser Asp His Ser            2580 2585 2590 Thr Leu Gly His Ala Ala Leu Gln Ile Gly Gln Gly Arg Thr Gln Leu        2595 2600 2605 Glu Gly Gln Ala Ala Ala Asp Phe Asn Lys Gln Asn Tyr Val Ser Trp    2610 2615 2620 Trp Pro Leu Gly Ser Lys Ser Ser Asn Ile Arg Asn Ile Phe Asn Val 2625 2630 2635 2640 Ala Thr Glu Asp Gln Pro Asp Leu Lys Leu Arg Trp Ser Asp Phe Ser                2645 2650 2655 Gln Pro Ala His Gln Asn Asp Thr Leu Glu His Asp Met Ala Ser Glu            2660 2665 2670 Glu Asn Asp Gly Phe Gly Leu Lys Asp Gly Glu Thr Lys Leu Lys Arg        2675 2680 2685 Phe Ile Glu Lys Leu Asn Ala Ala Lys Gly Ile Asp Ala Ser Tyr Lys    2690 2695 2700 Asp Ala Ser Glu Gly Tyr Ala Ser Val Leu Leu Gly Asn Pro Asp Met 2705 2710 2715 2720 Leu Ala Ser Thr Gly Ile Pro Ala His Val Phe Gln Pro Phe Val Asp                2725 2730 2735 Gln Trp Asn Asp Thr Ser Tyr Asp Met Met Asp Val Ala Asn Arg Phe            2740 2745 2750 Ala Glu Glu Leu Gln Lys Gln Ala Gln Ala Ser Gly Asp Pro Ala Leu        2755 2760 2765 Val Glu Lys Arg Ile Asp Asn Val Val Arg Leu Phe Ala Glu Arg Ala    2770 2775 2780 Leu Glu Glu Ile Glu Ala Phe Lys Ala Ser Gln Ala Asp Glu Gly Arg 2785 2790 2795 2800 Val Phe Arg Ile Asn Leu Glu Gly Leu Asp Val Ala Ala Met Gln Ala                2805 2810 2815 Glu Trp Lys Arg Leu Ser Asn Asp Pro Asp Ala Arg Tyr Gln Leu Leu            2820 2825 2830 Thr Lys Asn Cys Ser Ser Thr Val Ala Lys Val Leu Lys Ala Gly Gly        2835 2840 2845 Ala Asp Lys Leu Ile Gly His Thr Trp Arg Pro Lys Phe Gly Val Trp    2850 2855 2860 Thr Pro Thr Glu Leu Phe Asn Phe Gly Gln Ala Leu Gln Glu Ala Gln 2865 2870 2875 2880 Leu Glu Ile Ala Ala Lys Lys Gln Ser His Gln Val Thr Asp Val Leu                2885 2890 2895 Asp Ala Leu Ser Gly Asn Glu Lys His Lys Glu Asn Val Ala Ile Glu            2900 2905 2910 Asn Asp Gly Thr Pro Pro Arg Asp Lys Glu Ser Leu Ser Pro Leu Thr        2915 2920 2925 Arg Phe Leu Asn Asn Glu Leu Tyr Gly Glu Lys Asp Ala Arg Arg Lys    2930 2935 2940 Ile Gly Glu Ile Thr Gln Thr Leu Leu Asp His Ala Val Glu Asn Gly 2945 2950 2955 2960 Glu Ser Gln Lys Val Thr Leu Lys Gly Glu Val Gly Arg Leu Thr Gly                2965 2970 2975 Tyr Tyr His Gln Gly Ala Ala Ser Ser Glu Gly Glu Thr Ser Ala Thr            2980 2985 2990 Ser Gly Lys Val Val Leu Phe Leu His Gly Ser Gly Ser Ser Ala Glu        2995 3000 3005 Glu Gln Ala Ser Glu Ile Arg Asn His Tyr Gln Lys Gln Gly Ile Asp    3010 3015 3020 Met Leu Ala Val Asn Leu Arg Gly Tyr Gly Glu Ser Asp Gly Gly Pro 3025 3030 3035 3040 Ser Glu Lys Gly Leu Tyr Gln Asp Ala Arg Thr Met Phe Asn Tyr Leu                3045 3050 3055 Val Asn Asp Lys Gly Ile Asp Pro Ser Asn Ile Ile Ile His Gly Tyr            3060 3065 3070 Ser Met Gly Gly Pro Ile Ala Ala Asp Leu Ala Arg Tyr Ala Ala Gln        3075 3080 3085 Asn Gly Gln Ala Val Ser Gly Leu Leu Leu Asp Arg Pro Met Pro Ser    3090 3095 3100 Met Thr Lys Ala Ile Thr Ala His Glu Val Ala Asn Pro Ala Gly Ile 3105 3110 3115 3120 Val Gly Ala Ile Ala Lys Ala Val Asn Gly Gln Phe Ser Val Glu Lys                3125 3130 3135 Asn Leu Lys Gly Leu Pro Lys Glu Thr Pro Ile Leu Leu Leu Thr Asp            3140 3145 3150 Asn Glu Gly Leu Gly Glu Glu Gly Glu Lys Leu Arg Ala Lys Leu Ala        3155 3160 3165 Ile Ala Gly Tyr Asn Val Thr Gly Glu Gln Thr Phe Tyr Gly His Glu    3170 3175 3180 Ala Ser Asn Arg Leu Met Gly Gln Tyr Ala Asp Gln Ile Val Ser Gly 3185 3190 3195 3200 Leu Phe Asn Ala Glu Gln Ala Ala Val Glu Ala Gly Glu Val Leu Lys                3205 3210 3215 Gly Leu Glu Lys Asp Phe Lys Arg Tyr Gly Asp Ala Leu Lys Pro Asp            3220 3225 3230 Thr Ser Val Pro Gly Lys Ser Lys Asp Ile Arg Thr Thr Lys Asp Phe        3235 3240 3245 Leu Asn Gly Tyr Lys Asn Asp His Ala Lys Glu Ile Val Asp Gly Phe    3250 3255 3260 Arg Ser Asp Met Ser Ile Lys Gln Leu Val Asp Leu Phe Val Lys Gly 3265 3270 3275 3280 Ser Trp Ser Ala Gln Gln Lys Gly Ala Leu Ala Trp Glu Ile Glu Ser                3285 3290 3295 Arg Ala Leu Lys Val Thr Phe Gln Asn Lys Ser Glu Lys Tyr Asn Arg            3300 3305 3310 Leu Phe Arg Glu Ile Ala Ser Ala Gly Val Val Asp Ala Lys Ala Thr        3315 3320 3325 Glu Gln Leu Ala Pro Gln Leu Met Leu Leu Asn Leu Ser Asn Asp Gly    3330 3335 3340 Phe Gly Gly Arg Cys Asp Pro Leu Ser Lys Leu Val Leu Val Ala Lys 3345 3350 3355 3360 Gln Leu Glu Asn Asp Gly Gln Val Gly Val Ala Arg Gln Leu Leu Glu                3365 3370 3375 Lys Met Tyr Ser Ala Ala Ala Val Leu Ser Asn Pro Thr Leu Tyr Ser            3380 3385 3390 Asp Ser Glu Asn Ala Asn Ala Ser Lys Leu Leu Ser Ser Leu Ala Ala        3395 3400 3405 Ile His Ala Lys Asn Pro Met His Asp Thr Ser Met Lys Val Trp Gln    3410 3415 3420 Glu Lys Leu Lys Gly Lys Gln Ala Leu Thr Val Asn Gly Val Val Glu 3425 3430 3435 3440 Lys Ile Thr Asp Ala Ser Ala Asn Gly Lys Pro Val Leu Leu Glu Leu                3445 3450 3455 Asp Ala Pro Gly His Ala Met Ala Ala Trp Ala Lys Gly Ser Gly Asp            3460 3465 3470 Asp Arg Val Tyr Gly Phe Tyr Asp Pro Asn Ala Gly Ile Val Glu Phe        3475 3480 3485 Ser Ser Ala Glu Lys Phe Gly Asp Tyr Leu Thr Arg Phe Phe Gly Lys    3490 3495 3500 Ser Asp Leu Asn Met Ala Gln Ser Tyr Lys Leu Gly Lys Asn Asp Ala 3505 3510 3515 3520 Gly Glu Ala Ile Phe Asn Arg Val Val Val Met Asp Gly Asn Thr Leu                3525 3530 3535 Ala Ser Tyr Lys Pro Thr Phe Gly Asp Lys Thr Thr Met Gln Gly Ile            3540 3545 3550 Leu Asp Leu Pro Val Phe Asp Ala Thr Pro Met Lys Lys Pro Gly Thr        3555 3560 3565 Ser Asp Val Asp Gly Asn Ala Lys Ala Val Asp Asp Thr Lys Glu Ala    3570 3575 3580 Leu Ala Gly Gly Lys Ile Leu His Asn Gln Asn Val Asn Asp Trp Glu 3585 3590 3595 3600 Arg Val Val Val Thr Pro Thr Ala Asp Gly Gly Glu Ser Arg Phe Asp                3605 3610 3615 Gly Gln Ile Ile Val Gln Met Glu Asn Asp Asp Val Val Ala Lys Ala            3620 3625 3630 Ala Ala Asn Leu Ala Gly Lys His Pro Glu Ser Ser Val Val Val Gln        3635 3640 3645 Ile Asp Ser Asp Gly Asn Tyr Arg Val Val Tyr Gly Asp Pro Ser Lys    3650 3655 3660 Leu Asp Gly Lys Leu Arg Trp Gln Leu Val Gly His Gly Arg Asp Asp 3665 3670 3675 3680 Ser Glu Ser Asn Asn Thr Arg Leu Ser Gly Tyr Ser Ala Asp Glu Leu                3685 3690 3695 Ala Val Lys Leu Ala Lys Phe Gln Gln Ser Phe Asn Gln Ala Glu Asn            3700 3705 3710 Ile Asn Asn Lys Pro Asp His Ile Ser Ile Val Gly Cys Ser Leu Val        3715 3720 3725 Ser Asp Asp Lys Gln Lys Gly Phe Gly His Gln Phe Ile Asn Ala Met    3730 3735 3740 Asp Ala Asn Gly Leu Arg Val Asp Val Ser Val Arg Ser Ser Glu Leu 3745 3750 3755 3760 Ala Val Asp Glu Ala Gly Arg Lys His Thr Lys Asp Ala Asn Gly Asp                3765 3770 3775 Trp Val Gln Lys Ala Glu Asn Asn Lys Val Ser Leu Ser Trp Asp Glu            3780 3785 3790 Gln Gly Glu Val Val Ala Lys Asp Glu Arg Ile Arg Asn Gly Ile Ala        3795 3800 3805 Glu Gly Asp Ile Asp Leu Ser Arg Ile Gly Val Ser Asp Val Asp Glu    3810 3815 3820 Pro Ala Arg Gly Ala Ile Gly Asp Asn Asn Asp Val Phe Asp Ala Pro 3825 3830 3835 3840 Glu Lys Arg Lys Ala Glu Thr Glu Thr Ser Ser Ser Ser Ala Asn Asn                3845 3850 3855 Lys Leu Ser Tyr Ser Gly Asn Ile Gln Val Asn Val Gly Asp Gly Glu            3860 3865 3870 Phe Thr Ala Val Asn Trp Gly Thr Ser Asn Val Gly Ile Lys Val Gly        3875 3880 3885 Thr Gly Gly Phe Lys Ser Leu Ala Phe Gly Asp Asn Asn Val Met Val    3890 3895 3900 His Ile Gly Asn Gly Glu Ser Lys His Ser Phe Asp Ile Gly Gly Tyr 3905 3910 3915 3920 Gln Ala Leu Glu Gly Ala Gln Met Phe Ile Gly Asn Arg Asn Val Ser                3925 3930 3935 Phe Asn Leu Gly Arg Ser Asn Asp Leu Ile Val Met Met Asp Lys Ser            3940 3945 3950 Ile Pro Thr Pro Pro Leu Val Asn Pro Phe Asp Gly Ala Ala Arg Ile        3955 3960 3965 Ser Gly Val Leu Gln Ser Ile Ala Thr Ser Gly Glu Gly Gln Asp Trp    3970 3975 3980 Leu Ala Ala Gln Glu Gln Gln Trp Thr Leu Ser Gly Ala Lys Lys Phe 3985 3990 3995 4000 Val Lys Asp Met Ser Gly Leu Asp Gln Ser Ser Ser Val Asp Tyr Thr                4005 4010 4015 Ser Leu Val Glu Leu Asp Ser Gln Asn Glu Arg Ser Ser Arg Gly Leu            4020 4025 4030 Lys His Asp Ala Glu Ala Ala Leu Asn Lys Gln Tyr Asn Gln Trp Leu        4035 4040 4045 Ser Gly Asn Ser Asp Ser Asp Thr Ser Lys Leu Ser Arg Ala Asp Lys    4050 4055 4060 Leu Arg Gln Ala Asn Glu Lys Leu Ala Phe Asn Phe Ala Val Gly Gly 4065 4070 4075 4080 Gln Gly Ala Asp Ile Gln Val Thr Thr Gly Asn Trp Asn Phe Met Phe                4085 4090 4095 Gly Asp Asn Ile Gln Ser Ile Leu Asp Thr Asn Leu Gly Ser Leu Phe            4100 4105 4110 Gly Leu Met Thr Gln Gln Phe Ser Ala Thr Gly Gln Ala Lys Thr Thr        4115 4120 4125 Phe Thr Tyr Thr Pro Glu Asp Leu Pro Arg Gln Leu Lys Asn Lys Leu    4130 4135 4140 Leu Gly Gln Leu Ala Gly Val Gly Ala Glu Thr Thr Leu Ala Asp Ile 4145 4150 4155 4160 Phe Gly Val Asp Tyr Thr Ala Ser Gly Gln Ile Val Ser Arg Asn Gly                4165 4170 4175 Glu Ala Val Asp Gly Val Ala Ile Leu Lys Glu Met Leu Glu Val Ile            4180 4185 4190 Gly Glu Phe Ser Gly Asp Gln Leu Gln Ala Phe Val Asp Pro Ala Lys        4195 4200 4205 Leu Leu Asp Ser Leu Lys Ala Gly Ile Asn Met Gly Ala Asp Gly Ile    4210 4215 4220 Lys Ser Phe Ala Glu Thr His Gly Leu Lys Glu Lys Ala Pro Glu Glu 4225 4230 4235 4240 Glu Glu Asp Asn Ser Ser Val Ser Val Asn Gly Ala Ser Val Asn Ser                4245 4250 4255 Ala Gln Gly Ala Thr Val Ala Asp Gly Ser Thr Glu Thr Ala Glu Thr            4260 4265 4270 Pro Asp Arg Ala Phe Gly Phe Asn Ser Leu Asn Leu Pro Asn Leu Phe        4275 4280 4285 Ala Thr Ile Phe Ser Gln Asp Lys Gln Lys Glu Met Lys Ser Leu Val    4290 4295 4300 Glu Asn Leu Lys Glu Asn Leu Thr Ala Asp Leu Leu Asn Met Lys Glu 4305 4310 4315 4320 Lys Thr Phe Asp Phe Leu Arg Asn Ser Gly His Leu Gln Gly Asp Gly                4325 4330 4335 Asp Ile Asn Ile Ser Leu Gly Asn Tyr Asn Phe Asn Trp Gly Gly Asp            4340 4345 4350 Gly Lys Asp Leu Gly Ala Tyr Leu Gly Asp Asn Asn Asn Phe Trp Gly        4355 4360 4365 Gly Arg Gly Asp Asp Val Phe Tyr Ala Thr Gly Thr Ser Asn Ile Phe    4370 4375 4380 Thr Gly Gly Glu Gly Asn Asp Met Gly Val Leu Met Gly Arg Glu Asn 4385 4390 4395 4400 Met Met Phe Gly Gly Asp Gly Asn Asp Thr Ala Val Val Ala Gly Arg                4405 4410 4415 Ile Asn His Val Phe Leu Gly Ala Gly Asp Asp Gln Ser Phe Val Phe            4420 4425 4430 Gly Glu Gly Gly Glu Ile Asp Thr Gly Ser Gly Arg Asp Tyr Val Val        4435 4440 4445 Thr Ser Gly Asn Phe Asn Arg Val Asp Thr Gly Asp Asp Gln Asp Tyr    4450 4455 4460 Ser Val Thr Ile Gly Asn Asn Asn Gln Val Glu Leu Gly Ala Gly Asn 4465 4470 4475 4480 Asp Phe Ala Asn Val Phe Gly Asn Tyr Asn Arg Ile Asn Ala Ser Ala                4485 4490 4495 Gly Asn Asp Val Val Lys Leu Met Gly Tyr His Ala Val Leu Asn Gly            4500 4505 4510 Gly Glu Gly Glu Asp His Leu Ile Ala Ala Ala Ile Ser Lys Phe Ser        4515 4520 4525 Gln Phe Asn Gly Gly Glu Gly Arg Asp Leu Met Val Leu Gly Gly Tyr    4530 4535 4540 Gln Asn Thr Phe Lys Gly Gly Thr Asp Val Asp Ser Phe Val Val Ser 4545 4550 4555 4560 Gly Asp Val Ile Asp Asn Leu Val Glu Asp Ile Arg Ser Glu Asp Asn                4565 4570 4575 Ile Val Phe Asn Gly Ile Asp Trp Gln Lys Leu Trp Phe Glu Arg Ser            4580 4585 4590 Gly Tyr Asp Leu Lys Leu Ser Ile Leu Arg Asp Pro Ala Ser Asp Ser        4595 4600 4605 Asp Gln Ala Lys Phe Glu His Ile Gly Ser Val Thr Phe Ser Asp Tyr    4610 4615 4620 Phe Asn Gly Asn Arg Ala Gln Val Ile Ile Ala Met Gly Glu Lys Asp 4625 4630 4635 4640 Ala Thr Gly Glu Arg Glu Tyr Thr Thr Leu Ser Glu Ser Ala Ile Asp                4645 4650 4655 Ala Leu Val Gln Ala Met Ser Gly Phe Asp Pro Gln Ala Gly Asp Asn            4660 4665 4670 Gly Phe Ile Asp Asn Leu Asp Ser Lys Ser Arg Val Ala Ile Thr Thr        4675 4680 4685 Ala Trp Ala Asp Val Val His Lys Lys Gly Ile Thr Val    4690 4695 4700 <210> 5 <211> 3636 <212> DNA <213> Vibrio vulnificus <400> 5 gcagagaagt ttggcgacta cctaacgcgt ttcttcggca agtccgatct gaacatggct 60 caaagctata agctgggtaa aaatgacgca ggtgaagcaa tcttcaaccg cgtggtggta 120 atggatggca acacgttagc aagctacaag ccgaccttcg gtgacaagac caccatgcag 180 gggatcctag atctacctgt gtttgacgct acaccgatga aaaagcccgg tacttcagat 240 gtcgatggca atgcaaaagc cgtagatgat acgaaagaag cattggctgg tggaaagata 300 cttcacaacc aaaatgtgaa tgactgggaa cgtgttgttg tgactccgac agcggacggc 360 ggtgaaagcc gttttgatgg tcaaatcatc gtgcaaatgg agaacgatga tgtcgttgca 420 aaagccgctg cgaaccttgc gggtaagcac ccagaaagca gtgtggtggt gcagatcgat 480 tcagacggca actatcgcgt ggtgtatggc gatccgtcaa agctggatgg aaagctacgt 540 tggcagttag taggtcatgg tcgagatgac tcagaaagta acaacacgcg tttaagtggc 600 tacagtgccg acgagctggc agtgaaattg gccaagttcc aacagtcgtt taatcaagcg 660 gaaaacatca acaataagcc tgatcatatc agtattgttg gttgttcttt ggtgagtgac 720 gataagcaaa aaggctttgg tcatcagttt attaacgcga tggatgcgaa tggtcttcgt 780 gtcgatgtct ctgtacgcag ttctgaactg gccgtagacg aggcagggcg taaacatacc 840 aaggacgcga atggtgattg ggtccaaaaa gccgaaaaca acaaagtttc gctaagctgg 900 gacgagcaag gtgaagttgt tgccaaggat gaacgtattc gcaacggtat tgcggaaggc 960 gacatcgatc tctctcgtat tggtgtcagc gacgttgacg agccagctcg tggtgcaatc 1020 ggtgacaaca atgatgtgtt tgatgcgcca gaaaaacgca aagcggagac agaaacctca 1080 tcttcttctg caaacaataa actcagctac tcaggtaaca ttcaagtcaa tgtgggtgat 1140 ggtgagttta cggcagtgaa ctggggcaca tcgaatgtgg gcattaaagt cggcacgggt 1200 ggctttaagt cgctggcttt tggtgacaat aacgtcatgg ttcacatcgg caatggtgag 1260 agcaagcaca gcttcgatat tggtggttat caggcactgg aaggtgcgca aatgttcatc 1320 ggtaatcgta atgtgagctt caacttaggt cgaagtaatg atctgattgt gatgatggac 1380 aagtcgattc cgactccgcc attggttaat ccgttcgatg gtgccgctcg tatttcgggc 1440 gtactgcaaa gcattgccac ctcgggtgag ggccaagatt ggctagcggc tcaagagcag 1500 caatggacat tgtctggcgc caagaagttc gtcaaagata tgtctggttt ggatcagagc 1560 agcagtgttg attacaccag tttggttgaa ctggactcgc agaacgagcg aagcagccgt 1620 ggcttgaagc acgatgcaga agcggctctg aacaagcagt acaatcaatg gttaagtggc 1680 aatagcgatt ctgacaccag caagctcagc cgcgcagata agcttcgtca agccaatgaa 1740 aagcttgcgt ttaactttgc tgtgggtggt caaggtgcgg atatccaagt cacgacaggt 1800 aactggaact tcatgtttgg tgacaacatc cagtctattt tggataccaa cctaggttca 1860 ctgtttggcc tcatgacaca gcagttctct gctacgggtc aggccaagac aaccttcacc 1920 tacacgccag aagatttgcc tcgtcagctt aagaacaagc tacttgggca gttagcgggt 1980 gtaggagccg agaccacgct agcggatatt tttggtgtgg attacaccgc gtcaggtcaa 2040 attgtttcgc gtaatggtga agctgtcgat ggtgtagcga ttctcaaaga gatgttggag 2100 gtcattggtg agttcagtgg tgatcaactg caagcttttg tcgacccagc taagttactg 2160 gatagcttga aggcgggtat caacatgggt gcggatggca ttaagtcttt tgctgaaact 2220 catggactga aagagaaggc gccagaagag gaagaggaca actcttcggt ttctgttaat 2280 ggtgcgagcg taaacagtgc gcaaggcgcg acggtggctg atggcagcac tgaaacagca 2340 gaaacaccag atcgtgcctt tggctttaac tcgcttaacc tgccgaactt gttcgccact 2400 atctttagtc aagacaagca gaaagagatg aaatcgctgg tggaaaatct caaagagaat 2460 ctcaccgccg atctgctgaa tatgaaagag aaaacgtttg atttccttcg taacagtggt 2520 catctccaag gtgatggtga tatcaacatc tccctaggaa actacaactt caactggggt 2580 ggtgatggta aagatctcgg agcgtatcta ggagacaaca acaacttctg gggcggacga 2640 ggcgatgatg tgttctacgc aacaggcacg tcaaacatct tcacgggcgg cgaaggcaac 2700 gacatgggcg ttctgatggg acgtgaaaac atgatgtttg gcggtgatgg caacgacaca 2760 gcagtggttg caggacgcat taaccatgtc ttccttggtg ccggtgatga ccagtcgttt 2820 gtctttggtg agggcggtga aattgacacc ggttcaggcc gcgactacgt ggtgacgtct 2880 ggcaacttca accgtgtgga tacgggggac gatcaagact actccgtgac gattggcaac 2940 aacaaccaag tagagctagg cgctggcaat gactttgcta atgtcttcgg taactacaac 3000 cgtatcaatg caagcgctgg caacgatgtt gtgaagctaa tgggctatca cgccgtgttg 3060 aatggtggtg agggcgagga ccatctcatc gcagcggcca tctctaagtt cagtcaattc 3120 aacggtggcg aagggcgcga tctgatggtg ttgggtggtt atcaaaacac gttcaaaggt 3180 ggcacggatg tggacagctt tgtggtgagc ggtgatgtta tcgacaacct tgttgaagac 3240 attcgcagcg aagataacat tgtcttcaat ggcatcgatt ggcagaaact gtggttcgaa 3300 cgcagcggat atgacctgaa gttgtctatt cttcgtgacc cggcaagcga cagtgaccaa 3360 gcgaagtttg agcatattgg ttcggtgacg tttagtgatt actttaacgg taatcgagcg 3420 caggtgatca tcgcaatggg tgagaaagac gcgacgggtg aacgtgagta caccaccttg 3480 tctgagagtg caattgatgc gctggtacaa gccatgagtg gctttgaccc tcaggcgggt 3540 gacaatggat tcatcgataa cctagacagc aaatctcgcg tggcgattac cactgcgtgg 3600 gcagacgttg ttcataaaaa gggtataacg gtgcat 3636 <210> 6 <211> 1211 <212> PRT <213> Vibrio vulnificus <400> 6 Ala Glu Lys Phe Gly Asp Tyr Leu Thr Arg Phe Phe Gly Lys Ser Asp   1 5 10 15 Leu Asn Met Ala Gln Ser Tyr Lys Leu Gly Lys Asn Asp Ala Gly Glu              20 25 30 Ala Ile Phe Asn Arg Val Val Val Asp Gly Asn Thr Leu Ala Ser          35 40 45 Tyr Lys Pro Thr Phe Gly Asp Lys Thr Thr Met Gln Gly Ile Leu Asp      50 55 60 Leu Pro Val Phe Asp Ala Thr Pro Met Lys Lys Pro Gly Thr Ser Asp  65 70 75 80 Val Asp Gly Asn Ala Lys Ala Val Asp Asp Thr Lys Glu Ala Leu Ala                  85 90 95 Gly Gly Lys Ile Leu His Asn Gln Asn Val Asn Asp Trp Glu Arg Val             100 105 110 Val Val Thr Pro Thr Ala Asp Gly Gly Glu Ser Arg Phe Asp Gly Gln         115 120 125 Ile Ile Val Gln Met Glu Asn Asp Asp Val Val Ala Lys Ala Ala Ala     130 135 140 Asn Leu Ala Gly Lys His Pro Glu Ser Ser Val Val Val Gln Ile Asp 145 150 155 160 Ser Asp Gly Asn Tyr Arg Val Val Tyr Gly Asp Pro Ser Lys Leu Asp                 165 170 175 Gly Lys Leu Arg Trp Gln Leu Val Gly His Gly Arg Asp Asp Ser Glu             180 185 190 Ser Asn Asn Thr Arg Leu Ser Gly Tyr Ser Ala Asp Glu Leu Ala Val         195 200 205 Lys Leu Ala Lys Phe Gln Gln Ser Phe Asn Gln Ala Glu Asn Ile Asn     210 215 220 Asn Lys Pro Asp His Ile Ser Ile Val Gly Cys Ser Leu Val Ser Asp 225 230 235 240 Asp Lys Gln Lys Gly Phe Gly His Gln Phe Ile Asn Ala Met Asp Ala                 245 250 255 Asn Gly Leu Arg Val Asp Val Ser Val Arg Ser Ser Glu Leu Ala Val             260 265 270 Asp Glu Ala Gly Arg Lys His Thr Lys Asp Ala Asn Gly Asp Trp Val         275 280 285 Gln Lys Ala Glu Asn Asn Lys Val Ser Leu Ser Trp Asp Glu Gln Gly     290 295 300 Glu Val Val Ala Lys Asp Glu Arg Ile Arg Asn Gly Ile Ala Glu Gly 305 310 315 320 Asp Ile Asp Leu Ser Arg Ile Gly Val Ser Asp Val Asp Glu Pro Ala                 325 330 335 Arg Gly Ala Ile Gly Asp Asn Asn Asp Val Phe Asp Ala Pro Glu Lys             340 345 350 Arg Lys Ala Glu Thr Glu Thr Ser Ser Ser Ser Ala Asn Asn Lys Leu         355 360 365 Ser Tyr Ser Gly Asn Ile Gln Val Asn Val Gly Asp Gly Glu Phe Thr     370 375 380 Ala Val Asn Trp Gly Thr Ser Asn Val Gly Ile Lys Val Gly Thr Gly 385 390 395 400 Gly Phe Lys Ser Leu Ala Phe Gly Asp Asn Asn Val Met Val His Ile                 405 410 415 Gly Asn Gly Glu Ser Lys His Ser Phe Asp Ile Gly Gly Tyr Gln Ala             420 425 430 Leu Glu Gly Ala Gln Met Phe Ile Gly Asn Arg Asn Val Ser Phe Asn         435 440 445 Leu Gly Arg Ser Asn Asp Leu Ile Val Met Met Asp Lys Ser Ile Pro     450 455 460 Thr Pro Pro Leu Val Asn Pro Phe Asp Gly Ala Ala Arg Ile Ser Gly 465 470 475 480 Val Leu Gln Ser Ile Ala Thr Ser Gly Glu Gly Gln Asp Trp Leu Ala                 485 490 495 Ala Gln Glu Gln Gln Trp Thr Leu Ser Gly Ala Lys Lys Phe Val Lys             500 505 510 Asp Met Ser Gly Leu Asp Gln Ser Ser Ser Val Asp Tyr Thr Ser Leu         515 520 525 Val Glu Leu Asp Ser Gln Asn Glu Arg Ser Ser Arg Gly Leu Lys His     530 535 540 Asp Ala Glu Ala Ala Leu Asn Lys Gln Tyr Asn Gln Trp Leu Ser Gly 545 550 555 560 Asn Ser Asp Ser Asp Thr Ser Lys Leu Ser Arg Ala Asp Lys Leu Arg                 565 570 575 Gln Ala Asn Glu Lys Leu Ala Phe Asn Phe Ala Val Gly Gly Gln Gly             580 585 590 Ala Asp Ile Gln Val Thr Thr Gly Asn Trp Asn Phe Met Phe Gly Asp         595 600 605 Asn Ile Gln Ser Ile Leu Asp Thr Asn Leu Gly Ser Leu Phe Gly Leu     610 615 620 Met Thr Gln Gln Phe Ser Ala Thr Gly Gln Ala Lys Thr Thr Phe Thr 625 630 635 640 Tyr Thr Pro Glu Asp Leu Pro Arg Gln Leu Lys Asn Lys Leu Leu Gly                 645 650 655 Gln Leu Ala Gly Val Gly Ala Glu Thr Thr Leu Ala Asp Ile Phe Gly             660 665 670 Val Asp Tyr Thr Ala Ser Gly Gln Ile Val Ser Arg Asn Gly Glu Ala         675 680 685 Val Asp Gly Val Ala Ile Leu Lys Glu Met Leu Glu Val Ile Gly Glu     690 695 700 Phe Ser Gly Asp Gln Leu Gln Ala Phe Val Asp Pro Ala Lys Leu Leu 705 710 715 720 Asp Ser Leu Lys Ala Gly Ile Asn Met Gly Ala Asp Gly Ile Lys Ser                 725 730 735 Phe Ala Glu Thr His Gly Leu Lys Glu Lys Ala Pro Glu Glu Glu Glu             740 745 750 Asp Asn Ser Ser Val Ser Val Asn Gly Ala Ser Val Asn Ser Ala Gln         755 760 765 Gly Ala Thr Val Ala Asp Gly Ser Thr Glu Thr Ala Glu Thr Pro Asp     770 775 780 Arg Ala Phe Gly Phe Asn Ser Leu Asn Leu Pro Asn Leu Phe Ala Thr 785 790 795 800 Ile Phe Ser Gln Asp Lys Gln Lys Glu Met Lys Ser Leu Val Glu Asn                 805 810 815 Leu Lys Glu Asn Leu Thr Ala Asp Leu Leu Asn Met Lys Glu Lys Thr             820 825 830 Phe Asp Phe Leu Arg Asn Ser Gly His Leu Gln Gly Asp Gly Asp Ile         835 840 845 Asn Ile Ser Leu Gly Asn Tyr Asn Phe Asn Trp Gly Gly Asp Gly Lys     850 855 860 Asp Leu Gly Ala Tyr Leu Gly Asp Asn Asn Asn Phe Trp Gly Gly Arg 865 870 875 880 Gly Asp Asp Val Phe Tyr Ala Thr Gly Thr Ser Asn Ile Phe Thr Gly                 885 890 895 Gly Glu Gly Asn Asp Met Gly Val Leu Met Gly Arg Glu Asn Met Met             900 905 910 Phe Gly Gly Asp Gly Asn Asp Thr Ala Val Val Ala Gly Arg Ile Asn         915 920 925 His Val Phe Leu Gly Ala Gly Asp Asp Gln Ser Phe Val Phe Gly Glu     930 935 940 Gly Gly Glu Ile Asp Thr Gly Ser Gly Arg Asp Tyr Val Val Thr Ser 945 950 955 960 Gly Asn Phe Asn Arg Val Asp Thr Gly Asp Asp Gln Asp Tyr Ser Val                 965 970 975 Thr Ile Gly Asn Asn Asn Gln Val Glu Leu Gly Ala Gly Asn Asp Phe             980 985 990 Ala Asn Val Phe Gly Asn Tyr Asn Arg Ile Asn Ala Ser Ala Gly Asn         995 1000 1005 Asp Val Val Lys Leu Met Gly Tyr His Ala Val Leu Asn Gly Gly Glu    1010 1015 1020 Gly Glu Asp His Leu Ile Ala Ala Ala Ile Ser Lys Phe Ser Gln Phe 1025 1030 1035 1040 Asn Gly Gly Glu Gly Arg Asp Leu Met Val Leu Gly Gly Tyr Gln Asn                1045 1050 1055 Thr Phe Lys Gly Gly Thr Asp Val Asp Ser Phe Val Val Ser Gly Asp            1060 1065 1070 Val Ile Asp Asn Leu Val Glu Asp Ile Arg Ser Glu Asp Asn Ile Val        1075 1080 1085 Phe Asn Gly Ile Asp Trp Gln Lys Leu Trp Phe Glu Arg Ser Gly Tyr    1090 1095 1100 Asp Leu Lys Leu Ser Ile Leu Arg Asp Pro Ala Ser Asp Ser Asp Gln 1105 1110 1115 1120 Ala Lys Phe Glu His Ile Gly Ser Val Thr Phe Ser Asp Tyr Phe Asn                1125 1130 1135 Gly Asn Arg Ala Gln Val Ile Ile Ala Met Gly Glu Lys Asp Ala Thr            1140 1145 1150 Gly Glu Arg Glu Tyr Thr Thr Leu Ser Glu Ser Ala Ile Asp Ala Leu        1155 1160 1165 Val Gln Ala Met Ser Gly Phe Asp Pro Gln Ala Gly Asp Asn Gly Phe    1170 1175 1180 Ile Asp Asn Leu Asp Ser Lys Ser Arg Val Ala Ile Thr Thr Ala Trp 1185 1190 1195 1200 Ala Asp Val Val His Lys Lys Gly Ile Thr Val                1205 1210 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 acatgaattc ataccatggc agagaagttt ggcgactac 39 <210> 8 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 8 ccattctcga gctaatgatg atgatgatga tgcgtgcctg ttgcgtagaa cac 53 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 acatgaattc ataccatggc agagaagttt ggcgactac 39 <210> 10 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 10 ccattctcga gctaatgatg atgatgatga tgctcacccg aggtggcaat gc 52

Claims (10)

Hybridoma cells of Accession No. KCLRF-BP-00261. A monoclonal antibody that binds to Vibrio sepsis RtxA1 antigen and heat treated Vibrio sepsis RtxA1 antigen produced by hybridoma cells of Accession No. KCLRF-BP-00261. The method of claim 2,
Monoclonal antibody, characterized in that binding to amino acid sites 3981 to 4380 of the Vibrio sepsis RtxA1 antigen. A composition for detecting vibrio sepsis comprising the monoclonal antibody of claim 2. A diagnostic kit for detecting Vibrio sepsis comprising the monoclonal antibody of claim 2. A method for detecting vibrio sepsis by contacting a monoclonal antibody of claim 2 with a biological sample to detect antigen-antibody complex formation. A method for detecting vibrio sepsis by contacting the monoclonal antibody of claim 2 with a heat treated biological sample to detect antigen-antibody complex formation. 8. The method of claim 6 or 7, wherein said detection is by ELISA or Western blot. Vibrio sepsis treatment comprising the antibody of claim 2. (A) immunizing a mouse using a recombinant protein of SEQ ID NO: 6 derived from Vibrio septic bacterium RtxA1 antigen or a fusion protein thereof;
(B) fusion and culture of splenocytes of the immunized mice with myeloma cells;
(C) selecting the hybridoma cells of Accession No. KCLRF-BP-00261 that produce a monoclonal antibody that binds to the Vibrio sepsis RtxA1 antigen in the fused hybridoma cells; Method for preparing bridoma cells.

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