KR101553845B1 - Monoclonal Antibody Specifically Binding to Vibrio vulnificus RtxA1 protein and Its Use - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to Vibrio septicemic strain < RTI ID = 0.0 > (RtxA1) < / RTI > and uses thereof, and more particularly to a method of high sensitivity, A monoclonal antibody that specifically binds to Vibrio septicemic strain < RTI ID = 0.0 > (RtxA1) < / RTI > that can be usefully used in the prevention and treatment of diseases caused by Vibrio sepsis and related infections, And its use.

Description

Monoclonal Antibody Specific Binding to Vibrio vulnificus RtxA1 protein and Its Use < RTI ID = 0.0 >

The present invention relates to a monoclonal antibody that specifically binds to Vibrio septicemic strain < RTI ID = 0.0 > (RtxA1) < / RTI > and uses thereof, and more particularly to a method of high sensitivity, A monoclonal antibody that specifically binds to Vibrio septicemic strain < RTI ID = 0.0 > (RtxA1) < / RTI > that can be usefully used in the prevention and treatment of diseases caused by Vibrio sepsis and related infections, And its use.

Vibrio vulnificus (Vibrio vulnificus) is a rust-resistant pathogenic bacterium that is mainly associated with the seaside and adjacent to the river, and is associated with most seafood-related deaths. Although the vibrio-sepsis infectious disease has a relatively short history, it is one of the newly-attracted diseases in which clinical cases are continuously increasing worldwide due to global warming. In particular, the absolute epidemic worldwide is less than cholera or Salmonella food poisoning, but it causes serious social problems due to high mortality and tragic clinical symptoms.

Since the first report of the bacteriological characteristics of voriconic and pathogenic Vibrio isolates from humans for 11 years by Hollis et al. Of the Centers for Disease Control (CDC) in 1976 (Lactose-fermenting Vibrio or Lac (+)) because of its ability to degrade lactose. In 1979, Blake et al. Of the CDC epidemiologically analyzed the data of 39 patients reported on CDC and classified them as primary septicemia and wound infection 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 has reached today (Farmer, J. J. III, Lancet 2: 903, 1979).

Vibrio septicemia Bacillus sepsis has a short latency and mortality rate of more than 50% despite various antibiotic treatments. Vibrio septicemia Bacillary sepsis occurs mostly in men over 40 years old (about 90-95%) (90% or more), rarely seen in normal people, and mainly in patients with underlying diseases. In the case of primary sepsis, most patients have chronic diseases such as liver disease and drinking wall, and hepatic diseases include cirrhosis, chronic hepatitis and liver cancer. In addition, diabetes, pulmonary tuberculosis, chronic Osteomyelitis, and rheumatoid arthritis. However, in some cases below 5%, no specific underlying disease can be found. In the United States, the incidence is relatively high in patients with diabetes, malignant tumors, hemochromatosis, and Mediterranean anemia (thalassemia). Recently, AIDS patients are reported to report the occurrence of sepsis of Vibrio sepsis. In the United States CDC, AIDS patients are advised not to produce seafood such as oysters in summer. In addition, patients with chronic illnesses in the hurricane-affected area are advised to be aware of Vibrio-sepsis infection through wounding. Therefore, the development of new antibacterial substances is urgently required for the prevention and treatment of sepsis due to infections of vibrio sepsis and related bacteria.

Meanwhile, various virulence factors have been reported through a study on the pathogenesis of Vibrio sepsis after the mid 1980s. In other words, capsular polysaccharide (VvCPS), iron assimilation system, flagella, pili, VvhA, VvpE and RtxA1 are known (Jones M. K., Oliver J. D. Infect Immun. 77: 1723-1733. Among them, RtxA1 is a major virulence factor secreted early in the infection of Vibrio sepsis, and it plays an important role in the spread of Vibrio sepsis and the death of cells through the intestinal blood. This suggests that RtxA1 is a useful target for the development of new diagnostics and therapeutic agents for early infections.

Among the methods for detecting the Vibrio septicemia, a single-chain antibody specifically recognizing the outer membrane protein of the Vibrio septicemia bacteria and a method and a method for detecting the Vibrio bacteria using the antibody are disclosed in Patent No. 10-1192130 (Published Date: 2012.01.09) .

However, despite the conventional treatment with various antibiotics, there have been several attempts to lower the high mortality rate for infection of Vibrio sepsis strains with a mortality rate of 50% or more. For example, vaccine development using inactivated Vibrio sepsis, VvCPS, and VvpE has been under way (Kreger AS, Gray LD, Testa J. Infect Immun. 45: 537-543, 1984, Devi SJ, Hayat U., Frasch CE , et al., Infect Immun. 63: 2906-2911, 1995, Chen YC, Chang CC, Chang SY, et al., Letters in applied microbiology 50: 168-172. However, inactivated Vibrio paracchiasis and VvpE have potential side effects (Chang AK, Kim HY, Park JE, et al., J. Bacteriol 187: 6909-6916, 2005) (Devi SJ, Hayat U., Frasch CE, et al. Infect Immun. 63: 2906-2911, 1995).

Furthermore, despite the extremely high mortality rate in the case of infection with Vibrio sepsis, there is a problem that effective prevention and treatment of Vibrio septicemia can not be achieved at present.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and a first object of the present invention is to provide a method for the prevention of diseases caused by Vibrio-septicemia and related infections by specific binding ability of high- And a monoclonal antibody having excellent therapeutic effect, and a hybridoma cell line producing the same.

A second object of the present invention is to provide a therapeutic agent for Vibrio septicemia, a prophylactic agent for Vibrio septicemia, and a diagnostic kit for Vibrio septicemia comprising the monoclonal antibody of the present invention.

The present invention provides a hybridoma cell having a deposit number KCLRF-BP-00311, which is characterized in that it produces a monoclonal antibody against the Vtrioceptor RtxA1 protein.

In addition, the present invention provides a hybridoma cell having a deposit number KCLRF-BP-00309, which is characterized in that it produces a monoclonal antibody against the Vtrioceptor RtxA1 protein.

Furthermore, the present invention provides a monoclonal antibody or antigen-binding fragment thereof for the Vtrioceptor RtxA1 protein produced by a hybridoma cell with accession number KCLRF-BP-00311.

In addition, the present invention provides monoclonal antibodies or antigen-binding fragments thereof for the Vtrioceptor RtxA1 protein produced by hybridoma cells with accession number KCLRF-BP-00309.

According to a preferred embodiment of the present invention, the monoclonal antibody can specifically bind to the 3491 to 3980th amino acid sequence region of the RtxA1 protein of Sequence Listing 4.

In addition, the present invention provides a therapeutic agent for Vibrio septicemia comprising a monoclonal antibody or an antigen-binding fragment thereof.

The present invention also provides a prophylactic agent for Vibrio septicemia comprising a monoclonal antibody or an antigen-binding fragment thereof.

Further, the present invention provides a Vibrio septicemia diagnostic kit comprising a monoclonal antibody or antigen-binding fragment thereof.

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

The term "recombinant " as used in reference to a cell refers to that the cell expresses a peptide or protein that replicates the nucleic acid of the variant or is encoded by the variant nucleic acid. In addition, recombinant cells can express genes found in the original form of the cells, but modified genes may be reintroduced into cells by artificial methods.

The term "primer" of the present invention means a synthetic or natural oligonucleotide. The primer serves as a starting point for synthesis under conditions in which the synthesis of the primer extension product complementary to the template is induced, that is, the presence of a polymer such as nucleotides and DNA polymerase, and a 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, the oligonucleotides of the present invention can be synthesized by using a framework-modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexy Tolyl DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having C-7 substituents (the substituents being fluoro, bromo, chloro, bromo, chloro, , children Methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.

The term "vector" of the present invention refers to a DNA molecule as a carrier capable of stably transporting a foreign gene into a host cell. In order to become a useful vector, it must be replicable, have a means for influx into the host cell, and have means for detecting its presence. Here, the foreign gene means a sequence encoding the 3491-4701 amino acid region of the Vtrioceptor RtxA1 antigen or a sequence having a poly His-tag sequence added to the end of the sequence to facilitate purification.

The term "plasmid" of the present invention generally refers to a circular DNA molecule that is operatively linked to a vector so that the foreign gene can be expressed in the host cell. However, the plasmid can be used as a vector which is degraded by a specific restriction enzyme by gene recombination and introduces a new gene to produce a plasmid containing the desired gene. Thus, plasmids and vectors are used interchangeably herein, and those with ordinary knowledge in the field of genetic engineering will appreciate their meaning even if they do not distinguish their names.

The term "fusion protein" of the present invention refers to Vtrio lung-activated mutant RtxA1 having a His-tag attached at its C-terminus for convenience of protein purification. Therefore, although it is expressed in the present invention as "fusion protein ", it does not necessarily mean that the poly His-tag is attached, and fusion proteins may not be used if the inconvenience of protein purification is taken into consideration. Therefore, in the present invention, the term "recombinant vibrio pulmonary growth factor RtxA1" is defined as an abbreviation of "Vibrio sepsis RtxA1" or "Vibrio sepsis strains RtxA1 added at the N-terminus or C-terminus for convenience of purification.

The recombinant RtxA1 (3491-4701) according to the present invention provides a recombinant RtxA1 protein antigen which is very useful for polyclonal antibody production, monoclonal antibody production and Vibrio sepsis vaccine production of Vtrio sepsis RtxA1. It also provides recombinant RtxA1 (3491-4701) protein antigens that are very useful for diagnosis using Vtrioceptor RtxA1 and facilitates the mass expression and purification of recombinant RtxA1 (3491-4701) protein antigens.

In addition, the polyclonal antibody to the recombinant RtxA1 (3491-4701) protein according to the present invention has a specific binding ability of high affinity to Vibrio sepsis, and is very useful for quickly, accurately diagnosing, preventing and treating Vibrio sepsis infection . In addition, the recombinant protein RtxA1 antigen expressed in E. coli has excellent sensitivity and specificity and can be very usefully used in a diagnostic agent or a diagnostic kit for infection of Vibrio sepsis.

Furthermore, the monoclonal antibody produced in the hybridoma cells according to the present invention is highly useful for rapid, accurate diagnosis, prevention and treatment of vibriosal sepsis infection by the specific binding ability of high affinity to Vibrio sepsis strains . In addition, the recombinant protein RtxA1 antigen expressed in E. coli has excellent sensitivity and specificity and can be very usefully used in a diagnostic agent or a diagnostic kit for infection of Vibrio sepsis.

In addition, the monoclonal antibody of the present invention can be effectively used as a therapeutic agent for vibriosis, since it has excellent infection inhibitory effect on vibrio sepsis, excellent prevention and therapeutic effect on vibrio septicemia infection as well as excellent mouse survival rate and vaccination effect after infection with vibrio septicemia .

Figure 1 is a cleavage map of recombinant RtxA1 (3491-4701) expression vector incorporating recombinant RtxA1 (3491-4701).
Figure 2 shows the results of electrophoresis of purified recombinant RtxA1 (3491-4701) protein of Vibrio sepsis with 10% SDS-PAEG (lane 1: purified Glutathione S-transferase (GST) protein, lane 2: Purified RtxA1 (3491-4701) protein)
Figure 3 shows the results of electrophoresis on immunoblot analysis of the specificity of the purified recombinant RtxA1 (3491-4701) protein (lane 1: control GST protein, lane 2: recombinant RtxA1 (3491-4701) protein)
Fig. 4 is a diagram schematically showing a recombinant RtxA1 (3491-4701) fragment protein expressed in a transformed E. coli.
Figure 5 shows the results of electrophoresis in which the expression of the recombinant RtxA1 (3491-4701) fragment protein was confirmed by SDS-PAEG (lane 1: control recombinant protein, lane 2: recombinant RtxA1 (3491-4701) protein, lane 3: RtxA1 (3491-4380) protein and lane 4: recombinant RtxA1 (3491-3980) protein)
Figure 6 shows the results of electrophoresis on immunoblot analysis of the specificity of the expressed recombinant RtxA1 (3491-4701) fragment protein (lane 1: control recombinant protein, lane 2: recombinant RtxA1 (3491-4701) protein, lane 3 : Recombinant RtxA1 (3491-4380) protein and lane 4: recombinant RtxA1 (3491-3980) protein)
Fig. 7 is a diagram schematically showing a site where three groups of monoclonal antibodies bind to RtxA1 (3491-4701).
FIG. 8 is a graph showing the results of competitive binding analysis of monoclonal antibodies (13RA, 21RA, 24RA, 47RA) and 10RA and biotin-condensed 47RA used for investigating the therapeutic effect on Vibrio sepsis in Example 23.
9 is a graph showing the vaccine effect on the recombinant RtxA1 (3491-4701) protein. RtxA1-C and GST, denoted by solid and dashed lines, represent groups immunized with RtxA1 (3491-4701) and GST protein, respectively.
FIG. 10 is a graph showing the effect of vaccination of the recombinant RtxA1 (3491-4701) protein on the number of Vibrio septicemia in blood. RtxA1-C and GST, denoted by squares and circles, represent groups immunized with RtxA1 (3491-4701) and the GST protein, respectively.
FIG. 11 is a graph showing the proliferation rate of a polyclonal antibody against the recombinant RtxA1 (3491-4701) protein in terms of the survival rate of an experimental animal, against the infection with Vibrio septicemia. Α-RtxA1-C and α-GST, denoted by the solid and dashed lines, represent groups administered with RtxA1 (3491-4701) and GST protein polyclonal antibodies, respectively.
FIG. 12 is a graph showing the therapeutic effect of a polyclonal antibody against a recombinant RtxA1 (3491-4701) protein on the survival rate of an experimental animal for Vibrio septicemia infection. Α-RtxA1-C and α-GST, indicated by a straight line and a dotted line, represent a group administered RtxA1 (3491-4701) and a GST protein polyclonal antibody, respectively.
Fig. 13 is a graph showing the preventive effect of the monoclonal antibody against recombinant RtxA1 (3491-4701) protein against Vibrio septicemia infection.
FIG. 14 is a graph showing the therapeutic effect of monoclonal antibody against recombinant RtxA1 (3491-4701) protein on Vibrio septicemia infection as survival rate of experimental animals.

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

As described above, in case of infection with Vibrio sepsis, there is a problem that effective prevention and treatment of Vibrio septicemia can not be achieved despite the extremely high mortality rate.

Thus, according to a preferred embodiment of the present invention, there is provided a hybridoma cell having a deposit number KCLRF-BP-00309 and KCLRF-BP-00311, which is characterized by producing a monoclonal antibody against the Vtrioceptor RtxA1 protein, I sought to solve a problem.

The present invention relates to a method of immunizing a mouse with an immunogen, using recombinant RtxA1 (3491-4701) expressing Escherichia coli against the C-terminal region of RtxA1, which is determined as a specific toxin antigen secreted in the early stage of vibrio infection, And P3X63Ag8.653, which is a B-lymphoblast, were prepared, from which a monoclonal antibody against the Vibrio sepsis RtxA1 antigen was produced. The monoclonal antibody according to the present invention showed excellent reactivity to the Vtrioceptor RtxA1 antigen. 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 immunofluorescence antibody, immunoblot assay and enzyme immunoabsorbent assay. In addition, the polyclonal antibody and the monoclonal antibody against the recombinant RtxA1 (3491-4701) protein of the present invention have an excellent effect of inhibiting Vibrio septicemia infection, prevention and treatment of Vibrio septicemia infection as well as excellent survival rate of mouse after infection with Vibrio septicemia, It can be used as a treatment for sepsis.

Specifically, in order to selectively amplify the amino acid sequence at positions 3491 to -4701 of the amino acid sequence of Vtrio septicemia RtxA1 using the sequence represented by SEQ ID NO: 3 encoding the Vtrioceptic strain RtxA1 protein of SEQ ID NO: 4 as a template, PCR is carried out using forward and back primers of SEQ ID NOS: 1 and 2 as one primer sequence. (SEQ ID NO: 5) coding for the amino acid at positions 3491 to -4701 of the amino acid sequence of the amplified Vibrio sepsis strain RtxA1 or the sequence of the normal poly His-tag (6x -His) sequence was inserted into a plasmid vector and transformed into a host cell. The expressed protein is then purified and injected into the abdominal cavity of mice. The spleen cells are then harvested, fused with myeloma cells, and cultured to obtain a plurality of hybridoma cells. Thereafter, the monoclonal antibodies produced in the hybridoma cells were examined for the possibility of mass production according to isotype, competitive binding between monoclonal antibodies, prevention and therapeutic effect of Vibrio septicemia infection of monoclonal antibody, vaccine effect, And the survival rate of mice after infection were evaluated comprehensively. Thus, excellent 24RA and 47RA were deposited at Korean Cell Line Bank with the accession number KCLRF-BP-00311 and the accession number KCLRF-BP-00309, respectively. The 24RA and 47RA monoclonal antibodies thus produced can specifically bind to amino acid positions 3491 to 3980 of the amino acid sequence of Vtrioceptor RtxA1. However, it can be seen from Example 16 and FIG. 8 that it is a monoclonal antibody that binds to different epitopes.

The hybridoma cells that secrete the monoclonal antibodies of the present invention can be cultured in vitro or in vivo in vivo. The monoclonal antibody produced by the hybridoma cells may be used without purification. However, in order to obtain the best results, the monoclonal antibody may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention Is preferably used. Such purification techniques may be separated from the culture medium or ascites fluid using purification methods such as, for example, dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like.

(RIA), enzyme immunoabsorption (ELISA), immunofluorescence, Western blotting (RIA), immunoblot analysis, immunofluorescence, and immunohistochemistry to screen for monoclonal antibodies that selectively recognize Vtrioceptor RtxA1 protein. Western blotting, and flow cytometry, but the present invention is not limited thereto. According to one specific embodiment of the present invention, a single clone can be selected by enzyme immunoassay (ELISA).

Each of the monoclonal antibodies produced in 37 hybridized hybridoma cells using the recombinant RtxA1 protein as an immunogen is monoclonal antibodies binding to amino acids 3491 to 3980 according to the amino acid binding site of RtxA1 of Vibrio septicemia, Monoclonal antibodies binding to the 3981-4380th amino acid and monoclonal antibodies binding to the 4381-4701th amino acid. Among these monoclonal antibodies produced in 37 hybridized hybridoma cells using recombinant RtxA1 protein as an immunogen, the possibility of mass production by isotype, competitive binding between monoclonal antibodies, vibrio septicemia infection of monoclonal antibodies (3491-4701) protein monoclonal antibody 24RA (3491-4701) having different binding site, isotype subclass, and therapeutic effect of the antibody to the antigen, respectively, in consideration of the prophylactic and therapeutic effect on the antigen and the mouse survival rate after vibrio septicemia infection. And 47RA showed excellent effects. Thus, 24RA and 47RA were deposited at Korean Cell Line Bank with 241RA as deposit number KCLRF-BP-00311 and 47RA as deposit number KCLRF-BP-00309 (see Table 1).

According to another preferred embodiment of the present invention, a monoclonal antibody or an antigen-binding fragment thereof for the Vtrioceptor RtxA1 protein produced by a hybridoma cell having the accession number KCLRF-BP-00309 and the accession number KCLRF-BP- To solve the above-mentioned problem.

The monoclonal antibody of the present invention specifically binds to the Vtrioceptor RtxA1 protein with high affinity.

The monoclonal antibody of the present invention can specifically bind to the 3491 to 3980th amino acid sequence region of the RtxA1 protein of Sequence Listing 4.

The term "antibody" of the present invention means a specific antibody to the Vibrio lung activated RtxA1 protein, including not only the complete antibody form, but also the antigen binding fragment of the antibody molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

An antigen binding fragment of an antibody molecule means a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') 2, Fv and the like. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are described in PCT International Publication Nos. WO88 / 10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') 2 fragment can be obtained by cleavage with pepsin) Can be produced through recombinant DNA technology.

In the present invention, the antibody is preferably in the form of one of the group consisting of Fab, F (ab ') 2, F (ab') ₂ and Fv or a complete antibody form.

The "heavy chain" means both the variable length domain VH comprising the amino acid sequence with sufficient variable region sequence to confer specificity to the antigen and the full length heavy chain comprising the three constant region domains CH1, CH2 and CH3 and fragments thereof do.

Further, the "light chain" means both the full-length light chain and its fragment including the variable region domain VL and the constant region domain CL including the amino acid sequence having a sufficient variable region sequence to confer specificity to the antigen.

The term "monoclonal antibody" of the present invention means a highly specific antibody, as the term is known in the art, directed to a single antigenic site (epitope). Typically, unlike polyclonal antibodies that 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 that utilize antigen-antibody binding and are also produced by culturing hybridomas, so that they are easy to mass produce and are not contaminated by other immunoglobulins It has another advantage.

Monoclonal antibodies of the invention can be obtained from hybridoma cells produced by cell fusion methods known in the art. Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells and cancer cell lines from immunologically appropriate host animals, such as mice injected with antigen proteins. The fusion of these two cells is accomplished by fusion using a method known in the art using polyethyleneglycol, and the antibody producing cells are proliferated by a standard culture method. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antigen-specific antibody are cultured in vitro or in vivo. Examples of myeloma cells used for cell fusion include mouse-derived p3 / x63-Ag8, p3-U1, NS-1, MPC-11, SP-2/0, F0, P3x63Ag8.653, V653, S194, Can be used. The cell line used in the specific embodiment of the present invention is myeloma cell P3x63Ag8.653.

The monoclonal antibody of the present invention can bind to an antigen to inhibit or neutralize its action and further kill cells infected with pathogens and pathogens. For example, a monoclonal antibody that specifically recognizes RtxA1, a toxin portion of Vibrio sepsis, can bind to it to inhibit or neutralize its action, and an antigen-associated antibody can activate a complement, The complement may allow the antigen to be removed. In addition, monoclonal antibodies may bind to the antigen and cause the antigen to be better digested by the phagocytic cells. In addition, the antigenic cells to which the monoclonal antibody is bound are more easily killed by the natural killer cells (NK cells), so that the monoclonal antibodies can be used for the elimination of the antigen through the immune response and the vibriosis sepsis . Therefore, the result of the elimination and reduction of the antigen and the Vibrio sepsis by the immune reaction alone can be expected. Therefore, the antibody of the present invention can be used for diagnosis, prevention and / or treatment of Vibrio sepsis.

In addition, the monoclonal antibody of the present invention can be used as a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains, as long as it has the above- And / or diagnostic. The functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, and may include Fab, F (ab ') 2, F (ab') ₂ and Fv.

According to another preferred embodiment of the present invention, there is provided a therapeutic agent for Vibrio septicemia comprising the above-mentioned monoclonal antibody or antigen-binding fragment thereof, thereby solving the above-mentioned problem. Accordingly, the monoclonal antibody of the present invention exhibits a neutralizing ability of Vibrio sepsis, so that it can be used as a preventive and therapeutic composition for Vibrio septicemia infection together with an antibody alone or a conventional pharmaceutically acceptable carrier.

The therapeutic agent for Vibrio septicemia may be a pharmaceutical composition for preventing and treating Vibrio septicemia infection comprising the monoclonal antibody of the present invention. The pharmaceutical composition containing the monoclonal antibody of the present invention may be used in combination with antihistamines, And antibiotics, or may be used in combination.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above-mentioned components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, And the like. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. The pharmaceutical composition may also be administered by any device that allows the active agent to migrate to the target cell.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat infection with Vibrio septicemia.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, suppositories, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

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 formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Examples of the liquid preparation for oral administration 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.

Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The antibody composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

Antibodies can be injected into vivo in the form of antibody-therapeutic conjugates and used to treat bacterial infections. Therapeutic agents include chemotherapeutic agents, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents, and enzyme inhibitors. Methods for binding antibiotics to antibodies are described, for example, in G. Gregoriadies, ed., Academic Press London, (1979); Arnon et al., Recent Results in Cancer Res., 75: 236 (1980); And Moolton et al., Immunolog. Res., 62: 47 (1982)).

Preferred agents for coupling with the antibodies or antibody fragments of the invention are antibiotics, antiparasitic, antifungal and related agents and include, for example, sulfonamides, penicillins and cephalosporins, aminoglycosides, tetracyclines, chloramphenicol, 5-fluoro-cytosine, 5-iodo-2'-deoxyuridine, aminopyridine, aminopyridine, metroniazide, isoniazid, rifampin, streptomycin, sulfone, erythromycin, polymyxin, Adamantanamine, adenine arabinoside, amanidin, ribavirin and azatrimidine (AZT), preferably ribavirin. Various conditions suitable and desirable for targeting drugs to specific target sites are reported, for example, in Trouet et al., Plenum Press, New York and London, 19-30 (1982). By targeting an effective therapeutic agent directly to an infectious lesion using a highly specific antibody prepared against a microbial antigen, selective killing of the infectious agent can solve many problems that occur when treating a drug resistant infection. In addition, drugs targeted at lesions can increase the drug efficacy at high concentrations at the site of infection.

Immunomodulators that may be used as therapeutic agents in the antibody-therapeutic agent conjugate include, but are not limited to, lymphokines and cytokines.

In conclusion, the present invention provides a polyclonal antibody and a monoclonal antibody against a Vibrio sepsis RatxA1 protein and a hybridoma cell line producing the antibody, wherein the polyclonal antibody and the monoclonal antibody to the Vtrio sepsis RtxA1 protein of the present invention Has a specific binding ability of high affinity to the RtxA1 protein of Vibrio sepsis, and thus can be usefully used for the production of kits for the diagnosis and diagnosis of Vibrio septicemia. In addition, it can be used as a therapeutic agent for Vibrio septicemia because of its excellent effect of inhibiting Vibrio septicemia infection of polyclonal antibody and monoclonal antibody, prevention and treatment effect of Vibrio septicemia infection, and excellent survival rate of mouse after Vibrio septicemia infection.

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

[ Example ]

Example  1: Vibrio Sepsis  Recombination RtxA1  Oligo for synthesis Nucleotide  synthesis

The following two oligonucleotides having NheI and XhoI restriction enzyme recognition sites were synthesized. In addition, RA-4701R oligonucleotides contain six histidine (6x-His tags) to further refine the chromatographic purification using histidine-binding resin during the purification 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 nucleotides were used for the amplification of the following Vibrio sepsis RtxA1 gene (below Example 2) and for the production of recombinant vectors for recombinant RtxA1 protein production.

Example  2: Polymerase chain reaction  ( 중합체 chain reaction  ( PCR ))

(SEQ ID NO: 3, 14106 bp) - (NCBI accession No. 2) was added to the reaction solution in which the polymerase and the forward primer (SEQ ID NO: 1) and the back primer (SEQ ID NO: 2) synthesized in Example 1 were mixed. CP002470.1) was mixed with a template to carry out polymerase chain reaction to amplify amino acid sequence 3491 to 4701 of the Vtrioceptor RtxA1 (RtxA1 (3491-4701)). SEQ ID NO: 4 is the amino acid sequence- (NCBI accession No. YP_004191172.1) of the full-length Vibrio sepsis strain RtxA1. PCR was performed using a Thermalcycler (PTC-100, MJ Research, Inc., USA) at 94 ° C for 3 minutes, followed by denaturation at 94 ° C for 1 minute, Annealing at 55 ° C for 1 min and extension at 72 ° C for 4 min were repeated 30 times and finally further reaction was performed at 72 ° C for 10 min. To remove the following polymerase and nonspecific amplification products, electrophoresis was performed on the agarose gel, and the desired amplified product band was fragmented and purified using a gel extraction kit (Geneall, Korea).

Example  3: Recombination RtxA1 (3491-4701) Expression vector production

A recombinant RtxA1 plasmid vector having the cleavage map of Fig. 1 was prepared. Specifically, the amplified product of RtxA1 (3491-4701) in which the poly his tag sequence CATCATCATCATCATCAT (6x-His tag) was inserted at the C-terminal purified in Example 2 was digested with EcoRI (Fermentas, Canada) The cuts were filled in using an enzyme Klenow (New England Biolab, USA) and cut into XhoI (Fermentas, Canada). The vector pET-21 (a) (Novagen, USA) was digested with NheI (Fermentas, Canada) and then filled in with E. coli polymerase Klenow (New England Biolab, USA) , Canada). The cleaved fragments and the vector were purified using the gel extraction kit (Geneall, Korea) used in Example 2, and then 1 占 퐂 of the purified RtxA1 gene fragment and 30 ng of the pET-21 (a) fragment were mixed, (10 mM DTT, 100 mM MgCl ₂ , 10 mM ATP, 600 mM Tris-HCl, pH 7.5) and 10 units of T4 DNA ligase (Takara) And the reaction was carried out at room temperature for 16 hours to obtain a 9,027 bp annular plasmid (pET (SEQ ID NO: 5 + 6x-His tag) having the cleavage map of FIG. 1 inserted with the recombinant RtxA1 gene -RtxAl (3491-4701)). The following plasmid was amplified and inserted into E. coli DH5? (Invitrogen, USA) for pure isolation of only pET-RtxA1 (3491-4701) inserted with the RtxA1 (3491-4701) gene (SEQ ID NO: 5). After recombinant pET-RtxA1 (3491-4701) was re-extracted from the transformed E. coli, DNA sequencing and restriction enzyme digestion were performed and agarose gel electrophoresis was performed to obtain pET-RtxA1 (3491-4701) 3491-4701) genes were confirmed.

Example  4: Recombination RtxA1 (3491-4701) Expression of protein

(DE3) / pLyS (Invitrogen, USA) for the expression of the recombinant RtxA1 vector pET-RtxA1 (3491-4701) prepared in Example 3 above. The transformed Escherichia coli was cultured in a shaking incubator at 37 ° C. for 4 hours, and then 1 mM IPTG was added thereto, followed by further culturing for 3 hours. The transformed Escherichia coli thus cultivated was centrifuged at 8,000 rpm for 30 minutes and the Escherichia coli precipitate was suspended in an elution buffer (300 mM NaCl, 25 mM Tris-HCl (pH 7.5), 10% glycerol, 0.1% Tween-20) Respectively. Escherichia coli precipitates suspended for protein extraction were disrupted using an ultrasonic sonicator and centrifuged to collect supernatant protein extracts.

Example  5: Recombination RtxA1 (3491-4701) Protein purification

Proteins were extracted by the method of Example 4 in E. coli transformed with vector pET-RtxA1 (3491-4701) for production of recombinant RtxA1 (3491-4701), followed by Ni-NTA chromatography (Qiagen, USA) The recombinant RtxA1 (3491-4701) protein was then primarily purified from the protein extract. The recombinant RtxA1 (3491-4701) protein that was first purified using Ni-NTA chromatography was purified by secondary purification using gel filtration liquid chromatography (size exclusion-FPLC) (GE Healthcare, USA) to obtain recombinant RtxA1 3491-4701) protein (SEQ ID NO: 6 + 6x-His tag). Purification pattern and purification efficiency were confirmed by performing SDS-PAGE. The results are shown in Fig.

As shown in Figure 2, lane 1 is a purified control Glutathione S-transferase (designated GST) protein, and an asterisk indicates the position of the control protein GST. Also, lane 2 is the purified RtxA1 (3491-4701) protein in Ni-NTA chromatography and gel filtration liquid chromatography, and the arrow indicates the position of the purified recombinant RtxA1 (3491-4701) protein. The purified recombinant RtxA1 (3491-4701) protein showed 98% purity as confirmed in lane 2.

Example  6: Recombination RtxA1 (3491-4701) Analysis of specificity of protein

SDS-PAGE of the 130 kDa recombinant RtxA1 (3491-4701) protein and the control GST protein confirmed in Example 5 was carried out. After completion of the electrophoresis, the gel protein was purified using Towbin's (Towbin H, Staehelin T , Gordon J. Proc. Natl. Acad. Sci. USA 1979; 76: 4350-4354) and transferred to a nitrocellulose membrane (Bio-Rad). Nitrocellulose membranes from which proteins were transferred were blocked with phosphate buffer containing 5% skim milk. Serum from CD-1 mice infected with the Vibrio sepsis strain of Example 18 was diluted at a volume ratio of 1: 1000 and reacted at room temperature for 1 hour. Peroxidase-labeled IgG (Jackson labatory, USA) The antibody was diluted according to the manufacturer's instructions. After reacting with the primary and secondary antibodies, the cells were washed three times with phosphate buffered saline (PBS). The nitrocellulose membrane subjected to the secondary antibody reaction and washed was treated with an ECL western blotting substrate solution (Amersham, USA), and the result was measured using a luminescent image analyzer (Fujifilm, Japan) The results are shown in FIG. As shown in Figure 3, lane 1 is a control GST protein and lane 2 is a recombinant RtxA1 (3491-4701) protein.

As shown in FIG. 3, the recombinant RtxA1 (3491-4701) protein was positive for the wild type Vibrio sepsis infection sera, but the control GST was not reacted. Thus, in Example 5, the purified purified 130 kDa protein was confirmed to be a Vtrioceptor RtxA1 (3491-4701) protein.

As a result, the recombinant RtxA1 (3491-4701) expression vector according to the present invention provides a recombinant RtxA1 protein antigen highly useful for the production of monoclonal antibodies to the Vtrioceptor RtxA1 protein. It also provides a recombinant RtxA1 (3491-4701) protein, which is very useful for diagnosis using Vtrioceptor RtxA1, and facilitates the mass expression and purification of the recombinant RtxA1 (3491-4701) protein. Therefore, the recombinant RtxA1 (3491-4701) protein expressed in such transformed Escherichia coli is used as a diagnostic agent or a diagnostic kit for Vibrio sepsis, a vaccine for the prevention or treatment of Vibrio septicemia, and an antibody against Vibrio sepsis .

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

The RtxA1 (3491-4701) recombinant antigen protein (SEQ ID NO: 6 + 6x-His tag) prepared in Example 5 was mixed with Sigma adjuvant (Sigma, USA) in an equal volume (at a 1: 1 volume ratio) 200 [mu] l of BALB / C (female, 8 weeks old) mice were injected intraperitoneally four times at intervals. Three days after administration of the recombinant RtxA1 (3491-4701) protein purified into the tail vein one month after the fourth immunization, the spleen was harvested and used for cell fusion.

Example  8: B- B-lymphoblast P3X63Ag8 .653 culture

5 days before cell fusion, P3X63Ag8.653 (ATCC accession number CRL-1580), a myeloma stored in a vagina, was taken out and suspended in IMDM (Invitrogen, USA) supplemented with 10% fetal bovine serum , And centrifuged at 1,000 rpm for 5 minutes. The supernatant was discarded and the precipitated cells were carefully resuspended in IMDM supplemented with 10% fetal bovine serum and incubated in a 37 ° C incubator supplied with 5% carbon dioxide gas.

Example  9: cell fusion 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) as follows. Splenocytes from the spleen extracted in Example 7 were separated using a cell strainer (Falcon, USA) and diluted to a concentration of 1 x 10 ⁸ cells / ml. 1 × 10 ⁷ cells / ml of myeloma cells and 1 ml of PEG 1500 (Sigma, USA) were mixed and fused. The cells that had been fused were diluted in 200 ml of HAT (Sigma, USA) medium, and then 100 쨉 l was dispensed into a 96-well microplate and cultured in a 37 째 C incubator supplied with 5% carbon dioxide gas.

Example  10: Cell fusion Hybridoma  In a cell RtxA1 (3491-4701) Antibody production confirmation

Antibody production of the fused cells was confirmed by ELISA using a cell culture fluid fused with the recombinant RtxA1 (3491-4701) antigen expressed in Escherichia coli. The recombinant RtxA1 (3491-4701) antigen protein was diluted to a concentration of 10 μg / ml with carbonate buffer (pH 9.4) and then added to each well of Maxisorp ELISA plate (Nunc, USA) in an amount of 100 μl, Lt; 0 > C for 16 hours to coat the antigen. Each well coated with antigen was treated with Blocking Buffer (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactivated horse serum) containing 1% BSA and blocked for nonspecific reaction at 37 ° C for 1 hour . 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 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 was added to each well and reacted at 37 ° C for 1 hour. Washed three times. After washing, 100 μl of HRP (Horseradish peroxidase) -conjugated streptavidin (Invitrogen, USA) was added to each well, followed by reaction at 37 ° C for 1 hour, followed by washing three times with washing buffer solution. , 5,5'-tetramethylbenzidine (Sigma, USA) substrate solution was added to each well in an amount of 100 μl, followed by reaction in dark for 30 minutes to develop color, and the enzyme reaction was stopped by treating with 2N H ₂ SO ₄ . After the reaction, the absorbance was measured at 450 nm using an ELISA reader.

As a result, as shown in Table 1, 37 hybridoma cells were obtained which reacted with the Vtrio sepsis RtxA1 (3491-4701) antigen.

Example  11: Hybridoma  Cell Cloning

For cloning of the fused 37 hybridoma cells, a hybridoma reacting with the recombinant RtxA1 (3491-4701) antigen obtained in Example 5 was added to the first well of a 96-well plate to give a cell suspension , Followed by 2-fold stepwise dilution with a row, followed by 2-fold dilution with heat. The selected hybridomas were suspended in IMDM medium containing 30% fetal bovine serum and 7.5% DMSO (dimethyl sulfoxide) and stored in liquid nitrogen.

Example  12: Multiple of monoclonal antibody ( ascites Production and refining

For the multiple production of monoclonal antibodies, hybridomas producing monoclonal antibodies selected in Example 11 were cultured and BALB / C (female, 8 weeks old) mice primed with Pristene (Sigma, USA) 0.5 ml at a concentration of 1 x 10 < ⁶ > cells / ml was injected into the abdominal cavity of the mice. The harvested extracts were centrifuged at 1500 rpm for 10 minutes and the supernatant was harvested and stored at -80 ° C in a cryogenic freezer.

For purification of the monoclonal antibody, the hybridoma cells producing the monoclonal antibody selected in Example 11 were cultured in a 5% carbon dioxide gas thermostat at 37 DEG C, and the antibody was purified in the supernatant. Protein A or G chromatography (Peptron, Korea) was used for purification of the antibody, and the procedure was carried out according to the manufacturer's method.

Example  13: In isotyping  Screening of monoclonal antibodies by

The isotype of the monoclonal antibody was determined by ELISA.

For isotype determination, purified antibodies against the isotype of each mouse rabbit-fed mice were diluted to a concentration of 10 [mu] g / ml with carbonate buffer (pH 9.4), then resuspended in Maxisorp ELISA plates (Nunc, USA) was added at 100 ㎕ each well, followed by reaction at 4 째 C for 16 hours to coat the antibody. Each well coated with each isotype antibody was treated with Blocking Buffer (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactivated horse serum) containing 1% BSA and incubated for 1 hour at 37 ° C The reaction was blocked. 50 [mu] l of hybridoma cell culture solution producing monoclonal antibody was added to each well, followed by reaction at 4 [deg.] C for 1 hour, followed by washing three times with washing buffer solution (PBS, 0.05% Tween-20, 0.05% BSA). After washing, 100 μl of HRP (Horseradish peroxidase) -conjugated anti-mouse IgG + IgA + IgM antibody (1: 2000; Sigma, USA) diluted 1: 1000 was added to each well and incubated at 37 ° C for 1 hour And washed three times with wash buffer solution. After washing, 100 μl of TMB (3,3 ', 5,5'-tetramethylbenzidine) (Sigma, USA) substrate solution was added to each well, and the reaction was allowed to proceed for 30 minutes in darkness, followed by treatment with 2N H ₂ SO ₄ The enzyme reaction was stopped. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. The results are shown in Table 1.

As shown in Table 1, the isotype was classified according to a heavy chain subclass. 16 IgG1 subclasses, 18 IgG2a subclasses, and 3 IgG2b subclasses Of isotypes.

Example  14: affinity analysis of monoclonal antibodies and Antigenic determinant  Expression of RtxA1 (3491-4701) fragment for analysis

Recombinant RtxA1 (3491-4701) protein containing the amino acid sequence 3491 to 4701 of Vtrio sepsis RtxA1 was used which was expressed in Example 4. The gene region of RtxA1 (3491-4380) containing the amino acid sequence 3491 to 4380 of Vtrio septicemia RtxA1 contains two oligonucleotides (RA-3491F2 and RA-4380R) having EcoRI and XhoI restriction enzyme recognition sites and a polymerase Was amplified by the same method 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 region of RtxA1 (3491-3980) including the amino acid sequences 3491 to 3980 of RtxA1 is composed of two oligonucleotides (RA-3491F2 and RA-3980R) having EcoRI and XhoI restriction enzyme recognition sites and a polymerase chain Was amplified in the same manner as in Example 2.

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

RA-3980R: 5'-CCATTCTCGAGCTAATGATGATGATGATGATGCTCACCCGAGGTGGCAATGC-3 '(SEQ ID NO: 9, 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 E. coli transformed in the same manner as in Example 4 Recombinant RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins were expressed and extracted (Figure 5). The specificity of the extracted recombinant protein was confirmed using the same method as in Example 6 (Fig. 6). The results are shown in Figs. 5 to 6. Fig.

FIG. 4 schematically shows a recombinant RtxA1 (3491-4701) fragment protein expressed in a transformed E. coli.

5 shows the molecular weight magnitudes to the left of the figure and the first lane, the second lane, the third lane and the fourth lane correspond to the recombinant proteins RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 3491-3980). ≪ / RTI > Arrows indicate the positions of the expressed recombinant RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins.

Further, Figure 6 shows the results of analysis of the specificity of the recombinant RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins using antibodies against the Vtrioceptor RtxA1 protein. Lane 1, lane 2, lane 3 and lane 4 express the control recombinant protein, recombinant RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 (3491-3980), respectively, Lt; RTI ID = 0.0 > E. coli. ≪ / RTI > Arrows indicate the location of the RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins where antibodies against the RtxA1 protein are positive.

Thus, recombinant RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins were identified in Figures 5 and 6.

Example  15: Affinity analysis of monoclonal antibodies and Antigenic determinant  analysis

The affinity analysis and antigenic determinant analysis of the monoclonal antibodies used as information useful for the screening of monoclonal antibodies for diagnosis and treatment were carried out by ELISA and the fragment of recombinant RtxA1 (3491-4701) identified in Example 14.

The transformed E. coli extracts expressing the recombinant RtxA1 (3491-4701), RtxA1 (3491-4380) and RtxA1 (3491-3980) proteins were each diluted with carbonate buffer (pH 9.4) and then resuspended in Maxisorp ELISA plate Nunc, USA) were added to each well and reacted at 4 DEG C for 16 hours to coat each recombinant protein. Each well coated with each recombinant protein was treated with blocking buffer (PBS, 0.05% Tween-20, 1% BSA, 3% heat-inactivated horse serum) containing 1% BSA for nonspecific reaction Respectively. 50 μl of a cell culture solution containing a monoclonal antibody was added to each well, followed by reaction at 4 ° C for 1 hour, followed by washing 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 was added to each well and incubated at 37 ° C for 1 hour. ≪ / RTI > After washing, 100 μl of HRP (Horseradish peroxidase) -conjugated streptavidin was added to each well, followed by reaction at 37 ° C for 1 hour, followed by washing three times with washing buffer solution, followed by TMB (3,3 ', 5,5' -Tetramethyl-benzidine (Sigma, USA) substrate solution was added to each well in an amount of 100 μl, followed by reaction in dark for 30 minutes to develop color. The enzyme reaction was stopped by treating with 2N H ₂ SO ₄ . After the reaction, the absorbance was measured at 450 nm using an ELISA reader. The results are shown in Table 1.

As shown in the following Table 1, ten antigenic determinants of the monoclonal antibody strongly recognized the recombinant protein RtxA1 (3491-4701) antigen, 13 were RtxA1 (3491-4380) and RtxA1 (3491-4701 ) Antigen at the same time. In addition, 14 recognized all of the RtxA1 (3491-3980), RtxA1 (3491-4380) and RtxA1 (3491-4701) antigens.

Through this, 14 (2RA, 3RA, 8RA, 11RA, 19RA, 21RA, 22RA, 23RA, 24RA, 32RA, 40RA, 46RA, 47RA, 50RA) recognize RtxA1 (3491-3980) , 7RA, 9RA, 12RA, 13RA, 14RA, 20RA, 26RA, 28RA, 29RA, 38RA, 44RA recognize RtxA1 (3981-4380) , 45RA, 48RA, and 52RA recognized RtxA1 (4381-4701) (FIG. 7).

Therefore, it was possible to obtain three groups of monoclonal antibodies having different antigenic determinants according to the present invention, and monoclonal antibodies of each group were used for the development of monoclonal antibodies for the prevention and treatment of Vibrio sepsis and antigenic diagnostic agents for Vibrio sepsis However, it can be usefully used for basic studies on Vibrio sepsis using monoclonal antibodies.

Example  16: Between monoclonal antibodies  Competitive Combination Analysis

To determine whether the 47RA monoclonal antibody recognizing RtxA1 (3491-3980) was different from the binding site of the monoclonal antibodies of the three (13RA, 21RA, 24RA) and 10RA used for investigating the therapeutic effect in Example 23, Biotin Competitive binding assays between monoclonal antibodies were performed using labeled 47RA (designated Biotin-Conjugated 47RA).

To generate the biotin-condensed 47RA, 47RA purified in Example 12 and antibody Biotin labeling kit (FluoReporter Biotin-XX Protein labeling kit, Molecular Probes, USA) were used according to the manufacturer's instructions. Biotin-condensation 47RA Lt; / RTI >

For competitive binding assays between monoclonal antibodies 47RA and four monoclonal antibodies (10RA, 13RA, 21RA, 24RA), competitive ELISA using biotin-condensed 47RA and recombinant RtxA1 (3491-4701) purified in Example 5 Respectively.

The recombinant RtxA1 (3491-4701) protein purified in Example 5 was diluted to 2 [mu] g / ml with carbonate buffer (pH 9.4) and then added 55 [mu] l per well of a Maxisorp ELISA plate (Nunc, USA) , And reacted at 4 ° C for 16 hours to coat the recombinant RtxA1 (3491-4701) protein. Each well coated with recombinant protein was treated with blocking buffer (PBS, 0.05% Tween-20, 1% BSA, 3% heat inactivated horse serum) containing 1% BSA and blocked for nonspecific reaction at 37 ° C for 1 hour Respectively. 50 쨉 l of a cell culture solution containing a monoclonal antibody was added to each well, followed by reaction at 4 째 C for 1 hour, followed by addition of 15 쨉 l of 4 쨉 g / ml Biotin-condensed 47RA and reaction at 4 째 C for 1 hour . Then, 100 μl of HRP (Horseradish peroxidase) -conjugated streptavidin was added to each well, followed by reaction at 37 ° C for 1 hour, followed by washing with PBS (3, 3 ', 5, 5' -tetramethyl-benzidine) (Sigma, USA) was added to each well in an amount of 100 μl per reaction well. The enzyme reaction was stopped by treatment with 2N H ₂ SO ₄ . After the reaction, the absorbance at 450 nm was measured using an ELISA reader, and the biotin-condensation 47RA binding strength in the presence of each monoclonal antibody was expressed in%. The results are shown in Fig.

As shown in FIG. 8, as a result, Biotin-condensed 47RA did not competitively bind to four (10RA, 13RA, 21RA, 24RA) monoclonal antibodies and recombinant RtxA1 (3491-4701) proteins.

Thus, monoclonal antibody 47RA binds to different sites (epitopes) with the four (10RA, 13RA, 21RA, 24RA) monoclonal antibodies.

Example  17: Wild Vibrio Sepsis  culture

(Reddy GP, Hayat U, Abeygunawardana C, Fox C, et al., J Bacteriol. 174: 2620-2630, 1992) in a HI (heart infusion) medium containing 2.5% NaCl After inoculation, the cells were cultured in a 37 ° C shaking incubator for 16 hours. 1/200 of this culture was again inoculated into HI medium (Difco Co.) containing 2.5% NaCl, and then cultured for 4 hours in a 37 ° C shaking incubator.

Example  18: Wild Vibrio Sepsis  Infection Serum Generation

In a CD1 (female, 8 weeks old) mouse, 1 x 10 < ⁶ > / ml of the wild-type Vibrio sepsis cultured in Example 17 was intraperitoneally administered and after 3 weeks blood was obtained through incisional axillary vein, And centrifuged for 3 minutes to produce wild type Vibrio sepsis infected sera.

Example  19: Recombination RtxA1 (3491-4701) Vaccine effect of protein antigen

20 占 퐂 of the RtxA1 (3491-4701) recombinant antigen protein (SEQ ID NO: 6 + 6x-His tag tag) prepared in Example 5 was mixed with Sigma adjuvant (Sigma, USA) in the same manner as in Example 7, Two injections were made in the abdominal cavity of CD1 (female, 8 weeks old) mice at intervals. Fourteen days after the second immunization, 8 x 10 < ⁶ > / ml of the wild-type Vibrio sepsis strain cultivated in Example 17 was administered intraperitoneally and lethality was observed for 96 hours. The results are shown in Fig.

As shown in Fig. 9, mice vaccinated (or immunized) with the recombinant RtxA1 (3491-4701) protein (represented by "RtxA1-C" in FIG. 9) showed 93.3% survival rate and recombinant RtxA1 -4701) protein vaccine. However, the group immunized with the control recombinant GST protein showed a very low survival rate of 6.7%.

Example  20: Recombination RtxA1 (3491-4701) Protein Antigen Vaccination (or immunized)  Blood vibrio Sepsis  Effect on number

A mixture of recombinant RtxA1 (3491-4701) protein antigen and Sigma adjuvant (Sigma, USA) was injected into the abdominal cavity of CD1 (female, 8 weeks old) mice twice at 3-week intervals as in Example 19. After 14 days from the second immunization, 8 × 10 ⁶ / ml of the wild-type Vibrio sepsis cultured in Example 17 was intraperitoneally administered. After 150 minutes, the blood was obtained by axotomy and diluted 10-fold in phosphate buffer. The diluted samples were plated on solid HI medium to investigate the formation of colonies. The results are shown in Fig.

As shown in Fig. 10, only one mouse out of 9 mice vaccinated (or immunized) with the recombinant RtxA1 (3491-4701) protein antigen had 40 vibrio sepsis bacteria in 1 ml of blood. However, in the blood of all mice vaccinated (or immunized) with the control recombinant GST protein, a large number of virulence sepsis was observed at an average of 1 x 10 ⁵ / ml.

This means that mice vaccinated (or immunized) with recombinant RtxA1 (3491-4701) protein antigens have a vaccine effect by decreasing the amount of Vibrio sephacitis in the blood.

Example  21: RtxA1 (3491-4701) Prevention and treatment effect of polyclonal antibody against protein

20 μg of the recombinant RtxA1 (3491-4701) protein (SEQ ID NO: 6 + 6x-His tag) prepared in Example 5 was mixed with Sigma adjuvant (Sigma, USA) as in Example 7, (Female, 8 weeks old) mice. 14 days after the third immunization, blood was obtained through axillary vein incision and centrifuged at 12,000 rpm for 3 minutes to generate serum containing a polyclonal antibody against RtxA1 (3491-4701) protein.

To confirm the protective effect of the polyclonal antibody on the recombinant RtxA1 (3491-4701) protein, CD1 (female, 8 weeks old) mice were intraperitoneally injected with 200 쨉 l of serum containing a polyclonal antibody of RtxA1 (3491-4701) After 4 hours and 30 minutes, 2 x 10 < ⁶ > / ml of wild-type Vibrio sepsis cultured in Example 17 was administered intraperitoneally, and lethality was observed for 96 hours. The results are shown in Fig.

As shown in Fig. 11, mice administered with serum containing a polyclonal antibody against RtxA1 (3491-4701) protein before Vibrio sepsis infection showed 100% survival rate. However, mice receiving serum containing a polyclonal antibody to the control recombinant GST protein showed a very low survival rate of 10%.

In order to confirm the therapeutic effect of the polyclonal antibody against the recombinant RtxA1 (3491-4701) protein, CD1 (female, 8 weeks old) mice were intraperitoneally administered with 2 x 10 ⁶ / ml of the wild-type Vibrio sepsis cultured in Example 17 And 1 hour later, serum containing a polyclonal antibody against 200 μl of RtxA1 (3491-4701) protein was intraperitoneally administered and lethality was observed for 96 hours. The results are shown in Fig.

As shown in Fig. 12, mice administered with serum containing a polyclonal antibody against RtxA1 (3491-4701) protein after Vibrio sepsis infection showed 100% survival rate. However, mice receiving serum containing a polyclonal antibody to the control recombinant GST protein showed a very low survival rate of 10%.

Thus, it was confirmed that the polyclonal antibody against the recombinant RtxA1 (3491-4701) protein has excellent preventive and therapeutic effect on Vibrio paracrine infection.

Example  22: Recombination RtxA1 (3491-4701) Prophylactic effect of monoclonal antibodies against proteins on Vibrio septicemia infection

To confirm the preventive effect of the monoclonal antibody against the recombinant RtxA1 (3491-4701) protein, each monoclonal antibody obtained in Example 12 was injected into CD1 (female, 8 weeks old) mice in 200 ul / mouse Antibody, 1 mg / mosue (purified monoclonal antibody; "PUR" in Table 1 below)) was administered intraperitoneally and incubated in Example 17 at 4 hours and 30 minutes Wild-type Vibrio sepsis 1 × 10 ⁶ / ml was administered intraperitoneally, and lethality was observed for 96 hours. The results are shown in Table 1 and Fig.

Monoclonal antibody Iso
type Binding site Protective activity RtxA1
(3491-3980) RtxA1
(3491-4380) RtxA1
(3491-4701) % Surviving mice Antibody source PBS (control group) - - - - 30 1RA IgG1 - + + 20 ASC 2RA IgG2a + + + 60 ASC 3RA IgG1 + + + 70 ASC 4RA IgG1 - - + 50 PUR 5RA IgG2a - + + 60 ASC 7RA IgG1 - + + 40 ASC 8RA IgG2b + + + 30 ASC 9RA IgG1 - + + 20 ASC 10RA IgG1 - - + 20 PUR 11RA IgG2a + + + 80 ASC 12RA IgG2a - + + 60 ASC 13RA IgG2a - + + 100 ASC 14RA IgG1 - + + 50 ASC 16RA IgG2a - - + 40 ASC 19RA IgG2a + + + 70 ASC 20RA IgG1 - + + 30 ASC 21RA IgG2a + + + 100 ASC / PUR 22RA IgG2b + + + 30 ASC 23RA IgG2a + + + 20 ASC 24RA IgG2a + + + 100 ASC / PUR 26RA IgG1 - + + 30 ASC 27RA IgG2a - - + 30 ASC 28RA IgG2a - + + 30 ASC 29RA IgG1 - + + 50 ASC 30RA IgG1 - - + 50 ASC 32RA IgG2b + + + 30 ASC 38RA IgG2a - + + 40 ASC 40RA IgG1 + + + 30 ASC 41RA IgG1 - - + 50 ASC 42RA IgG2a - - + 70 ASC 44RA IgG1 - + + 60 ASC 45RA IgG2a - - + 90 ASC 46RA IgG2a + + + 90 ASC 47RA IgG2a + + + 90 PUR 48RA IgG1 - - + 40 ASC 50RA IgG2a + + + 100 ASC 52RA IgG1 - - + 60 ASC

As shown in Table 1 and FIG. 13, 13RA, 21RA, 24RA and 50RA showed 100% preventive effect against Vibrio septicemia infection, and 45RA, 46RA and 47RA showed 90% preventive effect. However, mice treated with the phosphate buffer solution as a control group showed a low survival rate of 30%.

Example  23: Recombination RtxA1 (3491-4701) Therapeutic effect of monoclonal antibody against protein on vibrio-sepsis infection

In order to confirm the therapeutic effect of the monoclonal antibody, CD1 (female, 8 weeks old) mice were intraperitoneally administered with 1 x 10 < ⁶ > / ml of the wild-type Vibrio sepsis strain cultured in Example 17, Each of the obtained monoclonal antibodies was intraperitoneally injected with 200 μl / mouse (monoclonal antibody obtained as a plurality) or 500 μg / mosue (purified monoclonal antibody), and lethality was observed for 96 hours. The results are shown in Fig.

As shown in FIG. 14, monoclonal antibody 24RA showed a therapeutic effect of 87% (P = 0.0001) and 47RA had a statistically significant therapeutic effect of 67% (P = 0.0207). However, mice treated with the phosphate buffer solution as a control group had a low survival rate of 32%.

As can be seen from Table 1, among monoclonal antibodies produced in 37 hybridoma cells fused using recombinant RtxA1 protein as an immunogen, the possibility of mass production according to isotype, competitive binding between monoclonal antibodies, In view of the prophylactic and therapeutic effects of the monoclonal antibody against Vibrio septicemia infection and the survival rate of mice after Vibrio septicemia infection, the binding site, isotype subclass, and recombinant RtxA1 (3491 -4701) protein, monoclonal antibodies 24RA and 47RA showed excellent effects. In particular, the monoclonal antibody 24RA showed a very high therapeutic effect against Vibrio septicemia infection.

Therefore, it can be usefully used for prevention and treatment of diseases caused by Vibrio sepsis and related infections, and can be used as a preventive and / or therapeutic agent for Vibrio septicemia by having an excellent vaccine effect.

Thus, the 24RA was deposited with the Korean Cell Line Bank under accession number KCLRF-BP-00311 and the 47RA with accession number KCLRF-BP-00309.

Korea Cell Line Research Foundation KCLRFBP00311BP 20140113 Korea Cell Line Research Foundation KCLRFBP00309BP 20140113

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Monoclonal Antibody Specific Binding to Vibrio vulnificus          RtxA1 protein and Its Use <130> 1040105 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 33 <212> DNA <213> oligonucleotide <400> 1 acatgaattc atgcagagaa gtttggcgac tac 33 <210> 2 <211> 51 <212> DNA <213> oligonucleotide <400> 2 ctcctcgagc taatgatgat gatgatgatg caccgttata ccctttttat g 51 <210> 3 <211> 14106 <212> DNA <213> Vibrio vulnificus RtxA1 <400> 3 atgggaaaac cattttggag aagtgttgaa tacttcttca cagggaacta ttccgccgac 60 gatggaaaca acagtattgt tgctatcggt tttggtggag aaatccatgc ctacggtggt 120 gatgatcatg tcactgtcgg atcgattggt gcaacggttt ataccggcag cggcaatgat 180 acggtcgtag gcggttcggc atatctaaga gtggaagaca ccacagggca cctttctgta 240 aaaggcgcag ctgggtatgc agacattaat aaaagtagcg atggcaatgt gtcattcgct 300 ggtgccgctg gcggtgtgtc gattgaccac ttaggtaatc atggtgatgt cagttacggc 360 ggtgccgcgg cttataatgg cattacccgc aaaggtttga gcggcaacgt cacctttaaa 420 ggcgctggcg gttacaatgc cctatggcat gaaaccaacc aaggtaatct ctcttttgct 480 ggcgcaggag cgggcaataa actagaccgt acttggttca accgttatca aggctcgcgt 540 ggtgatgtga cgtttgatgg cgctggcgcg gcaaacagca tcagttcacg tgttgacaac 600 attactttcc gtggggctgg tgctgacaac catttggtcc gtaaaggcaa agtgggcgac 660 attaccttgc aaggtgcggg cgcatcaaac cgcattgagc gtacacgcca agcggaagat 720 gtttacgcgc aaacacgcgg taacattcgc tttgaaggtg tcggtggtta caacagcctt 780 tactccgatg tggcacacgg tgacatccat ttctccggtg gcggtgctta taacaccatt 840 acacgaaaag gcagtggtag tagcttcgat gctcaaggta tggaatacgc gaaagcggaa 900 gacattgttc tcaccgcggc gcagatgcat ggtttatcga ttgataacgg caacaagttc 960 catgcggtta ccgctgtaaa atcagagcga gagccaaata cttatctgtt tgctatcgca 1020 gatggtactt acaccaaaat caacaaagtt cgtctctata acgatccaga aacgggcaaa 1080 ctcaaatact actctgaagc ttggttcaag cgcggtaatc atttagcgga gcttgctcgt 1140 tctgatgtct cttctgctgg tggttttgaa gtcaatccaa tcaatggtgg ttacaccctt 1200 tctaatattg ccgttgagca tcagcaatcg ttaaccgttc atgctgtcga gaaggacctg 1260 accgaatatg agtgggtgac ctacgccaat ggcgcactga ttgatgcgaa agatgtggca 1320 ttatcagatg cgaaaatggg cggtcacgct atctcgaccg atgggacgac agtcgatgtt 1380 caagcggtga aatcaaaccg aaaaccaaac acttatgttt acgcaaaagt gcttgggcct 1440 tacacaaaaa ttgtggtggt tgaacttgct aatgatccca aaacaggggc gcttaagtat 1500 caagctcgtt cttggtacaa agagggtaat cacaccgcaa acctagccaa tgaagatatt 1560 tcgtctgcaa acggatatca ctcgatgggc aaaggcggct actcactcag cgatctgcat 1620 tacagtgtga acgcggtaag aagcaccagc gaaaccgtgg ccgacatcga cgagtacacc 1680 gatcaaacct tgtttaagcc agcgaccgat agcggtgaaa gctctggtga tgtgcgcttc 1740 aatggtgcgg gtggcggtaa cgtcatcaag tccaatgtga ctcgcggcaa tgtctacttt 1800 aatggtggcg gtatcgctaa cgtcatcctc cacagttcgc aatttggtca taccgagttc 1860 aacggtggtg gcgcggcaaa cgtgattgtc aaaagtggcg aagaagggga tctcaccttc 1920 cgcggcgcgg gtttggcgaa cgtgttggta caccaaagca agcaaggcaa gatggatgtt 1980 tacgcgggcg gtgcggtaaa cgtactggtt cgtattggcg atggtcaata ccttgcgcat 2040 ttgctggctt acggcaacat ttccgtgcac aaaggcaatg gcaacagccg tgtcgttatg 2100 ctcggtggtt acaacaccca tacccaaatt ggctccggca acggcttgtg gttggcggcg 2160 ggcggcttca acgtgatgac gcaagtgggt aaaggtgatg tagcatcggt actggctggc 2220 ggcgccaacg tgctcaccaa agtgggtgac ggcgatctaa ccgcaggtat gttgggcggc 2280 gcaaacgtga ttacccgcat tagcggcgac aatgaaacct cgaataccac agccgtagcc 2340 ttaggcggtg ctaacattct caccaaaaaa ggcaaaggga atacccttgc ggtgatgggc 2400 ggtggcgcaa acgtgctgac gcatgtgggt gatggtacaa caactggcgt aatggtcggt 2460 ggtgcgaaca ttctcaccaa agtaggtaac ggcgacacga ccggcattat gcttggtgtt 2520 ggcaacgtgc taacgcatgt tggtgatggc caaactcttg gtgtgatggg cgcggcgggt 2580 tgatggaacc atcttcaccc atgtgggaga agggaatgct tgggcgctga tgggcggctt gggtaacgtc 2700 tttaccaaag tcggcaacgg tgatgctctc gcgttaatgg tggcagaagc caacgtcttc 2760 actcacattg gtgatggcat gtcggtcgca ctaatgttag ccaaaggcaa cgttgcgaca 2820 aaagtgggta atggaaccac actcgctgca atggttggta acgccaatat cttcactcac 2880 gtaggcagtg gcagcacgtt tgctgcaatg atcggccaag ccaacatcat gaccaaagtg 2940 ggcaatgatc tcaccgcggc actgatggtc ggtaaagcca acatctacac ccatgtcggt 3000 gatgggacca gcctaggaat attcgcgggc gaagtgaacg tgatgactaa ggtcggtaac 3060 ggcacaacct tggcggcgat gttcggtaaa gccaacatca tgacgcatgt cggcgatggt 3120 ctaacgggcg tgttagcact gggcgaagcc aatattgtca ccaaagtggg tgatgatttc 3180 atgggtgttg tggcggcagc aaaagccaac gtcgtgactc atgttggtga tgcaacaacc 3240 gcggctgtgt tggctggtaa aggcaatttc ctaaccaaag tgggtgaagg caccacggta 3300 gggttgttga tttccgatat cggcaacgta atgacccacg tcggagatgg cacgaccatt 3360 ggtattgcca aaggcaaggc aaacatcatt accaaagtcg gtgatggttt gggcgtcaat 3420 gtcgcttggg ggcaagcaaa cgtgtttacc caagtgggcg acggtgatcg ctataacttt 3480 gccaaaggtg aagccaacat catcaccaaa gtaggtgatg gccaagaagt ctccgtggtg 3540 caaggtaaag ctaacatcat tactcatgtg ggcaatggcg acgactacac cggagcttgg 3600 ggtaaagcga acgtcattac taaagtgggt aatggccgta acgtggtact ggcaaaaggt 3660 gaagccaaca tcgtgaccca agtgggggat ggcgacagct ttaacgcact ttggagcaaa 3720 ggcaacatcg tcaccaaagt gggtgatggt atgcaggtga cggcagcaaa aggcaaagcc 3780 aatatcacta ccaccgttgg taatggcttg agtgtgacaa ccgcctatgg tgatgccaac 3840 atcaatacca aagtcggtga tggcgtatcc gtcaacgtcg cttggggcaa atataacatc 3900 aacacgaaag tgggtgatgg cctgaatgtt gccgtgatga aaggcaaagc taacgccaat 3960 attcacgtgg gcgatggcct caacatcaat gcctcttacg cacaaaacaa cgttgcaatc 4020 aaagttggta acggcgattt ttacagtttg gctgttgcgt caagcaacac cagcagcaac 4080 aagctctcag cactgtttga taatatcaag caaaccgtac ttggtgtcgg tggtagccaa 4140 gccatcaact acttggttca aggtgatgaa gcatcgtcat ccggtacgca aaaaggccgt 4200 ggtgcgattg caacaccgga aatcaccaag ctggatggtt tccaaatgga agccattgaa 4260 gaagtgggct cggacttggg cgatagccta accggcagtg ttactaaggt agatacccca 4320 gatctcaaca agatgcaaaa tgcactcgac gttgacggat cttctgatca aactcaagct 4380 ccgaatctca ttgtgaatgg tgattttgag caaggtgatc gtggttggaa atcgacacac 4440 ggtgttgaag cttcctattc gggcaatgtc tatggcgtga acggcgaagg tcacggcgca 4500 cgtgtgacgg aactggatac ctacaccaac accagtctct atcaagatct gaccgatctt 4560 acggaaggtg aagtcattgc tgtgagcttt gattttgcga aacgtgcagg tctatcgaat 4620 aacgaaggca ttgaagttct ttggaatggc gaagtcgtct tctcatcgtc tggtgatgcc 4680 tctgcttggc agcaaaaaac cttaaaactg accgcgcatg ctggcagtaa ccgaatcgaa 4740 ttcaaagcca caggccacaa tgatggactg ggctatatcc tagataacgt cgtggcgaaa 4800 tcggaatcgt cgcaacaagc caacgcagtg agtgagcacg cgacacaaaa tcaagcgtcg 4860 cgaatgtcc tatcagataa agagcgcgca gaagcggatc gccaacgtct tgaacaagaa 4920 aagcagaaac agcttgatgc tgtcgcaggg tcacaaagtc agttggagtc gaccgaccaa 4980 caagcgctag gaaataacgg tcaagctcaa cgtgatgcgg tgaaagaaga gtcggaagcc 5040 gtgaccgccg agttgacaaa actggcgcaa ggtctcgatg tgcttgatgg ccaagcaacg 5100 catactggcg agtcaggcga ccaatggcgc aatgattttg ccggtggtct gctcgatggc 5160 gttcaaagcc agctcgacga tgccaagcaa ctcgcgaatg acaagattgc tgcagcgaag 5220 cagacgcagt ttgacaataa cagcaaagtc aaagaatccg ttgcgaaatc tgaagcgggt 5280 gtggcacaag gtgagcaaaa tcgcgcaggt gcagagcaag acattgccga agctaaagcc 5340 gatgcagaaa cacgcaaagc cgatgctgtg gcgaaaagta acgatgcgaa acaggccgaa 5400 tctgacgcac acagcgccgc aaacgacgcg caatcgcgtg gtgatcgcga tgcgatgaat 5460 gccgaaaaca aagtcaacca agcgcagaat gatgccaaag ggaccaagca aaacgaaggc 5520 gatcgccctg accgtgaagg cgtggctggc agcggtcttt caggtaacgc tcatcgcgtg 5580 gaaggcgccg gcgaaacggg cagtcatgtc aacaccgaca gtccaaccaa cgccgatggt 5640 cgattcagtg aaggtctgag cgaacaagag caagaagcgc tggaaggtgc gacaaacgca 5700 gtgaaccgtc tgcaaattaa cgcaggtatt cgcggaaaga acagcggtag cacaatcacc 5760 tcaatgttca ccgaaacaaa ctccgacagc attgtggtgc caaccactgc gtctcaggat 5820 gtggttcgaa aagagattcg catttctggg gtgaatttag aaggacttgg tgaagcttcg 5880 catgactctg ctgaatcgct tgtggctgct cgcgcggaaa aagtggcgaa tctctatcgc 5940 tggctagaca ctgataatga cgtggcgacg gacaagtatg tacctgtccc tggctttgaa 6000 cgcgtcgatg ttgatgtctc tgatgaagtc aagcagcgca tgattcaatc tatgagcggt 6060 tacatcgaac acaccgacaa tcaagtaccg aaagatcaag cggaagcatt agccactttg 6120 tttgttgaat cgacgctcga ttatgactgg gacaaacgcg ttgagtttct cactaagctt 6180 gagagctatg gttatagttt tgaagcgcca cacgcagaga aaagcattgt ttccttctgg 6240 tctggtaaaa acttcaagca ataccgagac attctcgaca atgcccagac tgatggcaaa 6300 aaggtcgtgt acgacatcga tgttaaaggc aacgcatttg caatcgacct gaataagcat 6360 ttaatgcgtt ggggcggcct gttccttgac ccagataacg ccgagcaaaa tcagttgaaa 6420 tcatcaatcg atgcagcaac gttcagcaat acaggttttt ggagttcggt ctatgcgacg 6480 ggggcacaaa atgatgtgta tgtcatcgca gaaggtggtg tgcgtcttgg caactatttc 6540 tggaatgttg agctacctgc attacgccaa ttgcagcgtg aagggttagt cggtgaaatt 6600 cgactgcttg ataagccagt atccgagtac aaagatctac cagcagacca gattggacgc 6660 cgacttaccg atgcaggtgt tgccgttaaa gtacgctttg atgcgttgag tcacgagaga 6720 caagctgagc ttttggccga caatccagat ggttacaaag ccgatacgct ggttgagttg 6780 gatgtgaagc taagtgcgat cgatagcatg ttgcgtgaat ctctaccatt ctattcactt 6840 cgtactgagc gcaacctctt agtgcaagaa ggcgaagaag gatttgaagt tcgctcatgg 6900 ccgggaatcg atgaaaagag caagactatt ctgcttgata acccagaaga tgcagctcaa 6960 caaaaatcca ttgagcgctt tatcttggca aacttcgaca acttcgagca gatgccagat 7020 gaacttttct tagtggataa caaggtgctc tcgcatcacg atggccgcac gcgaattata 7080 gcgcaaaaag aagacggtgc ttggacgtac aacaccaatg ttgaactgat gtctgtcact 7140 gagctactcg atgctgctca tgtgaacggt aaagttcgcg gtgatagtta ccaacaagtg 7200 atcgacgccc tgacggagta tcatgcgagt acggttgaac acgcagatta cgaactagaa 7260 tctgtggaaa aactgctcaa cctgcgtaaa cagattgaag gttatgtact cgggcatccg 7320 gattctggcc gtgtggaagc aatgaactca ctgttaaatc aggtgaattc tcgtctggaa 7380 gaagtctctg ttcttgcggt ctctgagcaa agcatcaaag cgcacgatag ctttagccgc 7440 ctatatgacc agcttgataa tgccaactta aaagaaagta aacatctgta tctggatgga 7500 aacggcgatt tcgtcactaa gggcaaaggc aacctagcca ctatcgatca gctaggtggt 7560 agcgatgctg tacttgaaaa agtaaaagca gccgtaactc atgagtatgg tcaagtcgtt 7620 gccgatacga tcttcgcaag gctttctgca aacgatctgg caaaagatgg taaaggcatt 7680 gatattgctg gtttaaacaa agtacaccaa gcgattgaac agcacatgtc gccagtgagc 7740 gcgaccatgt acatctggaa gccgagtgat cacagcacac taggtcatgc cgcattgcaa 7800 attggccaag gtcgcacgca gcttgaaggt caagccgctg ctgacttcaa taagcagaat 7860 tacgtgagtt ggtggccact cggtagcaag tcatccaata tccgcaatat cttcaacgtt 7920 gctacagaag atcagccgga tcttaaacta cgttggagcg atttcagcca acctgctcat 7980 caaaatgaca cacttgagca cgatatggca tctgaagaaa acgacggctt tggtttgaaa 8040 gatggtgaaa ctaagctgaa gcgctttatc gaaaaactca acgccgcgaa aggtattgat 8100 gcttcataca aagatgcttc tgaaggctat gcgagtgtac tactgggcaa tccggatatg 8160 cttgcatcga caggcattcc agcgcatgtt ttccagccat tcgtcgacca atggaatgac 8220 accagctacg acatgatgga cgtagcaaat cgttttgctg aagagctaca gaaacaagct 8280 caggcaagcg gtgacccagc ccttgtggaa aaacgtatcg acaacgtggt tcgtctgttt 8340 gctgaaagag cgctagaaga aatcgaagcc tttaaggcca gccaagcaga tgaaggccga 8400 gtgttccgca ttaacctaga agggttggat gtggcggcga tgcaagctga atggaaacgt 8460 ctaagcaacg atccggatgc tcgatatcag ttattgacca aaaactgctc aagtaccgtc 8520 gcaaaagtgt tgaaagcggg tggtgctgac aaacttatcg gccacacatg gcgacctaag 8580 tttggtgttt ggacgccgac agagcttttc aactttggtc aggcgctgca ggaagctcag 8640 cttgaaattg ccgcgaagaa gcaaagtcat caagtcactg atgtccttga tgccttgtca 8700 ggcaatgaga agcacaaaga aaacgtggcg attgaaaatg atggcacgcc accgcgcgat 8760 aaagaatctc ttagcccact gactcgattc ctcaacaatg agttatatgg tgagaaagac 8820 gcacgtcgta agattggcga aatcacgcaa accttacttg accatgcggt agaaaatgga 8880 gaatcgcaga aagttaccct caaaggggaa gtgggccgtc taacggggta ttaccatcag 8940 ggagcggctt caagcgaagg tgaaacaagc gcgacaagcg gcaaagtcgt gttgttcctg 9000 catggttctg gttcttctgc tgaagagcaa gcgagcgaga ttcgcaacca ctaccaaaaa 9060 caaggtatcg acatgctcgc agtcaacctg cgtggctatg gtgaaagcga cggcggacca 9120 agcgaaaagg gcttgtacca agacgctcgc accatgttca actacctagt gaacgacaag 9180 ggtattgatc caagcaatat catcattcac ggctactcaa tgggtggtcc aattgccgca 9240 gatttagcac gttatgccgc gcaaaatggc caagcggtgt ctggcttatt gcttgaccgt 9300 cctatgccaa gcatgaccaa agccatcacc gctcatgaag tggcgaatcc agcgggcatt 9360 gtaggggcta tcgcgaaagc ggttaacggt cagttctcag tggagaagaa cctaaaaggc 9420 ttgccaaaag agacgccgat tctgctgcta acggacaacg aaggtttagg cgaagaaggc 9480 gagaagctac gagctaaact cgcgattgct ggctacaacg tcacgggtga acaaaccttc 9540 tatggccacg aagcgagcaa ccgcttgatg ggtcagtacg cggatcaaat tgtctccggt 9600 ctgttcaatg cagagcaagc agcggtagaa gcgggcgaag tgctgaaagg actagagaaa 9660 gactttaaac gctatggcga cgcgctgaaa ccagatacga gcgtaccggg taaatcgaaa 9720 gacattcgca ccactaaaga tttcctaaat ggttacaaaa atgaccatgc aaaagaaatc 9780 gtggggct tccgctcaga tatgagtatc aagcaactgg tggatctgtt cgttaaaggt 9840 agctggagtg cacagcaaaa aggtgcgctt gcttgggaaa ttgaaagtcg tgcactgaaa 9900 gtgacgttcc agaacaagtc tgagaagtac aaccgattgt tccgtgagat tgcttctgct 9960 ggcgtggtgg atgcgaaagc gactgaacag cttgcgccac agttaatgct gttgaaccta 10020 tcgaatgacg gttttggtgg acgttgtgat ccactttcta aactcgtttt ggttgctaaa 10080 cagcttgaaa acgatggtca agttggcgtg gcaagacaac tgctagagaa gatgtactct 10140 gcggcagcgg tgctgagcaa tccaaccctt tactcagaca gtgaaaatgc caatgcaagc 10200 aagttgctca gcagcttggc ggccattcat gcgaagaacc caatgcatga tacgtcgatg 10260 aaagtgtggc aggaaaagct gaaaggaaag caagcactga ccgtaaacgg tgtggttgag 10320 aaaatcactg atgcatcggc taacggcaaa cctgtgttgt tggaacttga tgctccgggg 10380 catgcgatgg cagcttgggc aaaaggctca ggcgacgatc gtgtttacgg cttctacgat 10440 ccaaatgctg gcattgttga gttctcgtca gcagagaagt ttggcgacta cctaacgcgt 10500 ttcttcggca agtccgatct gaacatggct caaagctata agctgggtaa aaatgacgca 10560 ggtgaagcaa tcttcaaccg cgtggtggta atggatggca acacgttagc aagctacaag 10620 ccgaccttcg gtgacaagac caccatgcag gggatcctag atctacctgt gtttgacgct 10680 acaccgatga aaaagcccgg tacttcagat gtcgatggca atgcaaaagc cgtagatgat 10740 acgaaagaag cattggctgg tggaaagata cttcacaacc aaaatgtgaa tgactgggaa 10800 cgtgttgttg tgactccgac agcggacggc ggtgaaagcc gttttgatgg tcaaatcatc 10860 gtgcaaatgg agaacgatga tgtcgttgca aaagccgctg cgaaccttgc gggtaagcac 10920 ccagaaagca gtgtggtggt gcagatcgat tcagacggca actatcgcgt ggtgtatggc 10980 gatccgtcaa agctggatgg aaagctacgt tggcagttag taggtcatgg tcgagatgac 11040 tcagaaagta acaacacgcg tttaagtggc tacagtgccg acgagctggc agtgaaattg 11100 gccaagttcc aacagtcgtt taatcaagcg gaaaacatca acaataagcc tgatcatatc 11160 agtattgttg gttgttcttt ggtgagtgac gataagcaaa aaggctttgg tcatcagttt 11220 attaacgcga tggatgcgaa tggtcttcgt gtcgatgtct ctgtacgcag ttctgaactg 11280 gccgtagacg aggcagggcg taaacatacc aaggacgcga atggtgattg ggtccaaaaa 11340 gccgaaaaca acaaagtttc gctaagctgg gacgagcaag gtgaagttgt tgccaaggat 11400 gaacgtattc gcaacggtat tgcggaaggc gacatcgatc tctctcgtat tggtgtcagc 11460 gccgttgacg agccagctcg tggtgcaatc ggtgacaaca atgatgtgtt tgatgcgcca 11520 gaaaaacgca aagcggagac agaaacctca tcttcttctg caaacaataa actcagctac 11580 tcaggtaaca ttcaagtcaa tgtgggtgat ggtgagttta cggcagtgaa ctggggcaca 11640 tcgaatgtgg gcattaaagt cggcacgggt ggctttaagt cgctggcttt tggtgacaat 11700 aacgtcatgg ttcacatcgg caatggtgag agcaagcaca gcttcgatat tggtggttat 11760 caggcactgg aaggtgcgca aatgttcatc ggtaatcgta atgtgagctt caacttaggt 11820 cgaagtaatg atctgattgt gatgatggac aagtcgattc cgactccgcc attggttaat 11880 ccgttcgatg gtgccgctcg tatttcgggc gtactgcaaa gcattgccac ctcgggtgag 11940 ggccaagatt ggctagcggc tcaagagcag caatggacat tgtctggcgc caagaagttc 12000 gtcaaagata tgtctggttt ggatcagagc agcagtgttg attacaccag tttggttgaa 12060 ctggactcgc agaacgagcg aagcagccgt ggcttgaagc acgatgcaga agcggctctg 12120 aacaagcagt acaatcaatg gttaagtggc aatagcgatt ctgacaccag caagctcagc 12180 cgcgcagata agcttcgtca agccaatgaa aagcttgcgt ttaactttgc tgtgggtggt 12240 caaggtgcgg atatccaagt cacgacaggt aactggaact tcatgtttgg tgacaacatc 12300 cagtctattt tggataccaa cctaggttca ctgtttggcc tcatgacaca gcagttctct 12360 gctacgggtc aggccaagac aaccttcacc tacacgccag aagatttgcc tcgtcagctt 12420 aagaacaagc tacttgggca gttagcgggt gtaggagccg agaccacgct agcggatatt 12480 tttggtgtgg attacaccgc gtcaggtcaa attgtttcgc gtaatggtga agctgtcgat 12540 ggtgtagcga ttctcaaaga gatgttggag gtcattggtg agttcagtgg tgatcaactg 12600 caagcttttg tcgacccagc taagttactg gatagcttga aggcgggtat caacatgggt 12660 gcggatggca ttaagtcttt tgctgaaact catggactga aagagaaggc gccagaagag 12720 gaagaggaca actcttcggt ttctgttaat ggtgcgagcg taaacagtgc gcaaggcgcg 12780 acggtggctg atggcagcac tgaaacagca gaaacaccag atcgtgcctt tggctttaac 12840 tcgcttaacc tgccgaactt gttcgccact atctttagtc aagacaagca gaaagagatg 12900 aaatcgctgg tggaaaatct caaagagaat ctcaccgccg atctgctgaa tatgaaagag 12960 aaaacgtttg atttccttcg taacagtggt catctccaag gtgatggtga tatcaacatc 13020 tccctaggaa actacaactt caactggggt ggtgatggta aagatctcgg agcgtatcta 13080 ggagacaaca acaacttctg gggcggacga ggcgatgatg tgttctacgc aacaggcacg 13140 tcaaacatct tcacgggcgg cgaaggcaac gacatgggcg ttctgatggg acgtgaaaac 13200 atgatgtttg gcggtgatgg caacgacaca gcagtggttg caggacgcat taaccatgtc 13260 ttccttggtg ccggtgatga ccagtcgttt gtctttggtg agggcggtga aattgacacc 13320 ggttcaggcc gcgactacgt ggtgacgtct ggcaacttca accgtgtgga tacgggggac 13380 gatcaagact actccgtgac gattggcaac aacaaccaag tagagctagg cgctggcaat 13440 gactttgcta atgtcttcgg taactacaac cgtatcaatg caagcgctgg caacgatgtt 13500 gtgaagctaa tgggctatca cgccgtgttg aatggtggtg agggcgagga ccatctcatc 13560 gcagcggcca tctctaagtt cagtcaattc aacggtggcg aagggcgcga tctgatggtg 13620 ttgggtggtt atcaaaacac gttcaaaggt ggcacggatg tggacagctt tgtggtgagc 13680 ggtgatgtta tcgacaacct tgttgaagac attcgcagcg aagataacat tgtcttcaat 13740 ggcatcgatt ggcagaaact gtggttcgaa cgcagcggat atgacctgaa gttgtctatt 13800 cttcgtgacc cggcaagcga cagtgaccaa gcgaagtttg agcatattgg ttcggtgacg 13860 tttagtgatt actttaacgg taatcgagcg caggtgatca tcgcaatggg tgagaaagac 13920 gcgacgggtg aacgtgagta caccaccttg tctgagagtg caattgatgc gctggtacaa 13980 gccatgagtg gctttgaccc tcaggcgggt gacaatggat tcatcgataa cctagacagc 14040 aaatctcgcg tggcgattac cactgcgtgg gcagacgttg ttcataaaaa gggtataacg 14100 gtgtaa 14106 <210> 4 <211> 4701 <212> PRT <213> Vibrio vulnificus RtxA1 <400> 4 Met Gly Lys Pro Phe Trp Arg Ser Val Glu Tyr Phe Phe Thr Gly Asn   1 5 10 15 Tyr Ser Ala Asp Asp Gly Asn Asn Ser Ile Val Ala Ile Gly Phe Gly              20 25 30 Gly Ile His Ala Tyr Gly Gly Asp Asp His Val Thr Val Gly Ser          35 40 45 Ile Gly Ala Thr Val Tyr Thr Gly Ser Gly Asn Asp Thr Val Val Gly      50 55 60 Gly Ser Ala Tyr Leu Arg Val Glu Asp Thr Thr Gly His Leu Ser Val  65 70 75 80 Lys Gly Ala Ala Gly Tyr Ala Asp Ile Asn Lys Ser Ser Asp Gly Asn                  85 90 95 Val Ser Phe Ala Gly Ala Aly Gly Gly Val Ser Ile Asp His Leu Gly             100 105 110 Asn His Gly Asp Val Ser Tyr Gly Gly Ala Ala Ala Tyr Asn Gly Ile         115 120 125 Thr Arg Lys Gly Leu Ser Gly Asn Val Thr Phe Lys Gly Ala Gly Gly     130 135 140 Tyr Asn Ala Leu Trp His Glu Thr Asn Gln Gly Asn Leu Ser Phe Ala 145 150 155 160 Gly Ala Gly Ala Gly Asn Lys Leu Asp Arg Thr Trp Phe Asn Arg Tyr                 165 170 175 Gln Gly Ser Arg Gly Asp Val Thr Phe Asp Gly Ala Gly Ala Ala Asn             180 185 190 Ser Ile Ser Ser Arg Val Asp Asn Ile Thr Phe Arg Gly Ala Gly Ala         195 200 205 Asp His Leu Val Arg Lys Gly Lys Val Gly Asp Ile Thr Leu Gln     210 215 220 Gly Ala Gly Ala Ser Asn Arg Ile Glu Arg Thr Arg Gln Ala Glu Asp 225 230 235 240 Val Tyr Ala Gln Thr Arg Gly Asn Ile Arg Phe Glu Gly Val Gly Gly                 245 250 255 Tyr Asn Ser Leu Tyr Ser Asp Val Ala His Gly Asp Ile His Phe Ser             260 265 270 Gly Gly Gly Ala Tyr Asn Thr Ile Thr Arg Lys Gly Ser Gly Ser Ser         275 280 285 Phe Asp Ala Gln Gly Met Glu Tyr Ala Lys Ala Glu Asp Ile Val Leu     290 295 300 Thr Ala Ala Gln Met His Gly Leu Ser Ile Asp Asn Gly Asn Lys Phe 305 310 315 320 His Ala Val Thr Ala Val Lys Ser Glu Arg Glu Pro Asn Thr Tyr Leu                 325 330 335 Phe Ala Ile Ala Asp Gly Thr Tyr Thr Lys Ile Asn Lys Val Arg Leu             340 345 350 Tyr Asn Asp Pro Glu Thr Gly Lys Leu Lys Tyr Tyr Ser Glu Ala Trp         355 360 365 Phe Lys Arg Gly Asn His Leu Ala Glu Leu Ala Arg Ser Serp Val Ser     370 375 380 Ser Ala Gly Gly Ply Glu Val Asn Pro Ile Asn Gly Gly Tyr Thr Leu 385 390 395 400 Ser Asn Ile Ala Val Glu His Gln Gln Ser Leu Thr Val His Ala Val                 405 410 415 Glu Lys Asp Leu Thr Glu Tyr Glu Trp Val Thr Tyr Ala Asn Gly Ala             420 425 430 Leu Ile Asp Ala Lys Asp Val Ala Leu Ser Asp Ala Lys Met Gly Gly         435 440 445 His Ala Ile Ser Thr Asp Gly Thr Thr Val Asp Val Gln Ala Val Lys     450 455 460 Ser Asn Arg Lys Pro Asn Thr Tyr Val Tyr Ala Lys Val Leu Gly Pro 465 470 475 480 Tyr Thr Lys Ile Val Val Val Glu Leu Ala Asn Asp Pro Lys Thr Gly                 485 490 495 Ala Leu Lys Tyr Gln Ala Arg Ser Trp Tyr Lys Glu Gly Asn His Thr             500 505 510 Ala Asn Leu Ala Asn Glu Asp Ile Ser Ser Ala Asn Gly Tyr His Ser         515 520 525 Met Gly Lys Gly Gly Tyr Ser Leu Ser Asp Leu His Tyr Ser Val Asn     530 535 540 Ala Val Arg Ser Thr Ser Glu Thr Val Ala Asp Ile Asp Glu Tyr Thr 545 550 555 560 Asp Gln Thr Leu Phe Lys Pro Ala Thr Asp Ser Gly Glu Ser Ser Gly                 565 570 575 Asp Val Arg Phe Asn Gly Ala Gly Gly Gly Asn Val Ile Lys Ser Asn             580 585 590 Val Thr Arg Gly Asn Val Tyr Phe Asn Gly Gly Gly Ile Ala Asn Val         595 600 605 Ile Leu His Ser Ser Gln Phe Gly His Thr Glu Phe Asn Gly Gly Gly     610 615 620 Ala Ala Asn Val Ile Val Lys Ser Gly Glu Glu Asp Leu Thr Phe 625 630 635 640 Arg Gly Ala Gly Leu Ala Asn Val Leu Val His Gln Ser Lys Gln Gly                 645 650 655 Lys Met Asp Val Tyr Ala Gly Gly Ala Val Asn Val Leu Val Arg Ile             660 665 670 Gly Asp Gly Gln Tyr Leu Ala His Leu Leu Ala Tyr Gly Asn Ile Ser         675 680 685 Val His Lys Gly Asn Gly Asn Ser Arg Val Val Met Leu Gly Gly Tyr     690 695 700 Asn Thr His Thr Gln Ile Gly Ser Gly Asn Gly Leu Trp Leu Ala Ala 705 710 715 720 Gly Gly Phe Asn Val Met Thr Gln Val Gly Lys Gly Asp Val Ala Ser                 725 730 735 Val Leu Ala Gly Gly Ala Asn Val Leu Thr Lys Val Gly Asp Gly Asp             740 745 750 Leu Thr Ala Gly Met Leu Gly Aly Asn Val Ile Thr Arg Ile Ser         755 760 765 Gly Asp Asn Glu Thr Ser Asn Thr Thr Ala Val Ala Leu Gly Gly Ala     770 775 780 Asn Ile Leu Thr Lys Lys Gly Lys Gly Asn Thr Leu Ala Val Met Gly 785 790 795 800 Gly Gly Ala Asn Val Leu Thr His Val Gly Asp Gly Thr Thr Thr Gly                 805 810 815 Val Met Val Gly Gly Ala Asn Ile Leu Thr Lys Val Gly Asn Gly Asp             820 825 830 Thr Thr Gly Ile Met Leu Gly Val Gly Asn Val Leu Thr His Val Gly         835 840 845 Asp Gly Gln Thr Leu Gly Val Gly Ala Gly Asn Ile Phe Thr     850 855 860 Lys Val Gly Asp Gly Thr Ser Ile Ala Val Met Ile Gly Ala Gly Asn 865 870 875 880 Ile Phe Thr His Val Gly Glu Gly Asn Ala Trp Ala Leu Met Gly Gly                 885 890 895 Leu Gly Asn Val Phe Thr Lys Val Gly Asn Gly Asp Ala Leu Ala Leu             900 905 910 Met Val Ala Glu Ala Asn Val Phe Thr His Ile Gly Asp Gly Met Ser         915 920 925 Val Ala Leu Met Leu Ala Lys Gly Asn Val Ala Thr Lys Val Gly Asn     930 935 940 Gly Thr Thr Leu Ala Ala Met Val Gly Asn Ala Asn Ile Phe Thr His 945 950 955 960 Val Gly Ser Gly Ser Thr Phe Ala Ala Met Ile Gly Gln Ala Asn Ile                 965 970 975 Met Thr Lys Val Gly Asn Asp Leu Thr Ala Ala Leu Met Val Gly Lys             980 985 990 Ala Asn Ile Tyr Thr His Val Gly Asp Gly Thr Ser Leu Gly Ile Phe         995 1000 1005 Ala Gly Glu Val Asn Val Met Thr Lys Val Gly Asn Gly Thr Thr Leu    1010 1015 1020 Ala Ala Met Phe Gly Lys Ala Asn Ile Met Thr His Val Gly Asp Gly 1025 1030 1035 1040 Leu Thr Gly Val Leu Ala Leu Gly Aly Asn Ile Val Thr Lys Val                1045 1050 1055 Gly Asp Asp Phe Met Gly Val Val Ala Ala Ala Lys Ala Asn Val Val            1060 1065 1070 Thr His Val Gly Asp Ala Thr Thr Ala Ala Val Leu Ala Gly Lys Gly        1075 1080 1085 Asn Phe Leu Thr Lys Val Gly Glu Gly Thr Thr Val Gly Leu Leu Ile    1090 1095 1100 Ser Asp Ile Gly Asn Val Met Thr His Val Gly Asp Gly Thr Thr Ile 1105 1110 1115 1120 Gly Ile Ala Lys Gly Lys Ala Asn Ile Ile Thr Lys Val Gly Asp Gly                1125 1130 1135 Leu Gly Val Asn Val Ala Trp Gly Gln Ala Asn Val Phe Thr Gln Val            1140 1145 1150 Gly Asp Gly Asp Arg Tyr Asn Phe Ala Lys Gly Glu Ala Asn Ile Ile        1155 1160 1165 Thr Lys Val Gly Asp Gly Gln Glu Val Ser Val Val Gln Gly Lys Ala    1170 1175 1180 Asn Ile Ile Thr His Val Gly Asn Gly Asp Asp Tyr Thr Gly Ala Trp 1185 1190 1195 1200 Gly Lys Ala Asn Val Ile Thr Lys Val Gly Asn Gly Arg Asn Val                1205 1210 1215 Leu Ala Lys Gly Asp Gly Asp Ile Val Thr Gln Val Gly Asp Gly Asp            1220 1225 1230 Ser Phe Asn Ala Leu Trp Ser Lys Gly Asn Ile Val Thr Lys Val Gly        1235 1240 1245 Asp Gly Met Gln Val Thr Ala Ala Lys Gly Lys Ala Asn Ile Thr Thr    1250 1255 1260 Thr Val Gly Asn Gly Leu Ser Val Thr Thr Ala Tyr Gly Asp Ala Asn 1265 1270 1275 1280 Ile Asn Thr Lys Val Gly Asp Gly Val Ser Val Asn Val Ala Trp Gly                1285 1290 1295 Lys Tyr Asn Ile Asn Thr Lys Val Gly Asp Gly Leu Asn Val Ala Val            1300 1305 1310 Met Lys Gly Lys Ala Asn Ala Asn Ile His Val Gly Asp Gly Leu Asn        1315 1320 1325 Ile Asn Ala Ser Tyr Ala Gln Asn Asn Val Ala Ile Lys Val Gly Asn    1330 1335 1340 Gly Asp Phe Tyr Ser Leu Ala Val Ala Ser Ser Asn Thr Ser Ser Asn 1345 1350 1355 1360 Lys Leu Ser Ala Leu Phe Asp Asn Ile Lys Gln Thr Val Leu Gly Val                1365 1370 1375 Gly Gly Ser Gln Ala Ile Asn Tyr Leu Val Gln Gly Asp Glu Ala Ser            1380 1385 1390 Ser Ser Gly Thr Gln Lys Gly Arg Gly Ala Ile Ala Thr Pro Glu Ile        1395 1400 1405 Thr Lys Leu Asp Gly Phe Gln Met Glu Ala Ile Glu Glu Val Gly Ser    1410 1415 1420 Asp Leu Gly Asp Ser Leu Thr Gly Ser Val Thr Lys Val Asp Thr Pro 1425 1430 1435 1440 Asp Leu Asn Lys Met Gln Asn Ala Leu Asp Val Asp Gly Ser Ser Asp                1445 1450 1455 Gln Thr Gln Ala Pro Asn Leu Ile Val Asn Gly Asp Phe Glu Gln Gly            1460 1465 1470 Asp Arg Gly Trp Lys Ser Thr His Gly Val Glu Ala Ser Tyr Ser Gly        1475 1480 1485 Asn Val Tyr Gly Val Asn Gly Glu Gly His Gly Ala Arg Val Thr Glu    1490 1495 1500 Leu Asp Thr Tyr Thr Asn Thr Ser Leu Tyr Gln Asp Leu Thr Asp Leu 1505 1510 1515 1520 Thr Glu Gly Glu Val Ile Ala Val Ser Phe Asp Phe Ala Lys Arg Ala                1525 1530 1535 Gly Leu Ser Asn Asn Glu Gly Ile Glu Val Leu Trp Asn Gly Glu Val            1540 1545 1550 Val Phe Ser Ser Ser Gly Asp Ser Ser Ala Trp Gln Gln Lys Thr Leu        1555 1560 1565 Lys Leu Thr Ala His Ala Gly Ser Asn Arg Ile Glu Phe Lys Ala Thr    1570 1575 1580 Gly His Asn Asp Gly Leu Gly Tyr Ile Leu Asp Asn Val Val Ala Lys 1585 1590 1595 1600 Ser Glu Ser Ser Gln Gln Ala Asn Ala Val Ser Glu His Ala Thr Gln                1605 1610 1615 Asn Gln Ala Ser Gln Asn Val Leu Ser Asp Lys Glu Arg Ala Glu Ala            1620 1625 1630 Asp Arg Gln Arg Leu Glu Gln Glu Lys Gln Lys Gln Leu Asp Ala Val        1635 1640 1645 Ala Gly Ser Gln Ser Gln Leu Glu Ser Thr Asp Gln Gln Ala Leu Gly    1650 1655 1660 Asn Asn Gly Gln Ala Gln Arg Asp Ala Val Lys Glu Glu Ser Glu Ala 1665 1670 1675 1680 Val Thr Ala Glu Leu Thr Lys Leu Ala Gln Gly Leu Asp Val Leu Asp                1685 1690 1695 Gly Gln Ala Thr His Thr Gly Glu Ser Gly Asp Gln Trp Arg Asn Asp            1700 1705 1710 Phe Ala Gly Gly Leu Leu Asp Gly Val Gln Ser Gln Leu Asp Asp Ala        1715 1720 1725 Lys Gln Leu Ala Asn Asp Lys Ile Ala Ala Ala Lys Gln Thr Gln Phe    1730 1735 1740 Asp Asn Asn Ser Lys Val Lys Glu Ser Val Ala Lys Ser Glu Ala Gly 1745 1750 1755 1760 Val Ala Gln Gly Glu Gln Asn Arg Ala Gly Ala Glu Gln Asp Ile Ala                1765 1770 1775 Glu Ala Lys Ala Asp Ala Glu Thr Arg Lys Ala Asp Ala Val Ala Lys            1780 1785 1790 Ser Asn Asp Ala Lys Gln Ala Glu Ser Asp Ala His Ser Ala Ala Asn        1795 1800 1805 Asp Ala Gln Ser Arg Gly Asp Arg Asp Ala Met Asn Ala Glu Asn Lys    1810 1815 1820 Val Asn Gln Ala Gln Asn Asp Ala Lys Gly Thr Lys Gln Asn Glu Gly 1825 1830 1835 1840 Asp Arg Pro Asp Arg Glu Gly Val Ala Gly Ser Gly Leu Ser Gly Asn                1845 1850 1855 Ala His Arg Val Glu Gly Ala Gly Glu Thr Gly Ser His Val Asn Thr            1860 1865 1870 Asp Ser Pro Thr Asn Ala Asp Gly Arg Phe Ser Glu Gly Leu Ser Glu        1875 1880 1885 Glu Glu Glu Glu Ala Leu Glu Gly Ala Thr Asn Ala Val Asn Arg Leu    1890 1895 1900 Gln Ile Asn Ala Gly Ile Arg Gly Lys Asn Ser Gly Ser Thr Ile Thr 1905 1910 1915 1920 Ser Met Phe Thr Glu Thr Asn Ser Asp Ser Ile Val Val Pro Thr Thr                1925 1930 1935 Ala Ser Gln Asp Val Val Arg Lys Glu Ile Arg Ile Ser Gly Val Asn            1940 1945 1950 Leu Glu Gly Leu Gly Glu Ala Ser His Asp Ser Ala Glu Ser Leu Val        1955 1960 1965 Ala Ala Arg Ala Glu Lys Val Ala Asn Leu Tyr Arg Trp Leu Asp Thr    1970 1975 1980 Asp Asn Asp Val Ala Thr Asp Lys Tyr Val Pro Val Gly Phe Glu 1985 1990 1995 2000 Arg Val Asp Val Asp Val Ser Asp Glu Val Lys Gln Arg Met Ile Gln                2005 2010 2015 Ser Met Ser Gly Tyr Ile Glu His Thr Asp Asn Gln Val Pro Lys Asp            2020 2025 2030 Gln Ala Glu Ala Leu Ala Thr Leu Phe Val Glu Ser Thr Leu Asp Tyr        2035 2040 2045 Asp Trp Asp Lys Arg Val Glu Phe Leu Thr Lys Leu Glu Ser Tyr Gly    2050 2055 2060 Tyr Ser Phe Glu Ala Pro His Ala Glu Lys Ser Ile Val Ser Phe Trp 2065 2070 2075 2080 Ser Gly Lys Asn Phe Lys Gln Tyr Arg Asp Ile Leu Asp Asn Ala Gln                2085 2090 2095 Thr Asp Gly Lys Lys Val Val Tyr Asp Ile Asp Val Lys Gly Asn Ala            2100 2105 2110 Phe Ala Ile Asp Leu Asn Lys His Leu Met Arg Trp Gly Gly Leu Phe        2115 2120 2125 Leu Asp Pro Asp Asn Ala Glu Gln Asn Gln Leu Lys Ser Ser Ile Asp    2130 2135 2140 Ala Ala Thr Phe Ser Asn Thr Gly Phe Trp Ser Ser Val Tyr Ala Thr 2145 2150 2155 2160 Gly Ala Gln Asn Asp Val Tyr Val Ile Ala Glu Gly Gly Val Arg Leu                2165 2170 2175 Gly Asn Tyr Phe Trp Asn Val Glu Leu Pro Ala Leu Arg Gln Leu Gln            2180 2185 2190 Arg Glu Gly Leu Val Gly Glu Ile Arg Leu Leu Asp Lys Pro Val Ser        2195 2200 2205 Glu Tyr Lys Asp Leu Pro Ala Asp Gln Ile Gly Arg Arg Leu Thr Asp    2210 2215 2220 Ala Gly Val Ala Val Lys Val Arg Phe Asp Ala Leu Ser His Glu Arg 2225 2230 2235 2240 Gln Ala Glu Leu Leu Ala Asp Asn Pro Asp Gly Tyr Lys Ala Asp Thr                2245 2250 2255 Leu Val Glu Leu Asp Val Lys Leu Ser Ala Ile Asp Ser Met Leu Arg            2260 2265 2270 Glu Ser Leu Pro Phe Tyr Ser Leu Arg Thr Glu Arg Asn Leu Leu Val        2275 2280 2285 Glu Glu Gly Glu Glu Gly Phe Glu Val Arg Ser Trp Pro Gly Ile Asp    2290 2295 2300 Glu Lys Ser Lys Thr Ile Leu Leu Asp Asn Pro Glu Asp Ala Ala Gln 2305 2310 2315 2320 Gln Lys Ser Ile Glu Arg Phe Ile Leu Ala Asn Phe Asp Asn Phe Glu                2325 2330 2335 Gln Met Pro Asp Glu Leu Phe Leu Val Asp Asn Lys Val Leu Ser His            2340 2345 2350 His Asp Gly Arg Thr Arg Ile Ile Ala Gln Lys Glu Asp Gly Ala Trp        2355 2360 2365 Thr Asn Thr Asn Val Glu Leu Met Ser Val Thr Glu Leu Leu Asp    2370 2375 2380 Ala Ala His Val Asn Gly Lys Val Arg Gly Asp Ser Tyr Gln Gln Val 2385 2390 2395 2400 Ile Asp Ala Leu Thr Glu Tyr His Ala Ser Thr Val Glu His Ala Asp                2405 2410 2415 Tyr Glu Leu Glu Ser Val Glu Lys Leu Leu Asn Leu Arg Lys Gln Ile            2420 2425 2430 Glu Gly Tyr Val Leu Gly His Pro Asp Ser Gly Arg Val Glu Ala Met        2435 2440 2445 Asn Ser Leu Leu Asn Gln Val Asn Ser Arg Leu Glu Glu Val Val Ser    2450 2455 2460 Leu Ala Val Ser Glu Gln Ser Ile Lys Ala His Asp Ser Phe Ser Arg 2465 2470 2475 2480 Leu Tyr Asp Gln Leu Asp Asn Ala Asn Leu Lys Glu Ser Lys His Leu                2485 2490 2495 Tyr Leu Asp Gly Asn Gly Asp Phe Val Thr Lys Gly Lys Gly Asn Leu            2500 2505 2510 Ala Thr Ile Asp Glu Leu Gly Gly Ser Asp Ala Val Leu Glu Lys Val        2515 2520 2525 Lys Ala Ala Val Thr His Glu Tyr Gly Gln Val Val Ala Asp Thr Ile    2530 2535 2540 Phe Ala Arg Leu Ser Ala Asn Asp Leu Ala Lys Asp Gly Lys Gly Ile 2545 2550 2555 2560 Asp Ile Ala Gly Leu Asn Lys Val His Gln Ala Ile Glu Gln His Met                2565 2570 2575 Ser Pro Val Ser Ala Thr Met Tyr Ile Trp Lys Pro Ser Asp His Ser            2580 2585 2590 Thr Leu Gly His Ala Leu Gln Ile Gly Gln Gly Arg Thr Gln Leu        2595 2600 2605 Glu Gly Gln Ala Ala Asp Phe Asn Lys Gln Asn Tyr Val Ser Trp    2610 2615 2620 Trp Pro Leu Gly Ser Lys Ser Ser Asn Ile Arg Asn Ile Phe Asn Val 2625 2630 2635 2640 Ala Thr Glu Asp Gln Pro Asp Leu Lys Leu Arg Trp Ser Asp Phe Ser                2645 2650 2655 Gln Pro Ala His Gln Asn Asp Thr Leu Glu His Asp Met Ala Ser Glu            2660 2665 2670 Glu Asn Gly Phe Gly Leu Lys Asp Gly Glu Thr Lys Leu Lys Arg        2675 2680 2685 Phe Ile Glu Lys Leu Asn Ala Ala Lys Gly Ile Asp Ala Ser Tyr Lys    2690 2695 2700 Asp Ala Ser Glu Gly Tyr Ala Ser Val Leu Leu Gly Asn Pro Asp Met 2705 2710 2715 2720 Leu Ala Ser Thr Gly Ile Pro Ala His Val Phe Gln Pro Phe Val Asp                2725 2730 2735 Gln Trp Asn Asp Thr Ser Tyr Asp Met Met Asp Val Ala Asn Arg Phe            2740 2745 2750 Ala Glu Glu Leu Gln Lys Gln Ala Gln Ala Ser Gly Asp Pro Ala Leu        2755 2760 2765 Val Glu Lys Arg Ile Asp Asn Val Val Arg Leu Phe Ala Glu Arg Ala    2770 2775 2780 Leu Glu Glu Ile Glu Ala Phe Lys Ala Ser Gln Ala Asp Glu Gly Arg 2785 2790 2795 2800 Val Phe Arg Ile Asn Leu Glu Gly Leu Asp Val Ala Ala Met Gln Ala                2805 2810 2815 Glu Trp Lys Arg Leu Ser Asn Asp Pro Asp Ala Arg Tyr Gln Leu Leu            2820 2825 2830 Thr Lys Asn Cys Ser Ser Thr Val Ala Lys Val Leu Lys Ala Gly Gly        2835 2840 2845 Ala Asp Lys Leu Ile Gly His Thr Trp Arg Pro Lys Phe Gly Val Trp    2850 2855 2860 Thr Pro Thr Glu Leu Phe Asn Phe Gly Gln Ala Leu Gln Glu Ala Gln 2865 2870 2875 2880 Leu Glu Ile Ala Ala Lys Lys Gln Ser His Gln Val Thr Asp Val Leu                2885 2890 2895 Asp Ala Leu Ser Gly Asn Glu Lys His Lys Glu Asn Val Ala Ile Glu            2900 2905 2910 Asn Asp Gly Thr Pro Pro Arg Asp Lys Glu Ser Leu Ser Pro Leu Thr        2915 2920 2925 Arg Phe Leu Asn Asn Glu Leu Tyr Gly Glu Lys Asp Ala Arg Arg Lys    2930 2935 2940 Ile Gly Glu Ile Thr Gln Thr Leu Leu Asp His Ala Val Glu Asn Gly 2945 2950 2955 2960 Glu Ser Gln Lys Val Thr Leu Lys Gly Glu Val Gly Arg Leu Thr Gly                2965 2970 2975 Tyr Tyr His Gln Gly Ala Ala Ser Ser Glu Gly Glu Thr Ser Ala Thr            2980 2985 2990 Ser Gly Lys Val Val Leu Phe Leu His Gly Ser Gly Ser Ser Ala Glu        2995 3000 3005 Glu Gln Ala Ser Glu Ile Arg Asn His Tyr Gln Lys Gln Gly Ile Asp    3010 3015 3020 Met Leu Ala Val Asn Leu Arg Gly Tyr Gly Glu Ser Asp Gly Gly Pro 3025 3030 3035 3040 Ser Glu Lys Gly Leu Tyr Gln Asp Ala Arg Thr Met Phe Asn Tyr Leu                3045 3050 3055 Val Asn Asp Lys Gly Ile Asp Pro Ser Asn Ile Ile Ile His Gly Tyr            3060 3065 3070 Ser Met Gly Gly Pro Ile Ala Ala Asp Leu Ala Arg Tyr Ala Ala Gln        3075 3080 3085 Asn Gly Gln Ala Val Ser Gly Leu Leu Leu Asp Arg Pro Met Met Ser Ser    3090 3095 3100 Met Thr Lys Ala Ile Thr Ala His Glu Val Ala Asn Pro Ala Gly Ile 3105 3110 3115 3120 Val Gly Ala Ile Ala Lys Ala Val Asn Gly Gln Phe Ser Val Glu Lys                3125 3130 3135 Asn Leu Lys Gly Leu Pro Lys Glu Thr Pro Ile Leu Leu Leu Thr Asp            3140 3145 3150 Asn Glu Gly Leu Gly Glu Glu Gly Glu Lys Leu Arg Ala Lys Leu Ala        3155 3160 3165 Ile Ala Gly Tyr Asn Val Thr Gly Glu Gln Thr Phe Tyr Gly His Glu    3170 3175 3180 Ala Ser Asn Arg Leu Met Gly Gln Tyr Ala Asp Gln Ile Val Ser Gly 3185 3190 3195 3200 Leu Phe Asn Ala Glu Ala Gla Ala Val Glu Ala Gly Glu Val Leu Lys                3205 3210 3215 Gly Leu Glu Lys Asp Phe Lys Arg Tyr Gly Asp Ala Leu Lys Pro Asp            3220 3225 3230 Thr Ser Val Pro Gly Lys Ser Lys Asp Ile Arg Thr Thr Lys Asp Phe        3235 3240 3245 Leu Asn Gly Tyr Lys Asn Asp His Ala Lys Glu Ile Val Asp Gly Phe    3250 3255 3260 Arg Ser Asp Met Ser Ile Lys Gln Leu Val Asp Leu Phe Val Lys Gly 3265 3270 3275 3280 Ser Trp Ser Ala Gln Gln Lys Gly Ala Leu Ala Trp Glu Ile Glu Ser                3285 3290 3295 Arg Ala Leu Lys Val Thr Phe Gln Asn Lys Ser Glu Lys Tyr Asn Arg            3300 3305 3310 Leu Phe Arg Glu Ile Ala Ser Ala Gly Val Val Asp Ala Lys Ala Thr        3315 3320 3325 Glu Gln Leu Ala Pro Gln Leu Met Leu Leu Asn Leu Ser Asn Asp Gly    3330 3335 3340 Phe Gly Gly Arg Cys Asp Pro Leu Ser Lys Leu Val Leu Val Ala Lys 3345 3350 3355 3360 Gln Leu Glu Asn Asp Gly Gln Val Gly Val Ala Arg Gln Leu Leu Glu                3365 3370 3375 Lys Met Tyr Ser Ala Ala Ala Val Leu Ser Asn Pro Thr Leu Tyr Ser            3380 3385 3390 Asp Ser Glu Asn Ala Asn Ala Ser Lys Leu Leu Ser Ser Leu Ala Ala        3395 3400 3405 Ile His Ala Lys Asn Pro Met His Asp Thr Ser Met Lys Val Trp Gln    3410 3415 3420 Glu Lys Leu Lys Gly Lys Gln Ala Leu Thr Val Asn Gly Val Val Glu 3425 3430 3435 3440 Lys Ile Thr Asp Ala Ser Ala Asn Gly Lys Pro Val Leu Leu Glu Leu                3445 3450 3455 Asp Ala Pro Gly His Ala Met Ala Ala Trp Ala Lys Gly Ser Gly Asp            3460 3465 3470 Asp Arg Val Tyr Gly Phe Tyr Asp Pro Asn Ala Gly Ile Val Glu Phe        3475 3480 3485 Ser Ser Ala Glu Lys Phe Gly Asp Tyr Leu Thr Arg Phe Phe Gly Lys    3490 3495 3500 Ser Asp Leu Asn Met Ala Gln Ser Tyr Lys Leu Gly Lys Asn Asp Ala 3505 3510 3515 3520 Gly Glu Ala Ile Phe Asn Arg Val Val Val Met Asp Gly Asn Thr Leu                3525 3530 3535 Ala Ser Tyr Lys Pro Thr Phe Gly Asp Lys Thr Thr Met Gln Gly Ile            3540 3545 3550 Leu Asp Leu Pro Val Phe Asp Ala Thr Pro Met Lys Lys Pro Gly Thr        3555 3560 3565 Ser Asp Val Asp Gly Asn Ala Lys Ala Val Asp Asp Thr Lys Glu Ala    3570 3575 3580 Leu Ala Gly Gly Lys Ile Leu His Asn Gln Asn Val Asn Asp Trp Glu 3585 3590 3595 3600 Arg Val Val Thr Pro Thr Ala Asp Gly Gly Glu Ser Arg Phe Asp                3605 3610 3615 Gly Gln Ile Ile Val Gln Met Glu Asn Asp Asp Val Val Ala Lys Ala            3620 3625 3630 Ala Ala Asn Leu Ala Gly Lys His Pro Glu Ser Ser Val Val Val Gln        3635 3640 3645 Ile Asp Ser Asp Gly Asn Tyr Arg Val Val Tyr Gly Asp Pro Ser Lys    3650 3655 3660 Leu Asp Gly Lys Leu Arg Trp Gln Leu Val Gly His Gly Arg Asp Asp 3665 3670 3675 3680 Ser Glu Ser Asn Asn Thr Arg Leu Ser Gly Tyr Ser Ala Asp Glu Leu                3685 3690 3695 Ala Val Lys Leu Ala Lys Phe Gln Gln Ser Phe Asn Gln Ala Glu Asn            3700 3705 3710 Ile Asn Asn Lys Pro Asp His Ile Ser Ile Val Gly Cys Ser Leu Val        3715 3720 3725 Ser Asp Asp Lys Gln Lys Gly Phe Gly His Gln Phe Ile Asn Ala Met    3730 3735 3740 Asp Ala Asn Gly Leu Arg Val Asp Val Val Ser Arg Ser Ser Glu Leu 3745 3750 3755 3760 Ala Val Asp Glu Ala Gly Arg Lys His Thr Lys Asp Ala Asn Gly Asp                3765 3770 3775 Trp Val Gln Lys Ala Glu Asn Asn Lys Val Ser Leu Ser Trp Asp Glu            3780 3785 3790 Gln Gly Glu Val Val Ala Lys Asp Glu Arg Ile Arg Asn Gly Ile Ala        3795 3800 3805 Glu Gly Asp Ile Asp Leu Ser Arg Ile Gly Val Ser Asp Val Asp Glu    3810 3815 3820 Pro Ala Arg Gly Ala Ile Gly Asp Asn Asn Asp Val Phe Asp Ala Pro 3825 3830 3835 3840 Glu Lys Arg Lys Ala Glu Thr Glu Thr Ser Ser Ser Ala Asn Asn                3845 3850 3855 Lys Leu Ser Tyr Ser Gly Asn Ile Gln Val Asn Val Gly Asp Gly Glu            3860 3865 3870 Phe Thr Ala Val Asn Trp Gly Thr Ser Asn Val Gly Ile Lys Val Gly        3875 3880 3885 Thr Gly Gly Phe Lys Ser Leu Ala Phe Gly Asp Asn Asn Val Met Val    3890 3895 3900 His Ile Gly Asn Gly Glu Ser Lys His Ser Phe Asp Ile Gly Gly Tyr 3905 3910 3915 3920 Gln Ala Leu Glu Gly Ala Gln Met Phe Ile Gly Asn Arg Asn Val Ser                3925 3930 3935 Phe Asn Leu Gly Arg Ser Asn Asp Leu Ile Val Met Met Asp Lys Ser            3940 3945 3950 Ile Pro Thr Pro Pro Leu Val Asn Pro Phe Asp Gly Ala Ala Arg Ile        3955 3960 3965 Ser Gly Val Leu Gln Ser Ile Ala Thr Ser Gly Glu Gly Gln Asp Trp    3970 3975 3980 Leu Ala Ala Gln Glu Gln Gln Trp Thr Leu Ser Gly Ala Lys Lys Phe 3985 3990 3995 4000 Val Lys Asp Met Ser Gly Leu Asp Gln Ser Ser Ser Val Asp Tyr Thr                4005 4010 4015 Ser Leu Val Glu Leu Asp Ser Gln Asn Glu Arg Ser Ser Arg Gly Leu            4020 4025 4030 Lys His Asp Ala Glu Ala Ala Leu Asn Lys Gln Tyr Asn Gln Trp Leu        4035 4040 4045 Ser Gly Asn Ser Asp Ser Asp Thr Ser Lys Leu Ser Arg Ala Asp Lys    4050 4055 4060 Leu Arg Gln Ala Asn Glu Lys Leu Ala Phe Asn Phe Ala Val Gly Gly 4065 4070 4075 4080 Gln Gly Ala Asp Ile Gln Val Thr Thr Gly Asn Trp Asn Phe Met Phe                4085 4090 4095 Gly Asp Asn Ile Gln Ser Ile Leu Asp Thr Asn Leu Gly Ser Leu Phe            4100 4105 4110 Gly Leu Met Thr Gln Gln Phe Ser Ala Thr Gly Gln Ala Lys Thr Thr        4115 4120 4125 Phe Thr Tyr Thr Pro Glu Asp Leu Pro Arg Gln Leu Lys Asn Lys Leu    4130 4135 4140 Leu Gly Gln Leu Ala Gly Val Gly Ala Glu Thr Thr Leu Ala Asp Ile 4145 4150 4155 4160 Phe Gly Val Asp Tyr Thr Ala Ser Gly Gln Ile Val Ser Arg Asn Gly                4165 4170 4175 Glu Ala Val Asp Gle Val Ile Leu Lys Glu Met Leu Glu Val Ile            4180 4185 4190 Gly Glu Phe Ser Gly Asp Gln Leu Gln Ala Phe Val Asp Pro Ala Lys        4195 4200 4205 Leu Leu Asp Ser Leu Lys Ala Gly Ile Asn Met Gly Ala Asp Gly Ile    4210 4215 4220 Lys Ser Phe Ala Glu Thr His Gly Leu Lys Glu Lys Ala Pro Glu Glu 4225 4230 4235 4240 Glu Glu Asp Asn Ser Ser Val Ser Val Asn Gly Ala Ser Val Asn Ser                4245 4250 4255 Ala Gln Gly Ala Thr Ala Glu Thr            4260 4265 4270 Pro Asp Arg Ala Phe Gly Phe Asn Ser Leu Asn Leu Pro Asn Leu Phe        4275 4280 4285 Ala Thr Ile Phe Ser Gln Asp Lys Gln Lys Glu Met Lys Ser Leu Val    4290 4295 4300 Glu Asn Leu Lys Glu Asn Leu Thr Ala Asp Leu Leu Asn Met Lys Glu 4305 4310 4315 4320 Lys Thr Phe Asp Phe Leu Arg Asn Ser Gly His Leu Gln Gly Asp Gly                4325 4330 4335 Asp Ile Asn Ile Ser Leu Gly Asn Tyr Asn Phe Asn Trp Gly Gly Asp            4340 4345 4350 Gly Lys Asp Leu Gly Ala Tyr Leu Gly Asp Asn Asn Asn Phe Trp Gly        4355 4360 4365 Gly Arg Gly Asp Asp Val Phe Tyr Ala Thr Gly Thr Ser Asn Ile Phe    4370 4375 4380 Thr Gly Gly Glu Gly Asn Asp Met Gly Val Leu Met Gly Arg Glu Asn 4385 4390 4395 4400 Met Met Phe Gly Gly Asp Gly Asn Asp Thr Ala Val Val Ala Gly Arg                4405 4410 4415 Ile Asn His Val Phe Leu Gly Ala Gly Asp Asp Gln Ser Phe Val Phe            4420 4425 4430 Gly Glu Gly Gly Glu Ile Asp Thr Gly Ser Gly Arg Asp Tyr Val Val        4435 4440 4445 Thr Ser Gly Asn Phe Asn Arg Val Asp Thr Gly Asp Asp Gln Asp Tyr    4450 4455 4460 Ser Val Thr Ile Gly Asn Asn Asn Gln Val Glu Leu Gly Ala Gly Asn 4465 4470 4475 4480 Asp Phe Ala Asn Val Phe Gly Asn Tyr Asn Arg Ile Asn Ala Ser Ala                4485 4490 4495 Gly Asn Val Val Lys Leu Met Gly Tyr His Ala Val Leu Asn Gly            4500 4505 4510 Gly Glu Gly Glu Asp His Leu Ile Ala Ala Ala Ile Ser Lys Phe Ser        4515 4520 4525 Gln Phe Asn Gly Gly Glu Gly Arg Asp Leu Met Val Leu Gly Gly Tyr    4530 4535 4540 Gln Asn Thr Phe Lys Gly Gly Thr Asp Val Asp Ser Phe Val Val Ser 4545 4550 4555 4560 Gly Asp Val Ile Asp Asn Leu Val Glu Asp Ile Arg Ser Glu Asp Asn                4565 4570 4575 Ile Val Phe Asn Gly Ile Asp Trp Gln Lys Leu Trp Phe Glu Arg Ser            4580 4585 4590 Gly Tyr Asp Leu Lys Leu Ser Ile Leu Arg Asp Pro Ala Ser Asp Ser        4595 4600 4605 Asp Gln Ala Lys Phe Glu His Ile Gly Ser Val Thr Phe Ser Asp Tyr    4610 4615 4620 Phe Asn Gly Asn Arg Ala Gln Val Ile Ile Ala Met Gly Glu Lys Asp 4625 4630 4635 4640 Ala Thr Gly Glu Arg Glu Tyr Thr Thr Leu Ser Glu Ser Ala Ile Asp                4645 4650 4655 Ala Leu Val Gln Ala Met Ser Gly Phe Asp Pro Gln Ala Gly Asp Asn            4660 4665 4670 Gly Phe Ile Asp Asn Leu Asp Ser Lys Ser Arg Val Ala Ile Thr Thr        4675 4680 4685 Ala Trp Ala Asp Val Val His Lys Lys Gly Ile Thr Val    4690 4695 4700 <210> 5 <211> 3636 <212> DNA <213> Vibrio vulnificus RtxA1 <400> 5 gcagagaagt ttggcgacta cctaacgcgt ttcttcggca agtccgatct gaacatggct 60 caaagctata agctgggtaa aaatgacgca ggtgaagcaa tcttcaaccg cgtggtggta 120 atggatggca acacgttagc aagctacaag ccgaccttcg gtgacaagac caccatgcag 180 gggatcctag atctacctgt gtttgacgct acaccgatga aaaagcccgg tacttcagat 240 gtcgatggca atgcaaaagc cgtagatgat acgaaagaag cattggctgg tggaaagata 300 cttcacaacc aaaatgtgaa tgactgggaa cgtgttgttg tgactccgac agcggacggc 360 ggtgaaagcc gttttgatgg tcaaatcatc gtgcaaatgg agaacgatga tgtcgttgca 420 aaagccgctg cgaaccttgc gggtaagcac ccagaaagca gtgtggtggt gcagatcgat 480 tcagacggca actatcgcgt ggtgtatggc gatccgtcaa agctggatgg aaagctacgt 540 tggcagttag taggtcatgg tcgagatgac tcagaaagta acaacacgcg tttaagtggc 600 tacagtgccg acgagctggc agtgaaattg gccaagttcc aacagtcgtt taatcaagcg 660 gaaaacatca acaataagcc tgatcatatc agtattgttg gttgttcttt ggtgagtgac 720 gataagcaaa aaggctttgg tcatcagttt attaacgcga tggatgcgaa tggtcttcgt 780 gtcgatgtct ctgtacgcag ttctgaactg gccgtagacg aggcagggcg taaacatacc 840 aaggacgcga atggtgattg ggtccaaaaa gccgaaaaca acaaagtttc gctaagctgg 900 gacgagcaag gtgaagttgt tgccaaggat gaacgtattc gcaacggtat tgcggaaggc 960 gacatcgatc tctctcgtat tggtgtcagc gacgttgacg agccagctcg tggtgcaatc 1020 ggtgacaaca atgatgtgtt tgatgcgcca gaaaaacgca aagcggagac agaaacctca 1080 tcttcttctg caaacaataa actcagctac tcaggtaaca ttcaagtcaa tgtgggtgat 1140 ggtgagttta cggcagtgaa ctggggcaca tcgaatgtgg gcattaaagt cggcacgggt 1200 ggctttaagt cgctggcttt tggtgacaat aacgtcatgg ttcacatcgg caatggtgag 1260 agcaagcaca gcttcgatat tggtggttat caggcactgg aaggtgcgca aatgttcatc 1320 ggtaatcgta atgtgagctt caacttaggt cgaagtaatg atctgattgt gatgatggac 1380 aagtcgattc cgactccgcc attggttaat ccgttcgatg gtgccgctcg tatttcgggc 1440 gtactgcaaa gcattgccac ctcgggtgag ggccaagatt ggctagcggc tcaagagcag 1500 caatggacat tgtctggcgc caagaagttc gtcaaagata tgtctggttt ggatcagagc 1560 agcagtgttg attacaccag tttggttgaa ctggactcgc agaacgagcg aagcagccgt 1620 ggcttgaagc acgatgcaga agcggctctg aacaagcagt acaatcaatg gttaagtggc 1680 aatagcgatt ctgacaccag caagctcagc cgcgcagata agcttcgtca agccaatgaa 1740 aagcttgcgt ttaactttgc tgtgggtggt caaggtgcgg atatccaagt cacgacaggt 1800 aactggaact tcatgtttgg tgacaacatc cagtctattt tggataccaa cctaggttca 1860 ctgtttggcc tcatgacaca gcagttctct gctacgggtc aggccaagac aaccttcacc 1920 tacacgccag aagatttgcc tcgtcagctt aagaacaagc tacttgggca gttagcgggt 1980 gtaggagccg agaccacgct agcggatatt tttggtgtgg attacaccgc gtcaggtcaa 2040 attgtttcgc gtaatggtga agctgtcgat ggtgtagcga ttctcaaaga gatgttggag 2100 gtcattggtg agttcagtgg tgatcaactg caagcttttg tcgacccagc taagttactg 2160 gatagcttga aggcgggtat caacatgggt gcggatggca ttaagtcttt tgctgaaact 2220 catggactga aagagaaggc gccagaagag gaagaggaca actcttcggt ttctgttaat 2280 ggtgcgagcg taaacagtgc gcaaggcgcg acggtggctg atggcagcac tgaaacagca 2340 gaaacaccag atcgtgcctt tggctttaac tcgcttaacc tgccgaactt gttcgccact 2400 atctttagtc aagacaagca gaaagagatg aaatcgctgg tggaaaatct caaagagaat 2460 ctcaccgccg atctgctgaa tatgaaagag aaaacgtttg atttccttcg taacagtggt 2520 catctccaag gtgatggtga tatcaacatc tccctaggaa actacaactt caactggggt 2580 ggtgatggta aagatctcgg agcgtatcta ggagacaaca acaacttctg gggcggacga 2640 ggcgatgatg tgttctacgc aacaggcacg tcaaacatct tcacgggcgg cgaaggcaac 2700 gacatgggcg ttctgatggg acgtgaaaac atgatgtttg gcggtgatgg caacgacaca 2760 gcagtggttg caggacgcat taaccatgtc ttccttggtg ccggtgatga ccagtcgttt 2820 gtctttggtg agggcggtga aattgacacc ggttcaggcc gcgactacgt ggtgacgtct 2880 ggcaacttca accgtgtgga tacgggggac gatcaagact actccgtgac gattggcaac 2940 aacaaccaag tagagctagg cgctggcaat gactttgcta atgtcttcgg taactacaac 3000 cgtatcaatg caagcgctgg caacgatgtt gtgaagctaa tgggctatca cgccgtgttg 3060 aatggtggtg agggcgagga ccatctcatc gcagcggcca tctctaagtt cagtcaattc 3120 aacggtggcg aagggcgcga tctgatggtg ttgggtggtt atcaaaacac gttcaaaggt 3180 ggcacggatg tggacagctt tgtggtgagc ggtgatgtta tcgacaacct tgttgaagac 3240 attcgcagcg aagataacat tgtcttcaat ggcatcgatt ggcagaaact gtggttcgaa 3300 cgcagcggat atgacctgaa gttgtctatt cttcgtgacc cggcaagcga cagtgaccaa 3360 gcgaagtttg agcatattgg ttcggtgacg tttagtgatt actttaacgg taatcgagcg 3420 caggtgatca tcgcaatggg tgagaaagac gcgacgggtg aacgtgagta caccaccttg 3480 tctgagagtg caattgatgc gctggtacaa gccatgagtg gctttgaccc tcaggcgggt 3540 gacaatggat tcatcgataa cctagacagc aaatctcgcg tggcgattac cactgcgtgg 3600 gcagacgttg ttcataaaaa gggtataacg gtgtaa 3636 <210> 6 <211> 1211 <212> PRT <213> Vibrio vulnificus RtxA1 <400> 6 Ala Glu Lys Phe Gly Asp Tyr Leu Thr Arg Phe Phe Gly Lys Ser Asp   1 5 10 15 Leu Asn Met Ala Gln Ser Tyr Lys Leu Gly Lys Asn Asp Ala Gly Glu              20 25 30 Ala Ile Phe Asn Arg Val Val Val Met Asp Gly Asn Thr Leu Ala Ser          35 40 45 Tyr Lys Pro Thr Phe Gly Asp Lys Thr Thr Met Gln Gly Ile Leu Asp      50 55 60 Leu Pro Val Phe Asp Ala Thr Pro Met Lys Lys Pro Gly Thr Ser Asp  65 70 75 80 Val Asp Gly Asn Ala Lys Ala Val Asp Asp Thr Lys Glu Ala Leu Ala                  85 90 95 Gly Gly Lys Ile Leu His Asn Gln Asn Val Asn Asp Trp Glu Arg Val             100 105 110 Val Val Thr Pro Thr Ala Asp Gly Gly Glu Ser Arg Phe Asp Gly Gln         115 120 125 Ile Ile Val Gln Met Glu Asn Asp Val Val Ala Lys Ala Ala Ala     130 135 140 Asn Leu Ala Gly Lys His Pro Glu Ser Ser Val Val Val Gln Ile Asp 145 150 155 160 Ser Asp Gly Asn Tyr Arg Val Val Tyr Gly Asp Pro Ser Lys Leu Asp                 165 170 175 Gly Lys Leu Arg Trp Gln Leu Val Gly His Gly Arg Asp Asp Ser Glu             180 185 190 Ser Asn Asn Thr Arg Leu Ser Gly Tyr Ser Ala Asp Glu Leu Ala Val         195 200 205 Lys Leu Ala Lys Phe Gln Gln Ser Phe Asn Gln Ala Glu Asn Ile Asn     210 215 220 Asn Lys Pro Asp His Ile Ser Ile Val Gly Cys Ser Leu Val Ser Asp 225 230 235 240 Asp Lys Gln Lys Gly Phe Gly His Gln Phe Ile Asn Ala Met Asp Ala                 245 250 255 Asn Gly Leu Arg Val Asp Val Ser Val Arg Ser Ser Glu Leu Ala Val             260 265 270 Asp Glu Ala Gly Arg Lys His Thr Lys Asp Ala Asn Gly Asp Trp Val         275 280 285 Gln Lys Ala Glu Asn Asn Lys Val Ser Leu Ser Trp Asp Glu Gln Gly     290 295 300 Glu Val Val Ala Lys Asp Glu Arg Ile Arg Asn Gly Ile Ala Glu Gly 305 310 315 320 Asp Ile Asp Leu Ser Arg Ile Gly Val Ser Asp Val Asp Glu Pro Ala                 325 330 335 Arg Gly Ala Ile Gly Asp Asn Asn Asp Val Phe Asp Ala Pro Glu Lys             340 345 350 Arg Lys Ala Glu Thr Glu Thr Ser Ser Ser Ala Asn Asn Lys Leu         355 360 365 Ser Tyr Ser Gly Asn Ile Gln Val Asn Val Gly Asp Gly Glu Phe Thr     370 375 380 Ala Val Asn Trp Gly Thr Ser Asn Val Gly Ile Lys Val Gly Thr Gly 385 390 395 400 Gly Phe Lys Ser Leu Ala Phe Gly Asp Asn Asn Val Met Met Val Ile                 405 410 415 Gly Asn Gly Glu Ser Lys His Ser Phe Asp Ile Gly Gly Tyr Gln Ala             420 425 430 Leu Glu Gly Ala Gln Met Phe Ile Gly Asn Arg Asn Val Ser Phe Asn         435 440 445 Leu Gly Arg Ser Ser Asn Leu Ile Val Met Met Asp Lys Ser Ile Pro     450 455 460 Thr Pro Pro Leu Val Asn Pro Phe Asp Gly Ala Ala Arg Ile Ser Gly 465 470 475 480 Val Leu Gln Ser Ile Ala Thr Ser Gly Glu Gly Gln Asp Trp Leu Ala                 485 490 495 Ala Gln Glu Gln Gln Trp Thr Leu Ser Gly Ala Lys Lys Phe Val Lys             500 505 510 Asp Met Ser Gly Leu Asp Gln Ser Ser Ser Val Asp Tyr Thr Ser Leu         515 520 525 Val Glu Leu Asp Ser Gln Asn Glu Arg Ser Ser Arg Gly Leu Lys His     530 535 540 Asp Ala Glu Ala Ala Leu Asn Lys Gln Tyr Asn Gln Trp Leu Ser Gly 545 550 555 560 Asn Ser Asp Ser Asp Thr Ser Lys Leu Ser Arg Ala Asp Lys Leu Arg                 565 570 575 Gln Ala Asn Glu Lys Leu Ala Phe Asn Phe Ala Val Gly Gly Gln Gly             580 585 590 Ala Asp Ile Gln Val Thr Thr Gly Asn Trp Asn Phe Met Phe Gly Asp         595 600 605 Asn Ile Gln Ser Ile Leu Asp Thr Asn Leu Gly Ser Leu Phe Gly Leu     610 615 620 Met Thr Gln Gln Phe Ser Ala Thr Gly Gln Ala Lys Thr Thr Phe Thr 625 630 635 640 Tyr Thr Pro Glu Asp Leu Pro Arg Gln Leu Lys Asn Lys Leu Leu Gly                 645 650 655 Gln Leu Ala Gly Val Gly Ala Glu Thr Thr Leu Ala Asp Ile Phe Gly             660 665 670 Val Asp Tyr Thr Ala Ser Gly Gln Ile Val Ser Arg Asn Gly Glu Ala         675 680 685 Val Asp Gly Val Ala Ile Leu Lys Glu Met Leu Glu Val Ile Gly Glu     690 695 700 Phe Ser Gly Asp Gln Leu Gln Ala Phe Val Asp Pro Ala Lys Leu Leu 705 710 715 720 Asp Ser Leu Lys Ala Gly Ile Asn Met Gly Ala Asp Gly Ile Lys Ser                 725 730 735 Phe Ala Glu Thr His Gly Leu Lys Glu Lys Ala Pro Glu Glu Glu Glu             740 745 750 Asp Asn Ser Ser Val Ser Val Asn Gly Ala Ser Val Asn Ser Ala Gln         755 760 765 Gly Ala Thr Val Ala Asp Gly Ser Thr Glu Thr Ala Glu Thr Pro Asp     770 775 780 Arg Ala Phe Gly Phe Asn Ser Leu Asn Leu Pro Asn Leu Phe Ala Thr 785 790 795 800 Ile Phe Ser Gln Asp Lys Gln Lys Glu Met Lys Ser Leu Val Glu Asn                 805 810 815 Leu Lys Glu Asn Leu Thr Ala Asp Leu Leu Asn Met Lys Glu Lys Thr             820 825 830 Phe Asp Phe Leu Arg Asn Ser Gly His Leu Gln Gly Asp Gly Asp Ile         835 840 845 Asn Ile Ser Leu Gly Asn Tyr Asn Phe Asn Trp Gly Gly Asp Gly Lys     850 855 860 Asp Leu Gly Ala Tyr Leu Gly Asp Asn Asn Asn Phe Trp Gly Gly Arg 865 870 875 880 Gly Asp Asp Val Phe Tyr Ala Thr Gly Thr Ser Asn Ile Phe Thr Gly                 885 890 895 Gly Glu Gly Asn Asp Met Gly Val Leu Met Gly Arg Glu Asn Met Met             900 905 910 Phe Gly Gly Asp Gly Asn Asp Thr Ala Val Val Ala Gly Arg Ile Asn         915 920 925 His Val Phe Leu Gly Ala Gly Asp Asp Gln Ser Phe Val Phe Gly Glu     930 935 940 Gly Gly Glu Ile Asp Thr Gly Ser Gly Arg Asp Tyr Val Val Thr Ser 945 950 955 960 Gly Asn Phe Asn Arg Val Asp Thr Gly Asp Asp Gln Asp Tyr Ser Val                 965 970 975 Thr Ile Gly Asn Asn Asn Gln Val Glu Leu Gly Ala Gly Asn Asp Phe             980 985 990 Ala Asn Val Phe Gly Asn Tyr Asn Arg Ile Asn Ala Ser Ala Gly Asn         995 1000 1005 Asp Val Val Lys Leu Met Gly Tyr His Ala Val Leu Asn Gly Gly Glu    1010 1015 1020 Gly Glu Asp His Leu Ile Ala Ala Ala Ile Ser Lys Phe Ser Gln Phe 1025 1030 1035 1040 Asn Gly Gly Gly Gly Arg Asp Leu Met Val Leu Gly Gly Tyr Gln Asn                1045 1050 1055 Thr Phe Lys Gly Gly Thr Asp Val Asp Ser Phe Val Ser Ser Gly Asp            1060 1065 1070 Val Ile Asp Asn Leu Val Glu Asp Ile Arg Ser Glu Asp Asn Ile Val        1075 1080 1085 Phe Asn Gly Ile Asp Trp Gln Lys Leu Trp Phe Glu Arg Ser Gly Tyr    1090 1095 1100 Asp Leu Lys Leu Ser Ile Leu Arg Asp Pro Ala Ser Asp Ser Asp Gln 1105 1110 1115 1120 Ala Lys Phe Glu His Ile Gly Ser Val Thr Phe Ser Asp Tyr Phe Asn                1125 1130 1135 Gly Asn Arg Ala Gln Val Ile Ile Ala Met Gly Glu Lys Asp Ala Thr            1140 1145 1150 Gly Glu Arg Glu Tyr Thr Thr Leu Ser Glu Ser Ala Ile Asp Ala Leu        1155 1160 1165 Val Gln Ala Met Ser Gly Phe Asp Pro Gln Ala Gly Asp Asn Gly Phe    1170 1175 1180 Ile Asp Asn Leu Asp Ser Lys Ser Arg Val Ala Ile Thr Thr Ala Trp 1185 1190 1195 1200 Ala Asp Val Val His Lys Lys Gly Ile Thr Val                1205 1210 <210> 7 <211> 39 <212> DNA <213> oligonucleotide <400> 7 acatgaattc ataccatggc agagaagttt ggcgactac 39 <210> 8 <211> 53 <212> DNA <213> oligonucleotide <400> 8 ccattctcga gctaatgatg atgatgatga tgcgtgcctg ttgcgtagaa cac 53 <210> 9 <211> 52 <212> DNA <213> oligonucleotide <400> 9 ccattctcga gctaatgatg atgatgatga tgctcacccg aggtggcaat gc 52

Claims (8)

A hybridoma cell according to Accession No. KCLRF-BP-00311, characterized in that it produces a monoclonal antibody against the Vtrio sepsis RtxA1 protein. A hybridoma cell according to Accession No. KCLRF-BP-00309, characterized in that it produces monoclonal antibodies against the Vtrio sepsis RtxA1 protein. A monoclonal antibody or antigen-binding fragment thereof to a Vtrioceptor RtxA1 protein produced by a hybridoma cell with accession number KCLRF-BP-00311. Monoclonal antibody or antigen-binding fragment thereof to a Vtrioceptor RtxA1 protein produced by a hybridoma cell with accession number KCLRF-BP-00309. The monoclonal antibody or the antigen-binding fragment thereof according to claim 3 or 4, wherein the monoclonal antibody specifically binds to the 3491 to 3980th amino acid sequence region of the RtxA1 protein of SEQ ID NO: 4. A therapeutic agent for vibriosis septicemia comprising the monoclonal antibody or antigen-binding fragment thereof of claim 3 or 4. A prophylactic agent for Vibrio septicemia comprising the monoclonal antibody of claim 3 or 4 or an antigen-binding fragment thereof. 4. A Vibrio septicemia diagnostic kit comprising the monoclonal antibody of claim 3 or 4 or an antigen-binding fragment thereof.

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