KR20120002641A - Antibody against ompu of vibrio and method of detecting vibro using the same - Google Patents

Abstract

PURPOSE: An antibody which specifically recognizes Vibrio parahaemolyticus or Vibrio vulnificus is provided to prevent food poisoning in advance. CONSTITUTION: An antigen-binding polypeptide specifically binds to OMP(outer membrane protein) of vibrio and contains VH domain and VL domain. The antigen-binding polypeptide additionally contains a linker polypeptide. The linker polypeptide has sequences of sequence numbers 31, 32, or 33. The antigen-binding polypeptide contains an amino acid sequence of sequence numbers 34, 35, 36, or 37. A kit for detecting vibrio contains the polypeptide. The vibrio includes Vibrio parahaemolyticus or Vibrio vulnificus. A method for detecting vibrio comprises a step of detecting the OMP of vibrio using the antigen-binding polypeptide.

Description

Antibodies against outer membrane proteins of Vibrio bacteria and methods for detecting the same using the same

The present application relates to the field of detection of food poisoning bacteria using antibodies.

Despite the improved living standards and advances in medical technology, food poisoning is not significantly reduced. According to Article 2 (10) of the Food Sanitation Act of Korea, food poisoning refers to an infectious or toxin type disease caused by or believed to be caused by microorganisms or toxic substances harmful to the human body due to the ingestion of food. Food poisoning is mainly caused by parasites, viruses and bacteria, of which food poisoning by bacteria accounts for more than 50% (domestic and overseas food poisoning occurrence and management trends, agri-food safety information service operation / management business, April 2008). There are currently 10 types of bacterial food poisoning tests (Salmonella, Enteritis Vibrio, Staphylococcus aureus, Pathogenic Escherichia coli, Listeria monocytogenes, Bacillus cereus, Yexinia enterolytica, Campylobacter jejuni, Clostropium botulinum, Clostrium perfringe) Jens).

In recent years, food poisoning occurs regardless of season, and the scale of food poisoning is gradually increasing. Therefore, it is necessary to develop an accurate test method for monitoring and determining the cause of food poisoning bacteria to reduce and suppress food poisoning.

Methods for rapidly detecting food poisoning-inducing bacteria can be divided into antigen-antibody reaction-based, nucleic acid conjugation reactions and characterization of mycological properties through enrichment. The former detects proteins as analytes, and the latter detects nucleic acids that make up genes. In the immunological method based on antigen-antibody, the development of antibodies that can specifically recognize bacteria is key, but there are no antibodies that can specifically detect Vibrio bacteria at the species level.

The present application is to detect Vibrio bacteria, Vibrio parahaemolyticus and Vibrio vulnificus , specifically from the many kinds of food poisoning-inducing Gram-negative bacteria and Gram-positive bacteria. To develop a system.

The present application is an antigen-binding polypeptide that specifically binds to the outer membrane protein of Vibrio bacteria, wherein the polypeptide includes a VH domain and a VL domain, and the VH domain includes H-CDR1, H-CDR2, and H-CDR3. , Wherein each of H-CDR1, H-CDR2 and H-CDR3 is H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 6 and H-CDR3 of SEQ ID NO: 11; H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 7 and H-CDR3 of SEQ ID NO: 12; H-CDR1 of SEQ ID NO: 3, H-CDR2 of SEQ ID NO: 8 and H-CDR3 of SEQ ID NO: 13; H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 9 and H-CDR3 of SEQ ID NO: 14; And H-CDR1 of H-CDR1 of SEQ ID NO: 5, H-CDR2 of SEQ ID NO: 10, and H-CDR3 of SEQ ID NO: 15, wherein the VL domain is L-CDR1 L-CDR2 and L-CDR3, wherein each of L-CDR1, L-CDR2 and L-CDR3 comprises L-CDR1 of SEQ ID NO: 16, L-CDR2 of SEQ ID NO: 21 and L-CDR3 of SEQ ID NO: 26; L-CDR1 of SEQ ID NO: 17, L-CDR2 of SEQ ID NO: 22 and L-CDR3 of SEQ ID NO: 27; L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 23 and L-CDR3 of SEQ ID NO: 28; L-CDR1 of SEQ ID NO: 19, L-CDR2 of SEQ ID NO: 24 and L-CDR3 of SEQ ID NO: 29; And an L-CDR set selected from the group consisting of L-CDR1 of SEQ ID NO: 20, L-CDR2 of SEQ ID NO: 25, and L-CDR3 of SEQ ID NO: 30, an antigen binding polypeptide. .

In addition, the present application is an antigen-binding polypeptide comprising any one amino acid sequence selected from the group consisting of the following VH-VL combination: H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 6 and SEQ ID NO: A combination of a VH comprising H-CDR3 of 11 and a L-CDR1 of SEQ ID NO: 16, L-CDR2 of SEQ ID NO: 21, and a VL comprising L-CDR3 of SEQ ID NO: 26; VH comprising H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 7 and H-CDR3 of SEQ ID NO: 12, L-CDR1 of SEQ ID NO: 17, L-CDR2 of SEQ ID NO: 22, and L- of SEQ ID NO: 27 Combination of CDR3VL; VH comprising H-CDR1 of SEQ ID NO: 3, H-CDR2 of SEQ ID NO: 8 and H-CDR3 of SEQ ID NO: 13, L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 23, and L- of SEQ ID NO: 28 Combination of VLs comprising CDR3; VH comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 9 and H-CDR3 of SEQ ID NO: 14, L-CDR1 of SEQ ID NO: 19, L-CDR2 of SEQ ID NO: 24, and L- of SEQ ID NO: 29 Combination of VLs comprising CDR3; And a VH comprising H-CDR1 of SEQ ID NO: 5, H-CDR2 of SEQ ID NO: 10 and H-CDR3 of SEQ ID NO: 15, L-CDR1 of SEQ ID NO: 20, L-CDR2 of SEQ ID NO: 25, and L of SEQ ID NO: 30 Provide a combination of VLs including CDR3.

The application also provides an antigen binding polypeptide, wherein said polypeptide further comprises a linker polypeptide located between said VH and VH.

The application also provides an antigen binding polypeptide, wherein the linker polypeptide is any one of SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33.

The application also provides an antigen binding polypeptide wherein the antigen binding polypeptide is selected from the group consisting of SEQ ID NOs: 34, 35, 36 and 37.

The present disclosure also provides nucleotides encoding such antigen binding polypeptides.

The application also provides nucleotides, wherein the nucleotides are selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.

The present application also provides a kit for detecting vibrio comprising the antigen-binding polypeptide.

The present invention also Vibrio bacteria Vibrio enteritis bacteria ( Vibrio parahaemolyticus ) or Vibrio sepsis bacteria ( Vibrio vulnificus ) at least one of which provides a kit for detecting Vibrio bacteria.

The present invention also provides a Vibrio bacteria detection method comprising the step of detecting the outer membrane protein of Vibrio bacteria using the antigen-binding polypeptide.

The present invention also Vibrio bacteria Vibrio parahaemolyticus or Vibrio sepsis bacteria ( Vibrio vulnificus ), which provides at least one, Vibrio bacteria detection method.

Antibodies that specifically react to only OmpU of Vibrio bacteria and the compositions and methods of the present application can specifically detect specific Vibrio species from many kinds of food poisoning-inducing Gram-negative bacteria and Gram-positive bacteria. Food poisoning can be prevented in advance, as well as through the estimation of the cause of food poisoning through the accurate detection of the causative bacteria of food poisoning can be usefully used in the establishment of pre and post-management system of food poisoning.

1 is a photograph of polyacrylamide gel of recombinant protein OmpU isolated and purified using Ni-NTA + resin. Each lane is as follows: lane 1: protein size marker; lane 2: purified recombinant protein OmpU fraction # 1; lane 3: purified recombinant protein OmpU fraction # 2; lane 4: purified recombinant protein OmpU fraction # 3; lane 5: purified recombinant protein OmpU fraction # 4.
FIG. 2 is an 8% polyacrylamide gel photograph showing BstN1 pinkerprinting for scFv monophage clones (1E, 1G, 3B, 4E and 9C) selected for ompU-His. ScFv inserts from each clone were amplified and digested with BstN1 restriction enzymes, followed by electrophoresis on 8% polyacrylamide gels.
Figure 3 is the amino acid sequence of antibody protein (scFv) obtained from monophage clones for OmpU.
Figure 4 is a photo of polyacrylamide gel of the scFv-OmpU 1E antibody isolated and purified.
FIG. 5 shows Western blot results confirming the specificity of scFv-OmpU antibodies of 1E, 1G, 3B, 4E, and 9C clones.
FIG. 6 is a Western blot result showing the specificity for the vibriobacteria of the scFv-OmpU 1E antibody. The bacteria were as follows: Vv ( Vibrio vulnificus) MO6024 / O, Vv ATCC29307, Vv CMCP6, Pa ( Pseudomonas aeruginosa) pR01957, Bc ( Bacillus cereus ).
Figure 7 is the result of Western blot confirming the species detection susceptibility to the Vibrio bacteria and other Gram negative and positive bacteria of scFv-OmpU 1G, 3B, 4E, and 9C antibodies.

In one embodiment the present application relates to an antigen binding polypeptide that specifically binds to the outer membrane protein of Vibrio bacteria. More specifically the polypeptide comprises a VH domain and a VL domain, wherein the VH domain comprises H-CDR1, H-CDR2 and H-CDR3, wherein each of the H-CDR1, H-CDR2 and H-CDR3 is a sequence H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 6 and H-CDR3 of SEQ ID NO: 11; H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 7 and H-CDR3 of SEQ ID NO: 12; H-CDR1 of SEQ ID NO: 3, H-CDR2 of SEQ ID NO: 8 and H-CDR3 of SEQ ID NO: 13; H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 9 and H-CDR3 of SEQ ID NO: 14; And H-CDR1 of H-CDR1 of SEQ ID NO: 5, H-CDR2 of SEQ ID NO: 10, and H-CDR3 of SEQ ID NO: 15, wherein the VL domain is L-CDR1 L-CDR2 and L-CDR3, wherein each of L-CDR1, L-CDR2 and L-CDR3 comprises L-CDR1 of SEQ ID NO: 16, L-CDR2 of SEQ ID NO: 21 and L-CDR3 of SEQ ID NO: 26; L-CDR1 of SEQ ID NO: 17, L-CDR2 of SEQ ID NO: 22 and L-CDR3 of SEQ ID NO: 27; L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 23 and L-CDR3 of SEQ ID NO: 28; L-CDR1 of SEQ ID NO: 19, L-CDR2 of SEQ ID NO: 24 and L-CDR3 of SEQ ID NO: 29; And an L-CDR set selected from the group consisting of L-CDR1 of SEQ ID NO: 20, L-CDR2 of SEQ ID NO: 25, and L-CDR3 of SEQ ID NO: 30, an antigen binding polypeptide. .

Herein, OmpU is isolated from recombinant outer protein of Outer membrane Protein (OMP) of Vibrio bacteria that interacts specifically with host cells, and then the human non-exposed single chain variable fragment (SCF) pid library ( Human naive scFv phage library) was used to obtain a polypeptide that specifically binds to OmpU.

Gram-negative bacteria have a characteristic structure called the outer membrane (Outer Membrane). In addition to lipopolysaccharides, OM contains several types of proteins called Outer Membrane Proteins (OMPs). These OMPs are known to be associated with the pathogenicity of Vibrio bacteria and their attachment to other individuals (Beaubrun et al., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2008, p. 907-911; Goo et al., Infection and Immunity, Oct 2006, p. 5586-5594). OMPU is a 35KDa major protein found in the outer membrane of Vibrio bacteria, and binds to the host cell fibronectin to induce adhesion to the host cell, thereby playing a major role in vibrio showing pathogenicity. OMPU proteins look somewhat difference in Vibrio species present in comparison to the sequences of selected of which the high affinity V i brio vulnificus and V. parahaemolyticus thereof OMPU protein sequence was used as an antigen.

The term “human antibody” as used herein refers to Kabat et al (1991) Sequences of proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication Np. 91-3242) and antibodies having variable and constant regions corresponding to the human Germline immunoglobulin sequence as described. Human antibodies herein are random in in vitro and in Mutations in viv o may include amino acid residues that differ from the human pointline immunoglobulin sequence, such as the CDR (Complementarity Determining Region), CDR3.

The term antibody, as used herein, is used interchangeably with immunoglobulins and refers to complete antibodies (two light chains, two heavy chains), fragments of the antibody, eg, Fab, F (ab ') 2 fragments, scFv and Fv fragments. It includes. The antibodies herein also include any Ig class and any Ig subclass (eg IgG1 to IgG4 as subclasses of IgG, subclasses of IgA, IgA1, IgA2 and secreted IgA),

As used herein, the term “antigen-binding polypeptide” is used to mean a polypeptide fragment of an immunoglobulin, antibody or antibody like molecule that binds to an antigen or competes for binding to an antibody against the antigen. Such antigen binding polypeptides within the scope of this application are antibodies or fragments thereof that bind to OMPU proteins and / or epitopes, especially Vibrio's OMPU proteins and / or epitopes.

The antibody consists of four polypeptide chains, which are divided into two light chains (L) and two heavy chains (H). The heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH), and the latter is divided into CH1, CH2 and CH3. The light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL are again divided into three regions called Complementarity Determining Regions (CDRs) (each CDR1, CDR2 and CDR3, these three regions are located between the framework sequences) and four framework sequences (FR). Consists of. Each VH and VL is arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. The CDR is the part that participates in binding to the antigen and determines the specificity of antigen antibody binding. The CDRs of the heavy chain are called H-CDR1, H-CDR2, and H-CDR3, and the three CDRs present in the light chain are called L-CDR1, L-CDR2, and L-CDR3. Among them, the mutation of the CDR3 region is the most severe, and at least 2 amino acids, at least 26 or more, are composed of amino acid residues. The portions other than CDRs in VH and VL are skeletal residues. The backbone of the antigen-binding polypeptide of the present application can also be used as a common sequence of sequences found in human antibodies present in nature, or sequences found in various antibodies.

The most widely used system for numbering CDRs is the Kabat system ( ibid ). Kabat is based on sequence variability, where the CDRs of the light chain variable region are residues at positions 24 and 34 (CDR-1), residues at positions 50 and 56 (CDR-2) and residues at positions 89 and 97 (CDR- 3) surrounded by. The heavy chain variable region is surrounded by residues at positions 31 and 35 (CDR-1), residues at positions 50 and 65 (CDR-2) and residues at positions 95 and 102 (CDR-3).

As used herein, the term binding site or antigen-binding polypeptide includes those that have specific binding capacity to OMPU as a complete antibody, as well as fragments thereof. Antigen-binding polypeptides can be produced by recombinant DNA techniques, or by cleaving complete antibodies using compounds or enzymes. Binding fragments include Fab, Fab ', F (ab') 2 Fabc, Fd, dAb, Fv, single chain antibodies such as scFv, and single domain antibodies. The VL and VH regions are encoded by different genes, but are linked by linking molecules (linkers) that link the two, pairing the VL and VH regions to form a monovalent antigen-binding polypeptide, scFv (single chain fragment variable) This is the case.

 The peptide linker connects the VH and VL regions, and the linker connects the carboxy terminus of one variable region to the amino terminus of the other variable region without affecting the performance of the VH-VL pairing and antigen binding sites. Peptide linkers are from about 1 to about 25 amino acids in length and typically consist of the hydrophilic amino acids glycine (G) and serine (S). 0-4 amino acids in length form a multimer. In general, (GGGGS) 3 linkers are used for, but are not limited to, scFv. The linker may have an amino acid sequence that may advantageously work in post-operation such as after expression of the antibody in a suitable cell, isolation / purification / affinity to antigen, and the like. For example, if the linker contains metal-bonded amino acids such as histidine (H) and cysteine (C), it can increase the sensitivity of the antibody to be bound to the gold particle in the desired direction. When using a linker comprising a cationic moiety arginine (R) such as RGRGRGRGRSRGGGS, an antibody comprising the same can bind to the anionic surface, and all such linkers are included within the scope of this application. In one embodiment of the present disclosure, the sequence of the linker peptide used in the antigen-binding peptide of the present invention has GGGGSGGGGSGGS from the amino terminus to the carboxy terminus. In another embodiment the sequence of the linker peptide has GGGGSGGGGSGGGGS from the amino terminus to the carboxy terminus.

The various VLs and VHs disclosed herein may be paired with one another in various combinations to form antigen binding peptides, and such various combinations of VL-VHs are also included within the scope of this application, as compared to those of other combinations. It may have the same or different antigen binding specificities and / or affinity, which are included in the scope of this application. In one embodiment, the combination comprises a VH comprising H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 6 and H-CDR3 of SEQ ID NO: 11 and L-CDR1 of SEQ ID NO: 16, L- of SEQ ID NO: 21 A combination of CDR2 and VL comprising L-CDR3 of SEQ ID NO: 26; VH comprising H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 7 and H-CDR3 of SEQ ID NO: 12, L-CDR1 of SEQ ID NO: 17, L-CDR2 of SEQ ID NO: 22, and L- of SEQ ID NO: 27 Combination of CDR3VL; VH comprising H-CDR1 of SEQ ID NO: 3, H-CDR2 of SEQ ID NO: 8 and H-CDR3 of SEQ ID NO: 13, L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 23, and L- of SEQ ID NO: 28 Combination of VLs comprising CDR3; VH comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 9 and H-CDR3 of SEQ ID NO: 14, L-CDR1 of SEQ ID NO: 19, L-CDR2 of SEQ ID NO: 24, and L- of SEQ ID NO: 29 Combination of VLs comprising CDR3; And a VH comprising H-CDR1 of SEQ ID NO: 5, H-CDR2 of SEQ ID NO: 10, and H-CDR3 of SEQ ID NO: 15, L-CDR1 of SEQ ID NO: 20, L-CDR2 of SEQ ID NO: 25, or L of SEQ ID NO: 30 Is selected from, but is not limited to, a combination of VLs including CDR3.

In other embodiments, antigen-binding polypeptides herein include a linker, and the antigen-binding polypeptide comprising such linker is selected from, but is not limited to, SEQ ID NO: 34, 35, 36, and 37.

The application also provides a nucleotide encoding the antigen binding polypeptide. Nucleotides include both DNA, RNA and binding fragments, derivatives and variants thereof. In one embodiment, the nucleotide is SEQ ID NO: 38, 39, 40 or 41, but is not limited thereto.

In addition, the antigen-binding polypeptide of the present invention can be usefully used for the detection and / or quantification of Vibrio food poisoning bacteria. To this end, the antigen-binding polypeptide of the present disclosure may be used in the form of a single entity (monomer, monomer) or a multimer of several single entities, and is included in the scope of the present application. In addition, the antigen-binding polypeptides of the present disclosure can be used by themselves or in combination with other substances for the detection and quantification of Vibrio food poisoning bacteria. For example, the antibody of the present invention may be labeled with various chromophores including not only the fluorescent material and the luminescent material required for detection, or may be used in combination with nanoparticles such as liposomes and gold particles. Antigen-binding polypeptides in the form are also included within the scope of this application.

In this context, the present application provides a composition and kit for detecting Vibriobacteria, comprising the antigen-binding polypeptide. The composition of the present invention detects OMPU protein and can be used for the detection of various kinds of Vibrio bacteria. In one embodiment, the Vibrio Vibrio enterobacteriaceae ( Vibrio parahaemolyticus) or Vibrio sepsis bacteria (Vibrio vulnificus ).

The present application also provides a kit for detecting Vibrio bacteria comprising the above-mentioned antigen-binding polypeptide or a composition comprising the same. The kit may further comprise an information booklet relating to its use in addition to the above components. The kit detects OMPU proteins and can be used to detect a variety of Vibrio bacteria. In one embodiment, the Vibrio Vibrio enterobacteriaceae ( Vibrio parahaemolyticus) or Vibrio sepsis bacteria (Vibrio vulnificus ), but not limited thereto.

In another aspect the present application provides a method for detecting Vibrio bacteria using the above-mentioned antigen-binding polypeptide or a composition comprising the same. The method further comprises the step of reacting the sample with the antibody of the present application, detecting whether the antigen-antibody reaction in the reactant. The method is based on an antigen-antibody immune response, and can be carried out using a variety of known methods, including, but not limited to, Radioimmunoassay, ELISA, Gel diffusion / Immunomagnetic separation, Hemagglutination, Western blot, ELISA It is not limiting.

The method detects OMPU proteins and can be used to detect a variety of Vibrio strains. In one embodiment, the Vibrio Vibrio enterobacteriaceae ( Vibrio parahaemolyticus) or Vibrio sepsis bacteria (Vibrio vulnificus ), but not limited thereto. The above-mentioned bacteria are the main causative agents of food poisoning occurring in Korea and can be usefully used in various experiments for screening, detection and quantification of infection by these bacteria in various samples suspected of being infected. The method can be carried out on a variety of materials for which bacteria can be found, for example for food, feed or specimens, and methods for taking samples are known in the art. The detection method of bacteria using an antibody from a sample can be used, for example, in food poisoning bacteria test methods such as AOAC, the Korea Food and Drug Administration, and the US Food and Drug Administration. For example, the method generally comprises preculturing a sample suspected of having Vibrio bacteria in an appropriate medium, followed by further incubation or directly binding to the antibody of the present application, and detecting the antigen-antibody complex. It includes. Antigen-antibody complexes can be detected directly when the antibody itself is labeled with a chromophore such as a fluorescent substance, and further, a second antibody (labeled with a chromophore detecting substance such as a fluorophore capable of detecting the complex) ( For example, fluorescently labeled goat antihuman immunoglobulin) can be used. The method can include the use of a control group and can determine the presence and / or amount of Vibrio bacteria present in the sample as compared to the negative control group and the positive control group, all of which are apparent to those skilled in the art.

TABLE 1 Third and fourth panning single phage ELISA for antigen OmpU-His

Table 2 List of Monoclonal Antibodies Responding to OmpU-His

The present invention will be described in detail with reference to the following examples, which are illustrative only and are not intended to limit the scope of the present application in any way. The present invention may be practiced using conventional techniques that are within the skill of one of ordinary skill in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombination techniques unless otherwise noted. Also, a more detailed description of the general technology can be found in Molecular Biotechnology: (Bernard et al., ASM press 1994); Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); See DNA Cloning, Volumes I and II (Glover ed., 1985).

Example 1. OmpU  Isolation and Purification of Proteins

OmpU protein used as an antigen is Vibrio vulnificus Histidine-tagged recombinant protein from Park et al., 2006. Cyclo (Phe-Pro) modulates the expression of ompU in Vibrio spp. J. Bacteriol. It was prepared and used according to the method described in 188: 22142221. In summary two V. vulnificus primers ompU RF (5-ATGGGCCCAAAGTCATCTTTGG-3) and ompUR-R (5-GCGGATCCAATCTATCTAGAAT-3) PCR was performed using the genomic DNA of MO6-24 / O as a template to obtain a 1.63-kb DNA fragment containing the ompU gene . This was then cloned into GEMT-Easy vector (Promega) to obtain pDK. The pDK was then digested with EcoRI to clone 791-bp DNA fragment into pTrcHisB (Invitrogen) tagged with six histidines to yield pTrcHisB-ompU. The plasmid was then transformed into JM109 to express the protein, using His-Bind kit (Ni-NTA + resin) (Novagen) as recommended by the manufacturer. OmpU was purified. The purified protein was electrophoresed in 8% SDS-polyacrylamide and stained with Coomassie Brilliant Blue (Sigma). The results are described in FIG.

Example 2 OmpU  Protein-specific scFv  quest

2-1 human naive scFv library phage  Ready

Phage library for antibody screening using Human Naive scFv library phage that can make a human antibody (A & C Ceraputix). TG1 Escherichia coli containing the library was suspended in 2 × YT (+ chloramphenicol, +2 mM MgCl ₂ , +1 mM IPTG, + Kanamycin) medium, incubated at 30 ° C. for 16 hours, and then centrifuged (tabletop centrifuge x3500 rpm, 15 minutes) to obtain a culture solution. Subsequently, 5 × PEG / NaCl (20% Polyethylene Glycol) 6000 / 2.5M NaCl) was added to 1.2 ml of the culture at a rate of 500 μl, and the precipitate was placed on ice for 30 minutes and resuspended in PBS. Phages are poured with E. coli ER2738 on LB plates with IPTG and X-gal, incubated at 37 ° C and identified for blue plague. Quantitatively, a human naive scFv library cell of 2.7 x 10 ¹⁰ titer was obtained.

2-2 OmpU - His  Antigen specific scFv phage clones  quest

In order to select phage that selectively reacts with OmpU, biopanning was performed using the OmpU protein prepared in Example 1 as an antigen. The biopanning method was basically performed according to the method disclosed in Methods in Molecular Biology, Vol 408, pp 243-255, 2007. In summary, first 25 μg / ml Concentration of purified recombinant OmpU protein 150 μl After first adding to each well of a 96-well plate and fixing at 4 ° C. overnight, the protein-free portion was blocked with PBS containing 5% nonfat milk and washed with PBS. Thereafter, the phages of Example 2-1 were added to each well by 100 µl (+ 1% skim milk) and bound at room temperature for 1 hour. Subsequently, after washing with PBST (Phosphate buffered saline: 0.01 M phosphate buffer with 0.138 M NaCl. 0.0027 M KCl, 0.05% Tween 20), the phages were eluted with 2 ml of TEA at 100 mM concentration. The eluted phage was neutralized by adding 1 ml of 1M Tris buffer (pH 7.5). Thereafter, the eluted phage was infected with E. coli XL1-blue to collect E. coli expressing the antibody of the OmpU antigen. The collected E. coli was cultured again, and the biopanning process consisting of selective binding with the OmpU antigen and elution was repeated four times. As the biopanning process was repeated, the concentration of antibody phage binding to the OmpU antigen was increased, and it was confirmed that colony titer of phage to the antigen was amplified more than 1,000 times in the fourth step.

96 positive clones were selected first in 3rd and 4th panning. Subsequently, the binding ability of each OmpU antigen protein was confirmed by ELISA (Enzyme-Linked ImmunoSorbent Assay) method, and five of these clones were selected. Phages obtained at each biopanning order were bound to a 96 well plate coated with 100 ng / well of OmpU antigen at room temperature for 1 hour and washed with PBST. Again anti-M13-HRP (horseradish peroxidase) was bound for 1 hour at room temperature, washed with PBST. After 5-10 minutes of color development with OPD (o-phenylenediamine) substrate, 50 µl of 2.5M sulfuric acid (H ₂ SO ₄ ) was added to stop color development, and the absorbance (Optical Density, OD) value was measured at 490 nm wavelength. Table 1 shows the ELISA results of phage for OmpU antigen, anti-Myc antibody, His tag. Five antibody-expressing phages 1E, 1G, 3B, 4E, and 9C, which strongly bind to the OmpU antigen, were selected by comparing the expression levels of Myc tags among the total 96.

Then concentrating with PEG / NaCl from 5 FY clones as in Example 2-1, then with 100% EtOH to obtain DNA. The scFV inserts contained in the DNA were then amplified by PCR. Foward: 5'aatttctaga taacgagggc aaatcatgaa atacct-3 '; Reverse: 5'-ctcctcgaat tccacgaggc tggcggaa-3 '). Here, the concentrations of the sense primer and the antisense primer were 1 μM, the annealing temperature was 50 ° C., and the repetition frequency was 30 cycles. Subsequently, the amplified product was digested with BstN1 restriction enzyme and electrophoresed on 8% polyacrylamide gel, and then stained with EtBr. The results are described in FIG. As shown in Figure 2, the selected clones show that they contain different antibody sequences.

Subsequently, sequencing of the monoclonal (1E, 1G, 3B, 4E, and 9C) DNAs of the five OmpUs isolated above was performed and translated into amino acid sequences using the TRANSLATE program in ExPASy [Expert Protein Analysis System). CDR and backbone (FR) regions were determined using KABAT numbering (http://www.biochem.ucl.ac.uk/~martin/abs/GeneralInfo.html). The results are shown in Table 2 and in FIG. 3.

Example 3 scFv - OmpU Recombinant Protein Antibody Purification and Specificity Test

3-1. OmpU Monoclonal for Phage  From antibodies scFv form  transform

Monoclonal phage antibodies against OmpU were converted from phage to ScFv form. In Example 2-2, each DNA obtained from each phage was digested with NcoI-EcoRI to cut an scFv insert and cloned into a pET22b-S vector (Novagen).

3-2. scFv - OmpU  Recombinant protein purification of 1E, 1G, 3B, 4E, and 9C

Vectors expressing each scFv-OmpU antibody selected (1E, 1G, 3B, 4E, and 9C) were overexpressed in E. coli BL21, respectively, and the proteins were purified as follows (FIG. 4). Each antibody protein in the denatured state was renaturated using refolding buffer. The protein purification method is as follows: BL21 was incubated overnight, and 100 µl of IPTG (1M) was induced at OD ₆₀₀ at 0.5 to 0.6 to induce protein expression for 3 h, followed by centrifugation to obtain cell precipitates. Subsequently, PBS (15 ml) was added to the precipitate to suspend the cells, and the cells were crushed by treatment with ultrasound about 7 times. Subsequently, the supernatant was stored separately by centrifugation at 4 ° C. and 8000 rpm for 10 minutes, and the precipitate was resuspended in 1 ml of a melting buffer solution (PBS containing 0.5% Triton X-100). Subsequently, the supernatant was discarded by centrifugation again at 4 ° C. and 8000 rpm for 10 minutes, and the precipitate was suspended in 4 ml of buffer A (with 10 mM imidazole). This was further centrifuged at 4 ° C. and 8000 rpm for 10 minutes, and the upper layer was purified on a column. The column purification was as follows: washed with 10 ml of 10 vol buffer A (with 10 mM imidazole), washed with 10 ml of 10 vol buffer B (with 10 mM imidazole), with 10 ml of 10 vol buffer C (with 10 mM imidazole) After washing, washing with 10 ml of 10 vol buffer C (with 20 mM imidazole), eluting the protein with 500 μl of buffer C (with 500 mM imidazole), and confirming it by SDS-PAGE (FIG. 4).

Protein refolding was carried out as follows: Protein eluted in 100-fold volume refold buffer on ice was added dropwise. The refolded protein was concentrated using Centricon (Millipore) (5,000 rpm at 4 ° C), which was stored at -20 ° C until use.

3-3. Refined scFv - OmpU  Antibody Specificity Test (1)

Specificity for the OmpU protein of the antibody prepared in Example 3-2 was confirmed by Western blot. Vibrio The supernatants were obtained by crushing the cells of the vulnificus wild type MO6-24 / O and OmpU mutant by ultrasonication and centrifugation. Here, specificity of each scFv-OmpU antibody purified in Example 3-2 was measured by Western blot. 100 μg of each prepared protein was electrophoresed on SDS-PAGE, and the Hybond-P membrane to be used as a transition membrane was immersed in methanol for 10 minutes and distilled water for 10 minutes, and then immersed in a transfer buffer. The electrophoretic gel was transferred to the membrane and transferred at 120V for 30 minutes. The membrane was then treated with 10% blocking solution (10% skim milk in PBS) and Subsequently, 500 µg of the antibody prepared in 3-2 was treated thereto, washed with TBST, and then connected to alkaline phosphatase as a secondary antibody. Anti-poly-histidine (Sigma) 5㎕ after treatment with (15ml blocks of king-solution), washed with TBST color reaction (Alkaline phosphotase (AP) 10 ml + BCIP (50mg / ml) 15㎕ + NBT (50mg / ml) 30 μl). The result is in FIG. 5. OmpU as shown in Figure 5 In the mutant, OmpU band did not appear, and the 35KDa OmpU band in the wild type was identified.

3-4. Refined scFv - OmpU  1E Antibody Specificity Test (2)

Specificity confirmed scFv-OmpU antibody is Vibrio Whether the Gram-negative bacteria and Gram-positive bacteria other than vulnificus could be specifically recognized was examined for the strains described in FIG. 6 using a western blot as scFv-OmpU 1E antibody as in Example 3-3. Gram-negative bacterium Pseudomonas aeruginosa and gram-positive bacteria Bacillus For cereus , the band reacting with the scFv-OmpU 1E antibody was not detected, so the vibrio-antibody response showed species specificity (FIG. 6).

3-5. Refined scFv - OmpU  1G, 3B, 4E, and 9C Antibody Specificity Testing (3)

Specificity confirmed scFv-OmpU antibody is Vibrio We investigated whether Gram-negative and Gram-positive bacteria other than vulnificus and Vibrio parahaemolyticus could be specifically recognized. In V. haveyi , V. choleare and V. fischeri , which are evolutionarily similar species to the Vibrio bacterium, the bands that react with the other 1G, 3B, 4E, and 9C antibodies except for the scFv-OmpU 1E antibody are not detected. It can be seen that the antigen-antibody response of Vibrio shows species specificity (FIG. 7).

<110> Hankuk University of Foreign Studies Research and Industry-University Cooperation Foundation <120> Antibody against OMPU of Vibrio and method of detecting Vibrio          using the same <160> 41 <170> KopatentIn 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 1 Asp Phe Ala Met His   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 2 Gly Pro Tyr Met Ser   1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 3 Asn Tyr Ala Met Thr   1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 4 Asn Tyr Tyr Met His   1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 5 Gly Pro Tyr Met Ser   1 5 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 6 Gly Val Ser Trp Asn Gly Ala Val Ile Arg Tyr Ala Asp Ser Val Gln   1 5 10 15 Gly     <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 7 Leu Ile Ser Thr Gly Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys Gly   1 5 10 15 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 8 Ser Ile Ser Gly Ser Gly Asp Asn Thr Tyr His Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 9 Ile Ile Asn Pro Ser Ser Gly Ser Ala Arg Asn Ala Gln Asn Phe Gln   1 5 10 15 Gly     <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 10 Leu Ile Ser Thr Gly Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys Gly   1 5 10 15 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 11 Tyr Leu Arg Ala Tyr Gly Leu Asp Val   1 5 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 12 His Gly Asp Ala Asn Thr Tyr Tyr Tyr Gly Leu Asp Val   1 5 10 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 13 Gly Gly Gly Leu Asp Val   1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 14 Gly Trp Asp Leu Asp Tyr   1 5 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 15 His Gly Asp Ala Asn Thr Tyr Tyr Tyr Gly Leu Asp Val   1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 16 Arg Ala Ser Gln Gly Ile Gly Ser Arg Leu Ala   1 5 10 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 17 Ser Gly Asp Ala Leu Ser Lys Gln Tyr Ala Ser   1 5 10 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 18 Arg Ser Ser Gln Ser Leu Val Tyr Ser Asp Gly His Thr Tyr Leu Asn   1 5 10 15 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 19 Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Ser   1 5 10 15 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 20 Ser Gly Asp Ala Leu Ser Lys Gln Tyr Ala Ser   1 5 10 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 21 Ala Ala Ser Ser Leu Gln Ser   1 5 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 22 Lys Asp Thr Glu Arg Pro Ser   1 5 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 23 Lys Val Ser Lys Arg Asp Ser   1 5 <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 24 Lys Ile Ser Asn Arg Phe Ser   1 5 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 25 Lys Asp Thr Glu Arg Pro Ser   1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 26 Gln Gln Ala Asn Ser Leu Pro Ile Thr   1 5 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 27 Gln Ser Ile Thr Asp Lys Ser Gly Thr Asp Val Ile   1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 28 Met Gln Gly Thr His Trp Pro Gln Leu Thr   1 5 10 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 29 Met Gln Ala Thr Phe Phe Pro Phe Thr   1 5 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 30 Gln Ser Ile Thr Asp Lys Ser Gly Thr Asp Val Ile   1 5 10 <210> 31 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide <400> 31 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser   1 5 10 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 32 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 33 Arg Gly Arg Gly Arg Gly Arg Gly Arg Ser Arg Gly Gly Gly Ser   1 5 10 15 <210> 34 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> scFv 1E <400> 34 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly   1 5 10 15 Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly              20 25 30 Phe Thr Phe Asp Asp Phe Ala Met His Trp Val Arg Gln Val Pro Gly          35 40 45 Lys Gly Leu Glu Trp Val Ser Gly Val Ser Trp Asn Gly Ala Val Ile      50 55 60 Arg Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Val Ser Arg Asp Asn  65 70 75 80 Ala Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                  85 90 95 Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Leu Arg Ala Tyr Gly Leu Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Ile Thr Val Ser Ser Gly Leu Gly Gly         115 120 125 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ser Gly     130 135 140 Val Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala 145 150 155 160 Ser Val Gly Asp Ser Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile                 165 170 175 Gly Ser Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys             180 185 190 Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg         195 200 205 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser     210 215 220 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser 225 230 235 240 Leu Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Gly                 245 250 255 Gly Ala Ser Leu Val Glu Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp             260 265 270 Leu     <210> 35 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> scFv 3B <400> 35 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly   1 5 10 15 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly              20 25 30 Phe Thr Ile Ser Gly Pro Tyr Met Ser Trp Val Arg Gln Ala Pro Gly          35 40 45 Lys Gly Leu Glu Trp Val Ser Leu Ile Ser Thr Gly Gly Arg Thr Arg      50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Thr Ser  65 70 75 80 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr                  85 90 95 Ala Val Tyr Tyr Cys Ala Arg His Gly Asp Ala Asn Thr Tyr Tyr Tyr             100 105 110 Gly Leu Asp Val Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Gly         115 120 125 Leu Gly Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Ser Ser Gly Val Gly Ser Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val 145 150 155 160 Ser Met Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala                 165 170 175 Leu Ser Lys Gln Tyr Ala Ser Trp Tyr Gln Leu Lys Pro Gly Gln Ala             180 185 190 Pro Val Val Val Met Tyr Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro         195 200 205 Asp Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile     210 215 220 Ser Gly Val Gln Ala Glu Asp Gly Ala Asp Tyr Tyr Cys Gln Ser Ile 225 230 235 240 Thr Asp Lys Ser Gly Thr Asp Val Ile Phe Gly Gly Gly Thr Lys Leu                 245 250 255 Thr Val Leu Ser Gly Gly Ala Ser Leu Val Glu Phe Glu Gln Lys Leu             260 265 270 Ile Ser Glu Glu Asp Leu         275 <210> 36 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> scFv 4E <400> 36 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly   1 5 10 15 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly              20 25 30 Phe Thr Phe Asn Asn Tyr Ala Met Thr Trp Val Arg Gln Ala Pro Ala          35 40 45 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Asp Asn Thr      50 55 60 Tyr His Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn  65 70 75 80 Ser Arg Asn Leu Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                  85 90 95 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Gly Leu Asp Val Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Leu Gly Gly Leu Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ser Gly Val Gly Ser     130 135 140 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 145 150 155 160 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser                 165 170 175 Asp Gly His Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Pro             180 185 190 Pro Arg Arg Leu Ile Tyr Lys Val Ser Lys Arg Asp Ser Gly Val Pro         195 200 205 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile     210 215 220 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 225 230 235 240 Thr His Trp Pro Gln Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile                 245 250 255 Lys Arg Gly Gly Ala Ser Leu Val Glu Phe Glu Gln Lys Leu Ile Ser             260 265 270 Glu Glu Asp Leu         275 <210> 37 <211> 278 <212> PRT <213> Artificial Sequence <220> ScFv 9C <400> 37 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly   1 5 10 15 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly              20 25 30 Phe Thr Ile Ser Gly Pro Tyr Met Ser Trp Val Arg Gln Ala Pro Gly          35 40 45 Lys Gly Leu Glu Trp Val Ser Leu Ile Ser Thr Gly Gly Arg Thr Arg      50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Thr Ser  65 70 75 80 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr                  85 90 95 Ala Val Tyr Tyr Cys Ala Arg His Gly Asp Ala Asn Thr Tyr Tyr Tyr             100 105 110 Gly Leu Asp Val Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Gly         115 120 125 Leu Gly Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Ser Ser Gly Val Gly Ser Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val 145 150 155 160 Ser Met Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala                 165 170 175 Leu Ser Lys Gln Tyr Ala Ser Trp Tyr Gln Leu Lys Pro Gly Gln Ala             180 185 190 Pro Val Val Val Met Tyr Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro         195 200 205 Asp Arg Phe Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile     210 215 220 Ser Gly Val Gln Ala Glu Asp Gly Ala Asp Tyr Tyr Cys Gln Ser Ile 225 230 235 240 Thr Asp Lys Ser Gly Thr Asp Val Ile Phe Gly Gly Gly Thr Lys Leu                 245 250 255 Thr Val Leu Gly Gly Gly Ala Ser Leu Val Glu Phe Gln Gln Lys Leu             260 265 270 Ile Ser Glu Glu Asp Leu         275 <210> 38 <211> 822 <212> DNA <213> Artificial Sequence <220> <223> scFv 1E <400> 38 gcccagccgg ccatggccca ggtgcagctg gtacagtctg ggggaggctt ggtacagcct 60 ggcaggtccc ttagactctc ctgtgcagcc tctggattca cctttgatga ttttgccatg 120 cactgggtcc ggcaagttcc agggaagggc ctggagtggg tctcaggtgt tagttggaat 180 ggtgctgtca tccggtatgc ggactctgtg cagggccggt tcaccgtttc tagagacaac 240 gccaagaaca ccctatttct gcaaatgaac agtctgagag ccgaggacac ggccgtgtat 300 tactgtgcga gatatctaag agcatacggt ctggacgtct gggggcaagg gaccacgatc 360 accgtctcct caggcctcgg gggcctcgga ggaggaggta gtggcggagg aggctccggt 420 ggatccagcg gtgtgggttc cgacatccag atgacccagt ctccatcttc cgtgtctgca 480 tctgtaggag acagcgtcac catcacttgt cgggcgagtc agggtattgg cagccggtta 540 gcctggtatc agcagaaacc agggaaagcc cctaagctcc tgatctatgc tgcatccagt 600 ttgcaaagtg gggtcccatc aaggttcagc ggcagtggat ctgggacaga tttcactctc 660 accatcagca gcctgcagcc tgaagatttt gcaacttact attgtcaaca ggctaacagt 720 ttaccgatca ccttcggcca agggacacga ctggagatca aacgtggagg agccagcctc 780 gtggaattcg agcagaagct gatctctgag gaagacctgt ag 822 <210> 39 <211> 837 <212> DNA <213> Artificial Sequence <220> <223> scFv 3B <400> 39 gcccagccgg ccatggccca ggtgcagctg gtggagtctg ggggcggcgt ggtccagcct 60 gggaggtccc tgagactctc ctgtgcagcc tctggattca ccatcagtgg cccctacatg 120 agttgggtcc gccaggctcc agggaagggg ctggagtggg tctcactcat ttctaccggt 180 ggtcgcacaa gatacgcgga ctccgtgaag ggccgattca gcatctccag agacacctcc 240 aagaacactc tgtatcttca aatgaacagt ctgagagccg aggacacggc cgtgtattac 300 tgtgcgagac atggcgatgc taacacctac tactatggtt tggacgtctg gggccaaggg 360 accaaggtca ccgtctcctc aggcctcggg ggcctcggag gaggaggtag tggcggagga 420 ggctccggtg gatccagcgg tgtgggttcc tcctatgagc tgacacagtc accctcggtg 480 tcaatgtccc caggacaaac ggccaggatc acctgttctg gagatgcttt gtcaaagcaa 540 tatgcttctt ggtaccagct gaagccaggc caggcccctg tggtggtgat gtataaagac 600 actgagaggc cctcagggat ccctgaccga ttctctggct ccagctccgg gacaacagtc 660 acgttgacca tcagtggagt ccaggcagaa gacggggctg attattactg tcaatcaata 720 acagacaaga gtggtactga tgtgatcttc ggcggaggga ccaagctgac cgtcctaagt 780 ggaggagcca gcctcgtgga attcgagcag aagctgatct ctgaggaaga cctgtag 837 <210> 40 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> scFv 4E <400> 40 gcccagccgg ccatggccca ggtgcagctg gtgcagtctg ggggaggctt ggtacagcct 60 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttaacaa ctatgccatg 120 acctgggtcc gccaggctcc agcgaagggg ctggagtggg tctcgtctat tagtggcagt 180 ggtgataaca cataccacgc agactccgtg aagggccggt tcgccatctc cagagacaat 240 tccaggaacc tactgtatct gcaaatgaac agtctgagag ccgaggacac ggccgtgtat 300 tactgtgcga gaggcggggg tttggacgtc tggggccaag ggaccacggt caccgtctcc 360 tcaggcctcg ggggcctcgg aggaggaggt agtggcggag gaggctccgg tggatccagc 420 ggtgtgggtt ccgatattgt gatgacccag actccactct ccctgcccgt cacccttgga 480 cagccggcct ccatctcctg caggtctagt caaagtctcg tatacagtga tggacacacc 540 tacttgaatt ggtttcagca gaggccaggc caacctccaa ggcgcctaat ctacaaggtt 600 tctaagcggg actctggggt cccagacaga ttcagcggca gtgggtcagg cactgatttc 660 acactgagaa tcagcagggt ggaggctgag gatgttgggg tttattactg catgcaaggg 720 acacactggc ctcagctcac tttcggcgga gggaccaagg tggatatcaa acgtggagga 780 gccagcctcg tggaattcga gcagaagctg atctctgagg aagacctgta g 831 <210> 41 <211> 837 <212> DNA <213> Artificial Sequence <220> ScFv 9C <400> 41 gcccagccgg ccatggccca ggtgcagctg gtggagtctg ggggcggcgt ggtccagcct 60 gggaggtccc tgagactctc ctgtgcagcc tctggattca ccatcagtgg cccctacatg 120 agttgggtcc gccaggctcc agggaagggg ctggagtggg tctcactcat ttctaccggt 180 ggtcgcacaa gatacgcgga ctccgtgaag ggccgattca gcatctccag agacacctcc 240 aagaacactc tgtatcttca aatgaacagt ctgagagccg aggacacggc cgtgtattac 300 tgtgcgagac atggcgatgc taacacctac tactatggtt tggacgtctg gggccaaggg 360 accaaggtca ccgtctcctc aggcctcggg ggcctcggag gaggaggtag tggcggagga 420 ggctccggtg gatccagcgg tgtgggttcc tcctatgagc tgacacagtc accctcggtg 480 tcaatgtccc caggacaaac ggccaggatc acctgttctg gagatgcttt gtcaaagcaa 540 tatgcttctt ggtaccagct gaagccaggc caggcccctg tggtggtgat gtataaagac 600 actgagaggc cctcagggat ccctgaccga ttctctggct ccagctccgg gacaacagtc 660 acgttgacca tcagtggagt ccaggcagaa gacggggctg attattactg tcaatcaata 720 acagacaaga gtggtactga tgtgatcttc ggcggaggga ccaagctgac cgtcctaggt 780 ggaggagcca gcctcgtgga attccagcag aagctgattt ctgaggaaga cctgtag 837

Claims (11)

An antigen-binding polypeptide that specifically binds to the outer membrane protein of Vibrio bacteria, wherein the polypeptide comprises a VH domain and a VL domain,
The VH domain comprises H-CDR1, H-CDR2 and H-CDR3, wherein each of H-CDR1, H-CDR2 and H-CDR3 is H-CDR1 of SEQ ID NO: 1, H-CDR2 and SEQ ID NO: 6 H-CDR3 of No. 11; H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 7 and H-CDR3 of SEQ ID NO: 12; H-CDR1 of SEQ ID NO: 3, H-CDR2 of SEQ ID NO: 8 and H-CDR3 of SEQ ID NO: 13; H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 9 and H-CDR3 of SEQ ID NO: 14; And an amino acid sequence selected from the group of H-CDR set consisting of H-CDR1 of SEQ ID NO: 5, H-CDR2 of SEQ ID NO: 10, and H-CDR3 of SEQ ID NO: 15,
The VL domain comprises L-CDR1, L-CDR2 and L-CDR3 and each of L-CDR1, L-CDR2 and L-CDR3 is L-CDR1 of SEQ ID NO: 16, L-CDR2 and SEQ ID NO: 21 26 L-CDR3; L-CDR1 of SEQ ID NO: 17, L-CDR2 of SEQ ID NO: 22 and L-CDR3 of SEQ ID NO: 27; L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 23 and L-CDR3 of SEQ ID NO: 28; L-CDR1 of SEQ ID NO: 19, L-CDR2 of SEQ ID NO: 24 and L-CDR3 of SEQ ID NO: 29; And an L-CDR set selected from the group consisting of L-CDR1 of SEQ ID NO: 20, L-CDR2 of SEQ ID NO: 25, and L-CDR3 of SEQ ID NO: 30, an antigen binding polypeptide.
The antigen binding polypeptide of claim 1, wherein the antigen binding polypeptide comprises one amino acid sequence selected from the group consisting of the following VH-VL combinations:
VH comprising H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 6 and H-CDR3 of SEQ ID NO: 11, L-CDR1 of SEQ ID NO: 16, L-CDR2 of SEQ ID NO: 21, and L- of SEQ ID NO: 26 Combination of VLs comprising CDR3;
VH comprising H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 7 and H-CDR3 of SEQ ID NO: 12, L-CDR1 of SEQ ID NO: 17, L-CDR2 of SEQ ID NO: 22, and L- of SEQ ID NO: 27 Combination of CDR3VL;
VH comprising H-CDR1 of SEQ ID NO: 3, H-CDR2 of SEQ ID NO: 8 and H-CDR3 of SEQ ID NO: 13, L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 23, and L- of SEQ ID NO: 28 Combination of VLs comprising CDR3;
VH comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 9 and H-CDR3 of SEQ ID NO: 14, L-CDR1 of SEQ ID NO: 19, L-CDR2 of SEQ ID NO: 24, and L- of SEQ ID NO: 29 Combination of VLs comprising CDR3; And
VH comprising H-CDR1 of SEQ ID NO: 5, H-CDR2 of SEQ ID NO: 10 and H-CDR3 of SEQ ID NO: 15, L-CDR1 of SEQ ID NO: 20, L-CDR2 of SEQ ID NO: 25, and L- of SEQ ID NO: 30 Combination of VLs comprising CDR3.
The antigen binding polypeptide of claim 1, wherein the antigen binding polypeptide further comprises a linker polypeptide located between the VH and VH.
The antigen binding polypeptide of claim 3, wherein the linker polypeptide is any one of SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33. 4.
The antigen binding polypeptide of claim 4, wherein the antigen binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37. 6.
A nucleotide encoding the antigen binding polypeptide of claim 1.
The nucleotide of claim 6, wherein the nucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41. 8.
Vibrio detection kit comprising the antigen-binding polypeptide of any one of claims 1 to 5.
The kit for detecting Vibrio bacteria according to claim 8, wherein the Vibrio bacteria are at least one of Vibrio parahaemolyticus or Vibrio vulnificus .
Vibrio bacteria detection method comprising the step of detecting the outer membrane protein of Vibrio bacteria using the antigen-binding polypeptide of any one of claims 1 to 5.
According to claim 10, wherein the Vibrio bacteria Vibrio parahaemolyticus or Vibrio sepsis bacteria ( Vibrio vulnificus ) is one or more, Vibrio bacteria detection method.

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