KR101372766B1 - Recombinant recombinant vector for producing antigen for Vibrio vulnificus diagnosis and Monoclonal antibody specific to Vibrio vulnificus RtxA1 - Google Patents

Abstract

The present invention relates to recombinant proteins and expression vectors for the production of monoclonal antibodies that bind to the Vibrio sepsis RtxA1 antigen and to provide diagnostic antigens. More specifically, the present invention relates to Vibrio sepsis RtxA1 and thermodenatured RtxA1, Expression of monoclonal antibody specific for Vibrio sepsis RtxA1 that can be widely used in diagnostic methods, recombinant antigen for producing hybridomas secreting it, and recombinant protein which can be useful for diagnosing, preventing and treating Vibrio sepsis infection It relates to a recombinant expression vector.

Description

Recombinant recombinant vector for producing antigen for Vibrio vulnificus diagnosis and Monoclonal antibody specific to Vibrio vulnificus RtxA1}

The present invention relates to a recombinant protein and an expression vector for producing a monoclonal antibody that binds to the Vibrio sepsis bacteria RtxA1 antigen and to provide a diagnostic antigen, and more particularly, to Vibrio sepsis bacteria RtxA1 and heat-denatured RtxA1, having high reactivity and various Expression of a monoclonal antibody specific to Vibrio sepsis bacteria RtxA1 that can be widely used in diagnostic methods, a recombinant antigen to produce hybridomas that secrete it, and a recombinant protein useful for diagnosis, prevention and treatment of Vibrio sepsis bacteria infection. It relates to a recombinant expression vector.

Vibrio vulnificus is a basophilic pathogenic bacterium that inhabits estuaries near the sea and is associated with most of the deadly diseases associated with seafood. Although Vibrio septicemia infection has a relatively short history, it is one of the newly attracting attention due to global warming, which continues to increase clinical cases worldwide. In particular, the absolute number of cases worldwide is less than that of cholera or salmonella food poisoning, but it causes serious social problems due to the high mortality rate and tragic clinical symptoms.

Vibrio sepsis was first reported in 1976 by Hollis of the Centers for Disease Control (hereinafter referred to as CDC) and the bacteriological properties of the basophilic, pathogenic Vibrio bacteria isolated from humans for 11 years. , It was named lactose-fermenting Vibrio or Lac(+)) because of its ability to break down lactose. In 1979, CDC's Blake et al. analyzed the data of 39 patients reported to the CDC epidemiologically and classified them into primary septicemia group and wound infection group 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 named Vibrio vulnificus (vulnus=wound, ficus=forming) as a new species, and is reaching today (Farmer, J.J. III, Lancet 2:903, 1979).

V. vulnificus sepsis has a short incubation period, and once an onset develops, it is easy to miss the timing of effective antimicrobial treatment. V. vulnificus sepsis occurs mostly in men (90% or more) in their 40s or older (approximately 90-95%), rarely seen in normal people, and mainly occurs in patients with underlying diseases. Analysis of cases around the world shows that in the case of primary sepsis, most patients have chronic diseases such as liver disease and alcohol barrier, and liver cirrhosis, chronic hepatitis, and liver cancer are the main types, and diabetes, pulmonary tuberculosis, chronic liver disease, etc. Underlying diseases such as osteomyelitis and rheumatoid arthritis have been identified. However, there are cases where no specific underlying disease can be found in less than 5%. In the United States, diabetes, malignancies, hemochromatosis, and thalassemia are relatively high in frequency. Recently, there have been reports of V. vulnificus sepsis in AIDS patients, and the US CDC recommends that AIDS patients do not eat seafood such as oysters in summer. Therefore, in order to quickly determine the infection of Vibrio sepsis, prevent the rapid spread of the disease, and maximize the efficiency of the initial treatment, there is an urgent need to develop an effective and rapid diagnostic method capable of early diagnosis of infection.

Several virulence factors have been reported through the study of the pathogenesis of Vibrio sepsis, which began after the mid-1980s. That is, hemolytic toxin (Kreger, A, Lockwood, D, Infect. Immun. 33:583-590, 1981), metalloproteinase (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 membrane (Wright, AC, Simpson, LM, Oliver JD, Morris , JG, Jr., Infect.Immun. 58:1769-1773, 1990), Sideropore (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) are known. Among them, RtxA1 is a major virulence factor secreted at the beginning of Vibrio sepsis infection, and plays an important role in the spread of Vibrio sepsis bacteria and cell death through the intestine. This shows that RtxA1 is a useful target for the development of new diagnostic methods and therapeutics for early infections.

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

Furthermore, when a sample is collected and analyzed from a patient to diagnose Vibrio sepsis, there is a problem in that the tester is infected with Vibrio sepsis during the examination and processing process.

The present invention was conceived to solve the above-described problem, and the first object of the present invention is a monoclonal antibody capable of specifically binding to Vibrio sepsis bacteria RtxA1 and also capable of binding to high cross-reactivity and heat-denatured RtxA1, and high production thereof. It is to provide a recombinant expression vector that provides an antigenic protein that induces a high immune response for the production of bridoma cells.

The second object of the present invention is to provide a recombinant protein and expression vector that can be used for diagnosis, prevention, and treatment of Vibrio sepsis bacteria of the present invention.

In order to solve the first problem of the present invention, a sequence encoding amino acid region 3491 to 4701 (SEQ ID NO: 6) of the Vibrio sepsis bacteria RtxA1 antigen or a sequence encoding the fusion protein is included, and binds to the Vibrio sepsis bacteria RtxA1 antigen. A recombinant expression vector for producing a monoclonal antibody is provided.

According to a preferred embodiment of the present invention, the recombinant plasmid vector may have a cleavage map of FIG. 1.

According to another preferred embodiment of the present invention, (1) preparing the recombinant expression vector of the present invention described above; (2) transforming the recombinant expression vector into a host cell; And (3) culturing the transformed host cell to produce the recombinant protein of SEQ ID NO: 6 or a fusion protein thereof.

According to another preferred embodiment of the present invention, PCR may be performed using a pair of forward primers represented by SEQ ID NO: 1 and rear primers represented by SEQ ID NO: 2 before step (1).

According to another preferred embodiment of the present invention, after the step (3); (4) Immunizing the mice using the Vibrio sepsis bacteria RtxA1 recombinant protein; (5) culturing the splenocytes of the immunized mouse by fusion with Myeloma cells; (6) selecting hybridoma cells that produce monoclonal antibodies that bind to Vibrio sepsis RtxA1 antigen from the fused hybridoma cells.

According to another preferred embodiment of the present invention, there is provided a Vibrio sepsis infection diagnostic kit comprising the recombinant RtxA1 protein of SEQ ID NO: 6 or a fusion protein thereof. In addition, the recombinant protein RtxA1 (3491-4701) expressed in the RtxA1 recombinant expression vector according to the present invention has excellent specificity for serum containing polyclonal antibodies.

According to another preferred embodiment of the present invention, there is provided a composition for diagnosing Vibrio sepsis infection, comprising the recombinant RtxA1 protein of SEQ ID NO: 6 or a fusion protein thereof and a carrier.

Hereinafter, terms used in the present specification will be briefly described.

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

It includes known analogues of tide, and unless otherwise specified, specific nucleic acid sequences include their complementary sequences.

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

The term'recombinant' as used in connection with a cell refers to a cell that replicates a heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid. Also, recombinant cells can express genes found in the original form of cells, but modified genes are sometimes reintroduced into cells by artificial methods.

'Primer' means a synthetic or natural oligonucleotide. The primer serves as a starting point of synthesis under conditions in which the synthesis of a primer extension product complementary to the template is induced, that is, the presence of a polymer such as nucleotide and DNA polymerase, and a suitable temperature and pH condition. For maximum efficiency of amplification, preferably the primer is single-stranded. Preferably, the primer is a deoxyribonucleotide. The primers of the present invention may include naturally occurring dNMPs (ie dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include a ribonucleotide. For example, the oligonucleotide of the present invention is a backbone modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365:566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoroamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexy Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidine (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, and Tityl-, propynyl-, alkynyl-, thiazoryl-, imidazoryl-, and pyridyl- included), 7-deazapurine having a C-7 substituent (substituents are fluoro-, bromo-, chloro- , 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. In order to be a useful vector, it must be able to be replicated, must be equipped with a way to enter the host cell, and must have a means to detect its existence. Herein, the foreign gene refers to a sequence encoding the 3491-4701 amino acid region of the Vibrio septicemia RtxA1 antigen or a sequence to which a His-tag sequence is added to facilitate purification at the end of the sequence.

'Plasmid' generally refers to a circular DNA molecule formed by operatively linking to a vector so that a foreign gene can be expressed in a host cell. However, the plasmid can be degraded by a specific restriction enzyme by gene recombination and used as a vector for introducing a new gene to construct a plasmid containing a desired gene. Therefore, in the present application, plasmids and vectors are used interchangeably, and those of ordinary skill in the field of genetic engineering will fully understand their meaning even if they do not distinguish between their names.

"Fusion protein" refers to Vibrio pneumococcal RtxA1, which has a His-tag attached to the C-terminus, for the convenience of protein purification in the future. it means. Therefore, although expressed as "fusion protein" in the present invention, it does not necessarily mean that the His-tag is attached, and if the inconvenience of protein purification is tolerated, the fusion protein may not be used. Therefore, in the present invention, the term "recombinant Vibrio pneumococcal RtxA1" is defined as an abbreviation of "Vibrio sepsis bacteria RtxA1" or "Vibrio sepsis bacteria RtxA1 in which a chain for convenience of purification is added to the N-terminus or C-terminus".

The term "monoclonal antibody" of the present invention refers to a highly specific antibody directed against a single antigenic site (epitope) as a term known in the art. Typically, unlike polyclonal antibodies, which contain 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 of not being contaminated by other immunoglobulins because they are produced by cultivation of hybridomas. .

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

The term "antigen-antibody complex" as used herein refers to a combination of the Vibrio pneumococcal RtxA1 protein and a monoclonal antibody reacted to confirm the expression of Vibrio pneumococcal RtxA1 in a biological sample.

Recombinant RtxA1 (3491-4701) according to the present invention provides a recombinant expression vector containing a recombinant RtxA1 protein antigen that is very useful for the production of monoclonal antibodies of Vibrio septicemia RtxA1. In addition, it provides a very useful recombinant RtxA1 (3491-4701) protein antigen for diagnosis and prevention using Vibrio septicemia RtxA1, and facilitates mass expression and purification of the recombinant RtxA1 (3491-4701) protein antigen.

The monoclonal antibody produced from the hybridoma clone according to the present invention has excellent sensitivity and specificity, and can be very useful for rapid and accurate diagnosis and prevention of infection with Vibrio sepsis. In addition, the recombinant protein RtxA1 antigen expressed in E. coli has excellent sensitivity and specificity and can be very useful for diagnosis and prevention of infection of Vibrio sepsis.

On the other hand, the monoclonal antibody derived from the recombinant RtxA1 expressed in the recombinant expression vector of the present invention not only has excellent cross-reactivity against other types of Vibrio sepsis bacteria, but also can bind to the RtxA1 antigen of heat-treated Vibrio sepsis bacteria. In addition to diagnosing sepsis, it is very safe because an examiner can perform a diagnosis of infection after removing the risk of infection by heat treatment of a biological sample infected with Vibrio sepsis bacteria to kill Vibrio sepsis bacteria.

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

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

The present inventors prepared a recombinant expression vector comprising a sequence encoding the amino acid region 3491 to 4701 (SEQ ID NO: 6) of the Vibrio sepsis RtxA1 antigen or a sequence encoding the fusion protein, and transformed it into a host cell. By expression, a recombinant RtxA1 protein was prepared. The recombinant RtxA1 protein was used as an immunogen to immunize mice, and a hybridoma clone was prepared in which the splenocytes of the immunized mouse and P3X63Ag8.653, which are B-lymphoblasts, are fused, from which Vibrio sepsis bacteria RtxA1 The present invention was completed by confirming the sensitivity and specificity of the recombinant RtxA1 protein expressed in the recombinant expression vector of the present invention by producing a monoclonal antibody against the antigen. Some monoclonal antibodies produced using the recombinant protein according to the present invention as an antigen showed excellent reactivity to the Vibrio sepsis bacteria RtxA1 antigen, and a cross-reaction with the heat-denatured RtxA1 antigen and several Vibrio sepsis strains. This can be used as a therapeutic agent since the monoclonal antibody of the present invention can be used in various diagnostic methods such as an indirect immunofluorescent antibody, an immunoblot method, and an enzyme immunological antibody method, and a neutralizing antibody of the virulence factor RtxA1. In addition, the recombinant RtxA1 (3491-4701) protein, a vibrio sepsis bacterium virulence factor of the present invention, can be used to develop a therapeutic agent using bacterial toxin.

Meanwhile, each monoclonal antibody produced in 10 hybridoma cells fused using the recombinant RtxA1 protein as an immunogen is a monoclonal antibody that binds to amino acids 3491 to 3980, depending on the amino acid binding site of RtxA1 in Vibrio sepsis bacteria. They can be classified into monoclonal antibodies that bind to amino acids 3981 to 4380 and monoclonal antibodies that bind to amino acids 4381 to 4701. Among these, the possibility of mass production according to the isotype, reactivity to heat-denatured antigens, affinity with RtxA1 antigen, and cross-ability to other types of Vibrio sepsis, etc., are summarized as 5RA (binding to amino acids 3981 ~ 4380), 11RA. (Bound to amino acids 3491 ~ 3980) showed the best effect. Accordingly, the 5RA was deposited with the Korean Cell Line Research Foundation under the accession number KCLRF-BP-00261, and the 11RA was deposited with the accession number KCLRF-BP-00262 (see Table 1).

Specifically, according to a preferred embodiment of the present invention, as the step (1), the sequence represented by SEQ ID NO: 3 encoding the Vibrio sepsis bacteria RtxA1 protein of SEQ ID NO: 4 as a template, and 3491 of the amino acid sequence of Vibrio sepsis bacteria RtxA1 In order to selectively amplify the amino acid site at position ~ 4701, PCR is performed using the forward and rear primers of SEQ ID NOs: 1 and 2, which are novel primer sequences.

Preferably, the primer used in the present invention may be hybridized or annealed at one site of the template to form a double-chain structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are Joseph Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

Preferably, various DNA polymerases can be used in the amplification step of the present invention, E. coli "Klenow" fragments of DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which comprises Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Includes. Most of these polymerases can be isolated from the bacteria themselves or can be purchased commercially. Further, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase.

When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with the components necessary for the reaction in excess. The excessive amount of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component. So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is preferred.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Next, as step (2), a sequence encoding the amino acid at positions 3491 to 4701 of the amplified Vibrio sepsis RtxA1 amino acid sequence (SEQ ID NO: 5) or a conventional His The sequence to which the -tag sequence has been added is inserted into the expression vector.

On the other hand, the recombinant expression vector of the present invention is a strong promoter capable of proceeding transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL promoter, pR promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter) , trp promoter and T7 promoter, etc.), ribosome binding site for initiation of translation, and transcription/translation termination sequence, and most preferably, when E. coli is used as a host cell, E. coli tryptophan Promoter and operator sites of the biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left-handed promoter of phage (pL promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as a regulatory site.

Preferred expression vectors of the present invention are plasmids often used in the art (e.g. pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phage (e.g., gt4?B, -Charon, z1 and M13, etc.) ) Or virus (eg, SV40, etc.), but most preferably, it is most advantageous for mass production to use pET-21(a) having a cleavage map of FIG. 1.

It is most advantageous for mass production to use E. coli as the host cell transformed with the recombinant expression vector of the present invention described above.

Meanwhile, in order to facilitate the purification of the recombinant RtxA1 protein expressed therefrom, the recombinant expression vector of the present invention has glutathione S-transferase (GST), maltose binding protein (MBP), FLAG, 5 to 7x inside the recombinant expression vector. A nucleotide sequence encoding His (hexahistidine), NusA (N utilization substance A) or Trx (Thioredoxin) may be additionally included. However, most preferably, when a nucleotide sequence encoding 6x His (hexahistidine) is additionally included at the C-terminus of the recombinant RtxA1 protein, the expression efficiency and purification efficiency can be maximized. Because of the additional sequence for the purification, the protein expressed in the host can be quickly and simply purified through affinity chromatography or the like.

On the other hand, the recombinant expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, and for example, there is a resistance gene for ampicillin.

Next, as step (3), the recombinant expression vector host cell is transformed to produce a recombinant RtxA1 protein.

Specifically, more preferably, the recombinant expression vector used in the mass production method of the present invention is the same as described above, so the description thereof will be replaced. On the other hand, the method of transporting the recombinant expression vector into the host cell, E. coli, can be carried out by a conventional method, and in detail, the CaCl ₂ method (Cohen, SN etal., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580 (1983)) and an electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)).

Then, in order to obtain the fusion protein expressed in the cultured host cell, the host cell (E. coli) may be disrupted. The crushing method may be performed using a method known in the art, for example, a French press, ultrasonic crushing, a bead mill, or a Manton-Gaulin homogenizer. Subsequently, the expressed protein may be purified by Ni-NTA affinity chromatography, but is not limited thereto.

Afterwards, the recombinant RtxA1 (3491-4701) protein, which was first purified by Ni-NTA affinity chromatography, was secondarily purified using gel filtration liquid chromatography (size exclusion-HPLC) and injected into the peritoneal cavity of the mouse. The splenocytes are excised, fused with Myeloma cells, and cultured to obtain a plurality of hybridoma cells. Subsequently, the possibility of mass production, cross-reactivity, and binding ability of the heat-treated Vibrio sepsis bacteria RtxA1 protein were comprehensively evaluated for the monoclonal antibodies produced in the hybridoma cells, and the excellent sensitivity and specificity of the recombinant RtxA1 protein antigen were evaluated. .

In addition, the present invention provides a diagnostic kit for detecting infection of Vibrio sepsis in humans, mammals, and fish and shellfish, including the recombinant protein or variant. In the diagnostic kit of the present invention, the recombinant protein or variant is preferably prepared by the method according to the present invention. In addition, the detection method used in the diagnostic kit of the present invention is preferably an ELISA method, a sandwich ELISA method, or a rapid immunochromatographic flow assay (RFA).

In the case of the ELISA kit, after reacting a sample as a primary antibody to a plate coated with the recombinant protein antigen of the present invention, a secondary antibody labeled with peroxidase was bound, and then OPD (orange), ABTS (green) or TMB (pink) ) Can be configured to react with a substrate. In the case of a rapid immunochromatographic flow assay (RFA) kit, after adsorbing and immobilizing the recombinant protein antigen of the present invention on gold, it is dropped onto a membrane on a glass pad, and horseradish peroxidase or alkaline phosphatase is added thereto. After instillation of the bound secondary antibody, it can be configured to react by dropping a sample of serum onto a glass pad.

The diagnostic kit of the present invention may further include one or more recombinant antigens in order to diagnose pathogens other than Vibrio sepsis bacteria together.

Meanwhile, monoclonal antibodies can be produced by a fusion method well known in the art (Kohler et al., European Journal of Immunology 6;511-519). In general, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells and cancer cell lines from an immunologically suitable host animal, such as a mouse injected with an antigenic protein. The fusion of these two populations of cells can be fused using a method known in the art, such as polyethylene glycol, and the antibody-producing cells can be proliferated by standard culture methods. After subcloning by limited dilution is performed to obtain a uniform cell population, hybridoma cells capable of producing antigen-specific antibodies can be cultured in large quantities in vitro or in vivo.

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

Hybridomas that secrete monoclonal antibodies can be cultured in large quantities in vitro or in vivo. The monoclonal antibody produced by the hybridoma may be used without purification, but in order to obtain the best result, it is purified to high purity (eg, 95% or more) according to a method well known in the art. It is preferable to use. As such a purification technique, it can be separated from a culture medium or an ascites fluid using a purification method such as gel electrophoresis, dialysis, salt precipitation, chromatography, or the like.

The monoclonal antibody of the present invention binds to an antigen, inhibits or neutralizes its action, and can further kill pathogens and cells infected with pathogens. For example, a monoclonal antibody that specifically recognizes RtxA1, a toxin moiety of Vibrio septicemia, binds to it and can inhibit or neutralize its action, and an antibody bound to an antigen can activate complement and activate it. The antigen can be removed by complement. In addition, monoclonal antibodies may bind to antigens and make the antigens more eaten 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 are used to eliminate antigens and Vibrio sepsis bacteria that produce these antigens through immune reactions. I can. Therefore, the antibody of the present invention can be used for diagnosis or treatment of Vibrio septicemia, since the antigen and Vibrio sepsis can be eliminated and reduced through an immune response by itself.

Those skilled in the art will readily understand that the monoclonal antibodies according to the present invention can be converted into chimeric antibodies, humanized antibodies, and human monoclonal antibodies with reduced immunogenicity for human application. Such chimeric antibodies are obtained by recombining the variable region of the monoclonal antibody of the present invention with the constant region of a human antibody, and the humanized antibody is a complementarity determining region that directly binds to an antigen in the variable region of the monoclonal antibody of the present invention. regions: CDRs) or complementarity determining regions, only amino acid residues involved in antigen binding specificity (specificity determining residues: SDRs) were grafted into human antibodies.

Human antibodies replace the heavy chain variable region or light chain variable region of the variable region of the monoclonal antibody of the present invention in place of the heavy chain variable region or light chain variable region of a human antibody, and as a result, a hybrid showing antigen-binding ability (hybrid: mouse heavy chain/human light chain Alternatively, from the mouse light chain/human heavy chain) antibody, the mouse heavy chain variable region or light chain variable region is replaced with a human heavy chain variable region or light chain variable region to select a fully human antibody variable region that exhibits antigen-binding ability, and then connect it to the human antibody constant region. It can be easily prepared from the monoclonal antibody of the present invention by using a known method such as lethal, and it is natural that such a variant belongs to the scope of the present invention. The chimeric antibody, humanized antibody and human monoclonal antibody prepared as described above can be produced in animal cells using a known method.

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 a total length of two heavy and two light chains, but also as a functional fragment of an antibody molecule for the treatment and diagnosis of Vibrio sepsis. Can be used. The functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may include Fab, F(ab'), F(ab')2, Fv, and the like.

According to a preferred embodiment of the present invention, the present invention provides a kit for diagnosing Vibrio sepsis comprising the monoclonal antibody described above. The monoclonal antibody used in the diagnostic kit for the detection of Vibrio sepsis of the present invention may also be a fragment of a monoclonal antibody 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.

The Vibrio sepsis diagnostic kit of the present invention may include a monoclonal antibody or fragment thereof that selectively recognizes the RtxA1 antigen, and tools/reagents used for immunological analysis.

Tools/reagents used for immunological analysis may include a suitable carrier, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, and the like. In addition, when the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction terminator may be included.

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, polyester, Polyacrylonitrile, fluorine

Resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

The assay system for use in the detection method and diagnostic kit for antigen-antibody complex formation of the present invention includes, but is not limited to, an ELISA plate, a dip-stick device, an immunochromatographic test strip, and a radiation split immunoassay device, and 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, There are Complement Fixation Assay, FACS, and protein chip, but are not limited thereto.

The pharmaceutical composition containing 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 used.

The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt thereof, 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 is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. I can. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, sucrose ( sucrose), lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

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

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Synthesis of Oligonucleotides for Synthesis of Recombinant RtxA1 in Vibrio Sepsis

The following two oligonucleotides having NheI and XhoI restriction enzyme recognition sites were synthesized. In addition, the RA-4701R oligonucleotide contains 6 histidines to further include the purification process by chromatograph using a histidine-binding resin during the purification process of recombinant RtxA1.

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

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

The nucleotide was used for the subsequent amplification of the Vibrio sepsis bacterium RtxA1 gene (Example 2 below) and for the production of a recombinant vector for the production of the recombinant RtxA1 protein.

<Example 2> Polymerase chain reaction (PCR)

Vibrio sepsis bacterium RtxA1 full-length DNA (SEQ ID NO: 3, 14106bp) was used as a template in a reaction solution in which the polymerase and the forward primer (SEQ ID NO: 1) and the rear primer (SEQ ID NO: 2) synthesized in Example 1 were mixed. By mixing and carrying out polymerase chain reaction, the amino acid sequence 3491 to 4701 of Vibrio sepsis bacteria RtxA1 was amplified (referred to as RtxA1 (3491-4701)). SEQ ID NO: 4 is the amino acid sequence of full-length Vibrio sepsis bacteria RtxA1. For the neutralization chain reaction, a thermal cycler (PTC-100), MJ Research. Inc., USA was used, and after predenaturation at 94°C for 3 minutes, denaturation at 94°C for 1 minute, Annealing at 55° C. for 1 minute, and extension at 72° C. for 4 minutes were repeated 30 times, and finally, an additional reaction was performed at 72° C. for 10 minutes. Next, in order to remove the polymerase and non-specific amplification products, after electrophoresis on an agarose gel, the desired band of the desired amplification product was fragmented and purified using a gel extraction kit (Geneall, Korea).

<Example 3> Construction of vector for production of recombinant RtxA1

A recombinant RtxA1 plasmid vector having a cleavage map of FIG. 1 was constructed. Specifically, the amplification product of RtxA1 (3491-4701) in which the His-tag sequence, CATCATCATCATCAT, was inserted into the C-terminus purified in Example 2, was cut with EcoRI (Fermentas, Canada), and then E. coli polymerase Klenow (New England Biolab, USA) was used to fill in the cut surface and cut with XhoI (Fermentas, Canada). In addition, pET-21(a) (Novagen, USA) vector was digested with NheI (Fermentas, Canada), and then the cut surface was filled with E. coli polymerase Klenow (New England Biolab, USA), and XhoI (Fermentas, Canada). The cut fragment and vector were purified using the gel extraction kit used in Example 2 (Geneall, Korea), and then 1 μg of the purified RtxA1 gene fragment and 30 ng of the pET-21 (a) fragment were mixed, 10 times. Concentration of conjugation concentrate (10 mM DTT, 100 mM MgCl2, 10 mM ATP, 600 mM Tris-HCl, pH7.5) 1 µl and T4 DNA ligase (Takara) 10 units were added to make the total volume 10 Μl and reacted at room temperature for 16 hours, a circular plasmid having a cleavage map of 9,027 bp in which the recombinant RtxA1 gene (SEQ ID NO: 5 + His-tag sequence) is inserted into the pET-21 (a) vector (pET- RtxA1 (designated 3491-4701)) was prepared. Next, the plasmid was amplified and inserted into E. coli DH5α (Invitrogen, USA) to purely isolate pET-RtxA1 (3491-4701) into which the RtxA1 (3491-4701) gene (SEQ ID NO: 5) was inserted. After re-extraction of recombinant pET-RtxA1 (3491-4701) from the transformed E. coli, DNA sequencing, restriction enzyme treatment, and agarose gel electrophoresis were performed. -4701) The presence or absence 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, it was inserted into E. coli BL21 (DE3)/pLy S (Invitrogen, USA). The transformed E. coli was incubated for 4 hours in a shaking incubator at 37°C, and then 1mM IPTG was added to incubate for another 3 hours. After centrifuging the transformed E. coli cultured in this way at 8,000 rpm for 30 minutes, the E. coli precipitate was used as an elution buffer (300 mM NaCl, 25 mM Tris-HCl (pH 7.5), 10% glycerol, 0.1% Tween-20). Suspended. The E. coli precipitate suspended for protein extraction was crushed using a sonicator, and then the supernatant protein extract was collected by centrifugation, and 10% SDS-PAGE was performed on the cell lysate (FIG. 2A). In FIG. 2A, the molecular weight is indicated on the right side of FIG. 2A, and lanes 1 and 2 are proteins extracted before and after expressing the recombinant RtxA1 protein from 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. 2A, it can be seen that the recombinant RtxA1 (3491-4701) protein was expressed in E. coli transformed with pET-RtxA1 (3491-4701).

<Example 5> Purification of recombinant RtxA1 (3491-4701) protein

After extracting the protein from E. coli transformed with the recombinant RtxA1 (3491-4701) vector pET-RtxA1 (3491-4701) transformed by the method of Example 4, Ni-NTA chromatography (Qiagen, USA) was performed according to the manufacturer's instructions. The recombinant RtxA1 (3491-4701) protein was primarily purified from the protein extract. Recombinant RtxA1 (3491-4701) protein, which was first purified using Ni-NTA chromatography, was purified secondarily using gel filtration liquid chromatography (size exclusion-HPLC) (GE Healthcare, USA). 3491-4701) protein (SEQ ID NO: 6 + His-tag) was produced. Purification aspect and purification efficiency were confirmed by performing SDS-PAGE (FIG. 2A). In FIG. 2A, lanes 3 and 4 are proteins purified by Ni-NTA chromatography and gel filtration liquid chromatography, respectively, and arrows indicate positions of purified recombinant RtxA1 (3491-4701) protein. As confirmed in lane 4 of FIG. 2A, the purified recombinant RtxA1 (3491-4701) protein showed a purity of 98%.

<Example 6> Analysis of the specificity of the recombinant RtxA1 (3491-4701) protein

The 130 kDa recombinant RtxA1 (3491-4701) protein identified in Example 5 and the control bovine serum albumin (BSA) protein were subjected to SDS-PAGE, and after the electrophoresis was completed, the gel protein was added to the ( Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA 1979; 76:4350-4354) was transferred to a Nitrocellulose membrane (Bio-Rad) according to the method. The protein-transferred membrane was blocked with a phosphate buffer solution containing 5% skim milk to block non-specific reactions. Here, 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 weeks old) mice 4 times at 3 weeks intervals. One month later, a 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) The secondary antibody was diluted and used according to the manufacturer's instructions. After reacting with the first and second antibodies, they were washed three times with a phosphate buffer solution. The membrane after the secondary antibody reaction and washing was treated with an ECL western blot substrate solution (Amersham, USA), and the results were analyzed using a LAS-1000 luminescent image analyzer (Fujifilm, Japan). (Figure 2B). In FIG. 2B, lane 1 is a control bovine serum albumin (BSA) protein, and lane 2 is a prepared RtxA1 (3491-4701) protein. 2B, the recombinant RtxA1 (3491-4701) protein showed a positive reaction to the antibody against the Vibrio sepsis bacteria RtxA1 protein, but the control bovine iron albumin did not. Therefore, it was confirmed that the purified 130 kDa recombinant protein in Example 5 was RtxA1 (3491-4701) protein.

Consequently, the recombinant RtxA1 (3491-4701) according to the present invention provides a highly useful recombinant RtxA1 protein antigen for the production of monoclonal antibodies of Vibrio septicemia RtxA1. In addition, it provides a very useful recombinant RtxA1 (3491-4701) protein antigen for diagnosis using Vibrio septicemia RtxA1, and facilitates mass expression and purification of the recombinant RtxA1 (3491-4701) protein antigen. Therefore, the recombinant RtxA1 (3491-4701) protein expressed in the transformed E. coli can be used as a diagnostic agent or diagnostic kit for Vibrio sepsis, a vaccine for preventing or treating Vibrio sepsis, and the production of antibodies against Vibrio sepsis. I can.

<Example 7> Mouse immunization for production of monoclonal antibody-producing cell lines

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. Each 200 µl was injected into the abdominal cavity of BALB/C (female, 8 weeks old) mice. One month after the fourth immunization, the spleen was excised and used for cell fusion 3 days after administration of the purified recombinant RtxA1 (3491-4701) protein into the tail vein.

<Example 8> B-lymphoblast P3X63Ag8.653 culture

Take out myeloma cells P3X63Ag8.653 (ATCC accession number CRL-1580) from a nitrogen tank 4-5 days before cell fusion, and suspend them in IMDM (Invitrogen, USA) medium added with 10% fetal calf serum, and for 5 minutes at 1,000 rmp. Centrifuged. The supernatant was discarded, and the precipitated cells were carefully resuspended in IMDM to which 10% fetal calf serum was added, and cultured in an incubator at 37° C. supplied with 5% carbon dioxide gas.

<Example 9> Cell fusion

Cell fusion was performed as follows according to the general method using polyethylene glycol (Ed Harlow, David Lane: Antibodis, A laboratory manual. Cold Springs Harbor press, 1988 P139-244). Splenocytes from the spleen extracted in Example 7 were isolated using a cell strainer (Falcon, USA) and diluted to a concentration of 1 ^{×10 8 cells/ml.} 1×10 ⁷ cells/ml of myeloma cells and 1 ml of PEG1500 (Sigma, USA) were mixed in equal amounts and fused. After fusion was completed, 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> Investigation of RtxA1 (3491-4701) antibody production in cell fusion hybridoma

Whether the fused cells produced antibodies was investigated by ELISA using a cell culture medium fused with the recombinant RtxA1 (3491-4701) antigen expressed in E. coli. Recombinant RtxA1 (3491-4701) antigen protein was diluted with carbonate buffer (pH 9.4) to a concentration of 10 µg/ml, and then 100 µl per well of Maxisorp ELISA plate (Nunc, USA) was added, and 4℃ The reaction was carried out for 16 hours to coat the antigen. Blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) was treated in each antigen-coated well to block non-specific reactions at 37°C for 1 hour I did. 50 µl of the cell culture solution fused to each well was added, reacted at 4° C. for 1 hour, and washed 3 times with a washing buffer solution (PBS, 0.05% Tween-20, 0.05% BSA). After washing, a 1:1000-fold diluted Biotin-condensed anti-mouse IgG+IgA+IgM antibody (1:2,000; Sigma, USA) was added at a rate of 100 μl per well and reacted at 37° C. for 1 hour, followed by washing buffer solution. Washed 3 times. After washing, 100 μl of HRP (Horseradish peroxidase)-condensed streptavidin (Invitrogen, USA) was added to each well, reacted at 37° C. for 1 hour, washed 3 times with a washing buffer solution, and subsequently TMB (3,3', 5,5'-tetramethyl-benzidine) (Sigma, USA) substrate solution was added to each well by 100 µl, reacted in the dark for 30 minutes _{to develop color, and then treated with 2N H 2} SO ₄ to stop the enzyme reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. As a result, 10 hybridomas reacting with the antigen of Vibrio sepsis bacteria RtxA1 (3491-4701) were obtained (Table 1).

<Example 11> Cloning of hybridoma cells

For the cloning of 10 fused hybridoma cells, hybridomas that react with the Vibrio sepsis bacterium RtxA1 (3491-4701) antigen obtained in Example 5 were added to the first well of a 96-well plate so that about 10 cells could enter the cells. After the suspension was added, it was diluted twice in a row, and then cloned twice by diluting it in two-fold steps with a column. The selected hybridoma clones were suspended in IMDM medium containing 30% fetal calf serum and 7.5% DMSO (dimethyl sulfoxide) and stored in liquid nitrogen.

<Example 12> Purification of monoclonal antibody

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

<Example 13> Selection of monoclonal antibodies by isotyping

The determination of the isotype of the monoclonal antibody from the selected 10 hybridoma clones was investigated by performing ELISA. For isotype determination, purified antibodies to each mouse isotype fished in rabbits were diluted to 10 ㎍/㎖ concentration with carbonate buffer (pH 9.4), and then used in Maxisorp ELISA plate (Nunc, USA). 100 μl of each well was added and reacted at 4° C. for 16 hours to coat the antibody. Each isotype antibody-coated well was treated with a blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactivated horse serum) at 37°C for 1 hour. Blocking specific reactions. 50 µl of the cell culture solution fused to each well was added, reacted at 4° C. for 1 hour, and washed 3 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-fold after actual washing was added 100 μl per well and reacted at 37° C. for 1 hour Then, it was washed three times with a washing buffer solution. After washing, 100 µl of TMB (3,3',5,5'-tetramethylbenzidine) (Sigma, USA) substrate solution was added to each well, reacted in the dark for 30 minutes to develop color, and then treated with 2N H2SO4 to stop the enzyme reaction. Made it. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. As a result of determining the isotype, as summarized in Table 1 below, when classified according to the heavy chain subclass, 6 were IgG1 subclass, 3 were IgG2a subclass, and the other 1 was IgG2b subclass. In general, IgM antibodies have the property of non-specific adsorption with components other than the solid surface or target antigen used for the development of diagnostic agents. Therefore, all ten monoclonal antibodies obtained through the present invention were selected as antibodies that can be used as diagnostic monoclonal antibodies. Subsequently, in all experiments, characterization was performed using only this antibody.

<Example 14>: Expression of RtxA1 (3491-4701) fragment for affinity analysis and epitope analysis of monoclonal antibodies

Recombinant RtxA1 (3491-4701) protein containing amino acid sequences 3491 to 4701 of Vibrio sepsis bacterium RtxA1 was expressed in Example 4. The gene site of RtxA1 (3491-4380) containing amino acid sequences 3491 to 4380 of Vibrio sepsis bacteria RtxA1 is the oligonucleotide and polymerase of the following two (RA-3491F2 and RA-4308R) having EcoRI and XhoI restriction enzyme recognition sites. Amplification was performed in the same manner as in Example 2 using a chain reaction.

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

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

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

RA-3491F2: 5'-ACATGAATTCATACCATGGCAGAGAAGTTTGGCGACTAC-3'' (SEQ ID NO: 9, forward primer)

RA-3980R: 5'-CCATTCTCGAGCTAATGATGATGATGATGATGCTCACCCGAGGTGGCAATGC-3' (SEQ ID NO: 10, rear primer)

The amplified RtxA1 (3491-4380) and RtxA1 (3491-3980) gene sites were inserted into the pET-21(a) vector in the same manner as in Example 3, and transformed E. coli using the same method as in Example 4. Using the recombinant RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins were expressed and extracted (Fig. 4B). The specificity analysis of the extracted recombinant protein was confirmed using the same method as in Example 6 (Fig. 4C). Figure 4A schematically shows the recombinant RtxA1 (3491-4701) fragment protein expressed in E. coli. In Figure 4B, the molecular weight size is indicated on the left side of the figure, and lanes 1, 2, 3 and 4 are respectively the control recombinant protein and recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), RtxA1 (3491- 3980). Arrows indicate the positions of the expressed recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins. In FIG. 4C, the specificity of recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins was analyzed using an antibody against Vibrio septicemia RtxA1 protein. The molecular weight size is indicated on the left side of the figure, and lanes 1, 2, 3 and 4 are respectively a control recombinant protein, recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), RtxA1 (3491-3980). It is a protein extracted from expressing transformed E. coli. Arrows indicate the positions of RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins that show a positive reaction to the RtxA1 protein. Recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) were identified through FIGS. 4B and 4C.

<Example 15> Affinity analysis and epitope analysis of monoclonal antibodies

Affinity analysis and epitope analysis of monoclonal antibodies used as useful information for selection of diagnostic and therapeutic monoclonal antibodies were performed by fragmentation of recombinant RtxA1 (3491-4701) identified in Example 14 and ELISA. Transgenic E. coli extracts expressing recombinant RtxA1 (3491-4701), RtxA1 (3491-4380), and RtxA1 (3491-3980) proteins were diluted with carbonate buffer (pH 9.4), respectively, and then Maxisorp ELISA plates (Nunc , U.S.) was added at a rate of 100 μl per well, and reacted at 4° C. for 16 hours to coat each of the recombinant proteins. Each well coated with each recombinant protein was treated with a blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) at 37°C for 1 hour. The reaction was blocked. 50 μl of cell culture solution containing monoclonal antibody was added to each well, reacted at 4° C. for 1 hour, and washed 3 times with a washing buffer solution (PBS, 0.05% Tween-20, 0.05% BSA). After washing, a 1:1000-fold diluted Biotin-condensed anti-mouse IgG+IgA+IgM antibody (1: 2,000; Sigma, USA) was added at a rate of 100 μl per well and reacted at 37° C. for 1 hour, followed by washing buffer solution. Washed 3 times. After washing, 100 µl of HRP (Horseradish peroxidase)-condensed streptavidin was added to each well, reacted at 37°C for 1 hour, washed three times with a washing buffer solution, and subsequently TMB (3,3',5,5'- Tetramethyl-benzidine) (Sigma, USA) substrate solution was added to each well at a rate of 100 µl, reacted in the dark for 30 minutes _{to develop color, and then treated with 2N H 2} SO ₄ to stop the enzyme 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 the monoclonal antibody, as summarized in Table 1, five (5RA, 7RA, 8RA, 9RA, 11RA) strongly recognized the antigen of the recombinant protein RtxA1 (3491-4701). On the other hand, the 1RA and 10RA monoclonal antibodies recognized the RtxA1 (3491-4701) antigen with moderate intensity, and the 2RA, 3RA and 4RA monoclonal antibodies weakly recognized the RtxA1 (3491-4701) antigen. The 2RA, 3RA, 8RA and 11RA monoclonal antibodies recognized the amino acid sequence 3491 to 3980 of RtxA1, and the 1RA, 5RA, 7RA and 9RA monoclonal antibodies bound to the amino acid sequence 3981 to 4380 of RtxA1. Monoclonal antibodies to 4RA and 10RA recognized amino acid sequences 4381 to 4701 of RtxA1. Therefore, three groups of monoclonal antibodies with different epitopes were obtained through the present invention, and the monoclonal antibodies of each group can be usefully used in basic research on Vibrio sepsis bacteria as well as in the development of antigen diagnostic agents for Vibrio sepsis. have.

<Example 16> Analysis of the reactivity of a monoclonal antibody against the heat-modified RtxA1 (3491-4701) antigen.

Monoclonal antibodies specific to specific antigens can be used to develop diagnostic agents that detect antigens under denaturing conditions such as high temperatures in infected samples. Accordingly, in the present invention, the recombinant RtxA1 (3491-4071) antigen was heat-treated at 100° C. for 3 minutes to obtain a heat-denatured RtxA1 (3491-4701) antigen, and then ELISA was performed to investigate the reactivity of each monoclonal antibody. Heat denatured recombinant RtxA1 (3491-4701) antigen was diluted with carbonate buffer (pH 9.4), and then 100 µl per 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 a blocking buffer solution containing 1% BSA (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactive horse serum) at 37°C for 1 hour. The reaction was blocked. 50 μl of cell culture solution containing monoclonal antibody was added to each well, reacted at 4° C. for 1 hour, and washed 3 times with a washing buffer solution (PBS, 0.05% Tween-20, 0.05% BSA). After washing, a 1:1000-fold diluted Biotin-condensed anti-mouse IgG+IgA+IgM antibody (1:2,000, Sigma, USA) was added at a rate of 100 μl per well and reacted at 37° C. for 1 hour, followed by washing buffer solution. Washed 3 times. After washing, 100 µl of HRP (Horseradish peroxidase)-condensed streptavidin was added to each well, reacted at 37°C for 1 hour, washed three times with a washing buffer solution, and subsequently TMB (3,3',5,5'- Tetramethyl-benzidine) (Sigma, USA) substrate solution was added to each well at a rate of 100 μl, reacted in the dark for 30 minutes to develop color, and then treated with 2N H2SO4 to stop the enzyme reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. As a result of reaction stone analysis of the monoclonal antibody against the thermodenatured RtxA1 (3491-4701) antigen of each monoclonal antibody, 1RA, 2RA, 3RA, 5RA, 7RA, 8RA, 9RA and 11RA are thermally defined as shown in Table 1. Although the denatured RtxA1 (3491-4701) antigen was recognized, 4RA and 10RA did not recognize the thermodenatured RtxA1 (3491-4701) antigen. Therefore, the monoclonal antibody obtained in the present invention may be usefully used in the development of a diagnostic agent for detecting denatured antigens after high-temperature treatment of a test sample.

<Example 17> Reactivity analysis of monoclonal antibodies against wild species Vibrio sepsis.

Immunoblot was performed to analyze the reactivity of monoclonal antibodies against the RtxA1 toxin protein produced in wild species Vibrio sepsis. Wild species Vibrio septicemia 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) ) After inoculation in HI medium containing 2.5% NaCl, it was incubated for 16 hours in a shaking incubator at 37°C. After inoculating the culture solution at 1/200 in HI medium containing 2.5% NaCl, the main culture was performed in a shaking incubator at 37°C for 2 hours. The culture solution thus cultured 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 the precipitate was collected, and the precipitate was eluted in 20 µl 2x sample buffer, followed by 10% SDS-PAGE. After completion of SDS-PAGE, the gel-like protein was transferred to a Nitrocellulose membrane (Bio-Rad, USA) according to Tobin's (Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA 1979; 76:4350-4354) method. Moved to. The protein-transferred membrane was blocked with a phosphate buffer solution containing 5% skim milk to block non-specific reactions. Here, using a Vibrio sepsis bacterium RtxA1 monoclonal antibody diluted to 50 µg/ml, reacted for 1 hour at room temperature, and diluted peroxidase-labeled IgG (Jackson labatory, USA) secondary antibody according to the manufacturer's instructions. Was used. After reacting with the first and second antibodies, they were washed three times with a phosphate buffer solution. The membrane after the secondary antibody reaction and washing was treated with an 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, lane 1 is a control group, 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 a culture medium of wild species Vibrio sepsis. As can be seen in Figure 5, the 1RA, 5RA, and 11RA monoclonal antibodies showed a positive reaction at 130 kDa in the culture medium of Yangsaeng Vibrio sepsis bacteria. Therefore, 1RA, 5RA, 11RA monoclonal antibodies will be useful for the development of diagnostic agents for detection of Vibrio sepsis RtxA1 antigen.

<Example 18> Cross-reaction of monoclonal antibodies against several strains of Vibrio sepsis.

In order to develop an efficient and useful diagnostic agent for detection of Vibrio sepsis, a monoclonal antibody must show a cross-reactivity against several Vibrio sepsis strains, so the cross-reactivity of the monoclonal antibodies obtained in the present invention and several Vibrio sepsis strains was investigated. I did. To this end, the Vibrio sepsis strain isolated from the patient (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 TM, Smith B, Oliver JD. Appl Environ Microbiol 2006;72: 4356-4359.)) were inoculated in HI medium containing 2.5% NaCl, respectively, and cultured for 16 hours in a shaking incubator at 37°C. After inoculating the culture solution at 1/200 in HI medium containing 2.5% NaCl, the main culture was performed in a shaking incubator at 37°C for 2 hours. The culture solution thus cultured 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 the precipitate was collected, and the precipitate was eluted in 20 µl 2x sample buffer, followed by 10% SDS-PAGE. After completion of SDS-PAGE, the gel-like protein was transferred to a Nitrocellulose membrane (Bio-Rad, USA) according to Tobin's (Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA 1979; 76:4350-4354) method. Moved to. The protein-transferred membrane was blocked with a phosphate buffer solution containing 5% skim milk to block non-specific reactions. Here, using a Vibrio sepsis bacterium RtxA1 monoclonal antibody diluted to 50 µg/ml, reacted for 1 hour at room temperature, and diluted peroxidase-labeled IgG (Jackson labatory, USA) secondary antibody according to the manufacturer's instructions. Was used. After reacting with the first and second antibodies, they were washed three times with a phosphate buffer solution. The membrane after the secondary antibody reaction and washing was treated with an ECL western blot substrate solution (Amersham, USA), and the results were analyzed using a LAS-1000 luminescent image analyzer (Fujifilm, Japan). (Fig. 6). In Figures 6A and 6B, lane 1 is a culture medium of mutant Vibrio sepsis strain CMM744 from which RtxA1 inheritance has been deleted as a control, lane 2 is a culture medium of ATCC 29307 Vibrio sepsis strain, lane 3 is a culture medium of CMCP6 Vibrio sepsis strain, lane 4 is YJ016 Vibrio sepsis. The culture medium of the strain, lane 5, is the culture medium of the Env1 Vibrio sepsis strain. As can be seen in Figure 6A, the 5RA monoclonal antibody showed a positive reaction at a size of 130 kDa to ATCC 29307, CMCP6, and YJ016 Vibrio sepsis strain culture, and the 11RA monoclonal antibody in FIG. 6B was ATCC 29307, CMCP6, YJ016 and Env1 Vibrio. The culture medium of sepsis strains showed a positive reaction at 130 kDa. Therefore, 5RA and 11RA monoclonal antibodies will be useful for the development of diagnostic agents for detection of RtxA1 antigen in several Vibrio sepsis strains.

<Example 19> Reactivity analysis of monoclonal antibodies in cells infected with Vibrio sepsis

Until recently, immunofluorescence assay has been widely used as a standard diagnostic method for diagnosing bacterial tissue infections. Therefore, Vibrio sepsis bacteria were inoculated to investigate the reactivity of the monoclonal antibodies obtained in the present invention and Vibrio sepsis bacteria. Indirect immunofluorescent antibody method was performed using HeLa cells (ATCC, USA). Vibrio sepsis bacteria were inoculated into HeLa cells monolayer cultured on FA (fluorescence assay) slides (Nunc, USA) at 100 MOI for 1 hour, and cells infected with 3.7% formaldehyde were fixed. Immobilized Vibrio sepsis-infected cells were treated with 0.2% Triton X-100, and then each monoclonal antibody was treated with a phosphate buffer solution containing 1% BSA at a concentration of 10-50 µg/ml. It was diluted and treated, reacted at 37° C. for 30 minutes, and washed 3 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, it was washed three times with a phosphate buffer solution, mounted with a ProLong Gold anti-fade (Invitrgen, USA) solution containing DAPI, and observed under a fluorescence microscope. As shown in FIG. 7, the monoclonal antibody was negative in the RtxA1 mutant Vibrio sepsis strain CMM744, but a positive reaction was evident in cells infected with the wild species Vibrio sepsis. Therefore, 1RA, 4RA, 5RA, 11RA monoclonal antibodies will be useful for the development of diagnostic agents for detection of RtxA1 antigen in cells infected with Vibrio sepsis.

Monoclonal antibody
Isotype

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

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

Since the present invention can easily diagnose and treat Vibrio sepsis bacteria, it can be widely used in the medical industry.

Korea Cell Line Research Foundation KCLRFBP00261 20110328 Korea Cell Line Research Foundation KCLRFBP00262 20110328

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Claims (8)

A vaccine composition for preventing or treating vibrio sepsis, comprising the recombinant RtxA1 protein of SEQ ID NO: 6. The vaccine composition for preventing or treating vibrio sepsis according to claim 1, further comprising at least one selected from antihistamines, anti-inflammatory drugs, and antibiotics. The vaccine composition for preventing or treating vibrio sepsis according to claim 1, further comprising at least one selected from a carrier, an excipient, and a diluent. The vaccine composition of claim 1, wherein the recombinant RtxA1 protein of SEQ ID NO: 6 has a purity of 98% or more. The method according to claim 1, wherein the recombinant RtxA1 protein of SEQ ID NO: 6 is extracted from transformed E. coli,
Vibrio septic bacterium Vibrio sepsis prevention or treatment vaccine composition, characterized in that the recombinant expression vector comprising a sequence encoding amino acid sites 3491 to 4701 of the RtxA1 antigen transformed into E. coli. The vaccine composition according to claim 5, wherein the E. coli is Bl21 (DE3) / pLs S.
(1) preparing a recombinant expression vector comprising a sequence encoding amino acid sites 3491 to 4701 (SEQ ID NO: 6) of the Vibrio sepsis RtxA1 antigen or a sequence encoding the fusion protein thereof;
(2) transforming the recombinant expression vector into a host cell; And
(3) culturing the transformed host cell to produce a recombinant RtxA1 protein of SEQ ID NO: 6; And
(4) purifying the recombinant RtxA1 protein and preparing a vaccine composition; vibrio sepsis prevention or treatment method for producing a vaccine comprising the. The vaccine for preventing or treating Vibrio sepsis, according to claim 7, wherein the PCR is performed using a front primer pair represented by SEQ ID NO: 1 and a rear primer pair represented by SEQ ID NO: 2 before step (1). Manufacturing method.

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