KR101309386B1 - Monoclonal antibody specific to Vibrio vulnificus RtxA1, hybridoma producing the monoclonal antibody and diagnostic kit comprising the monoclonal antibody - Google Patents

Abstract

The monoclonal antibody produced in the hybridoma clone according to the present invention has excellent sensitivity and specificity, and can be very useful for quickly and accurately diagnosing and preventing and treating Vibrio sepsis infection. In addition, the recombinant protein RtxA1 antigen expressed in E. coli is excellent in sensitivity and specificity, and thus can be very useful for diagnosing, preventing, and treating an infection of Vibrio sepsis.
Meanwhile, since the monoclonal antibody of the present invention not only has excellent cross-reactivity to other types of vibrio sepsis, but also can bind to heat-treated vibrio sepsis, the examiner heat-processes the biological sample infected with Vibrio sepsis. It is very safe because it can eliminate the risk of infection by killing and then diagnose the infection.

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 primers 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. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. 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 may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, 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

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Claims (10)

A hybridoma cell of Accession No. KCLRF-BP-00261, which generates a monoclonal antibody that binds to amino acid sites 3981 to 4380 of the Vibrio sepsis RtxA1 antigen. A monoclonal antibody that binds to the amino acid sites 3981-4380 of the Vibrio sepsis RtxA1 antigen and the heat treated Vibrio sepsis RtxA1 antigen produced by the hybridoma cells of Accession No. KCLRF-BP-00261. delete 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; And
(C) selecting the hybridoma cells of Accession No. KCLRF-BP-00261 that produce a monoclonal antibody binding to amino acid sites 3981 to 4380 of the Vibrio sepsis RtxA1 antigen in the fused hybridoma cells;
Method for producing a hybridoma cell, characterized in that it comprises a.

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