CN120040584B

CN120040584B - Monoclonal antibodies against pathogenic Campylobacter and their application

Info

Publication number: CN120040584B
Application number: CN202510408874.9A
Authority: CN
Inventors: 罗威; 周筱洲; 周逸
Original assignee: Beijing Bioneovan Co ltd
Current assignee: Beijing Bioneovan Co ltd
Priority date: 2025-04-02
Filing date: 2025-04-02
Publication date: 2025-07-22
Anticipated expiration: 2045-04-02
Also published as: CN120040584A

Abstract

The invention discloses a monoclonal antibody CJ116 for resisting pathogenic campylobacter and application thereof. In the monoclonal antibody CJ116, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The CJ116 monoclonal antibody has high affinity and high specificity, can detect campylobacter jejuni and campylobacter coli with the sensitivity of 10 ² CFU/mL, and can detect campylobacter jejuni, campylobacter Uppsalanus and campylobacter gull with the sensitivity of 10 ³ CFU/mL, but has no cross reaction with other common enteropathogenic bacteria such as pathogenic escherichia coli, salmonella enteritidis, yersinia enterocolitica, shigella flexneri, shigella pallidus and staphylococcus aureus.

Description

Monoclonal antibody against pathogenic campylobacter and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody for resisting pathogenic campylobacter and application thereof.

Background

Infectious diarrhea is a common symptom caused by infection of the gastrointestinal tract with bacteria, viruses or parasites, and is the second leading cause of morbidity and mortality worldwide. In developing countries, acute infectious diarrhea is one of the main causes of morbidity and mortality in children aged 1-3 years, and is also the most common cause of malnutrition in children.

Campylobacter spp is a common food-borne zoonotic pathogen, an important pathogen causing infectious diarrhea worldwide, and species pathogenic to humans include Campylobacter jejuni Campylobacter jejuni, campylobacter colons Campylobacter coli, campylobacter foetidus Campylobacter fetus, campylobacter gull Campylobacter lari, campylobacter Uppsalaensis Campylobacter upsaliensis, and the like. Campylobacter jejuni and campylobacter coli are the major causative bacteria of human infection, accounting for 95% of all reported cases of campylobacter jejuni infection, with campylobacter jejuni infection exceeding 90%. Campylobacteriosis is a self-limiting gastroenteritis disease, with typical symptoms ranging from mild diarrhea to inflammatory diarrhea, including diarrhea, abdominal cramps, vomiting and fever, lasting about 5-7 days. In addition to acute gastroenteritis, campylobacter jejuni infection can lead to serious sequelae such as green-barre syndrome, reactive arthritis, irritable bowel syndrome, and the like.

In recent years, the incidence of campylobacteriosis has increased gradually, and about 1.66 million cases of diarrhea are caused by campylobacter jejuni each year worldwide, accounting for about 5-14% of diarrhea diseases. At present, in European and American countries, diarrhea caused by campylobacter jejuni infection is the first place of diarrhea caused by pathogenic bacteria infection, and part of countries are also only inferior to salmonella or shigella. In China, infants are highly-developed people suffering from campylobacter jejuni enteritis, and particularly in summer and autumn in the eastern coastal developed area, the infants are most likely to be infected by campylobacter jejuni to cause diarrhea. The Chinese recent monitoring results show that the detection rate of campylobacter jejuni for diarrhea infants is 4.0-17.7%, and the detection rate of diarrhea for adults is 3.85-9.61%. In addition, the campylobacter jejuni can be carried in healthy people, and the carrying rate is between 0.6 and 9.4 percent. Therefore, the rapid and accurate detection of campylobacter is of great importance for preventing and controlling the outbreak of campylobacter jejuni, early treatment of infected patients and prevention and treatment of serious complications.

Currently, methods for detecting campylobacter mainly include a separation culture method, a conventional biochemical method, a nucleic acid detection method, an immunodetection method, a mass spectrometry detection method and the like. The separation culture of pathogenic bacteria is a gold standard for clinical diagnosis of campylobacter infection, but the traditional separation culture method is time-consuming and labor-consuming and has lower sensitivity and can not meet clinical requirements due to high nutrition requirements and special culture conditions of campylobacter. The gold standard for identification of campylobacter at the species level is to differentiate bacteria by enrichment and isolation of the culture medium and then to perform biochemical experiments. However, some campylobacter bacteria are inert to biochemical reactions and may misdiagnose campylobacter jejuni as campylobacter coli, thus limiting the clinical application of biochemical methods. In recent years, molecular biological detection methods such as nucleic acid molecular hybridization and PCR provide a more reliable method for detecting campylobacter, but still cannot meet the requirement of large-scale rapid detection due to the complex operation and special equipment.

Immunological-based assays include antibody assays and antigen assays. There has been much research in the population before the generation of antibodies after infection with campylobacter, and it is generally believed that campylobacter infection stimulates the body to produce specific antibodies 5-7 days after the onset of the disease, and anti-campylobacter antibodies in serum can be maintained for a longer period of time, igG drops to baseline levels after 4-5 months, and IgM drops rapidly to baseline levels after 30-50 days. However, there have also been studies to find that there is no significant change in antibodies in diarrhea patients caused by campylobacter infection. Thus, current serological antibody detection of campylobacter is not a clinical diagnostic criterion for campylobacter infection.

The campylobacter antigen has a complex structure and wide antigen diversity or diversity. At present, monoclonal antibodies capable of identifying all main pathogenic campylobacter strains are lacking, the existing campylobacter antigen detection reagents are mainly used for detecting campylobacter jejuni and campylobacter coli, the detection sensitivity is respectively 10 ⁴ CFU/mL and 10 ⁵ CFU/mL, detection omission is easy to occur, and other pathogenic campylobacter strains such as fetal campylobacter can not be detected by the existing kit. Thus, there is a need in the art to prepare high coverage monoclonal antibodies capable of recognizing all major pathogenic campylobacter strains, as well as campylobacter infection detection products, in order to rapidly and effectively diagnose campylobacter infection.

Disclosure of Invention

Therefore, the present invention is directed to an anti-campylobacter monoclonal antibody prepared by using a fused hybridoma cell line, and the monoclonal antibody obtained by the experiment can identify 5 main pathogenic campylobacteria (campylobacter jejuni (Campylobacter jejuni), campylobacter coli (Campylobacter coli), campylobacter foetidus (Campylobacter fetus), campylobacter gull (Campylobacter lari) and campylobacter Uppsala (Campylobacter upsaliensis)), does not cross react with other non-campylobacter pathogenic bacteria, has a detection sensitivity of 10 ²～10³ CFU/mL for campylobacter with a polyclonal antibody, is significantly higher than that of the existing reagents, and can be used for detecting campylobacter in a fecal sample or a sample after fecal culture.

Accordingly, one aspect of the present invention relates to an anti-campylobacter monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 and a light chain variable region comprising CDR1, CDR2 and CDR3, wherein,

The amino acid sequence of the heavy chain CDR1 is a sequence shown as SEQ ID NO.2 or an amino acid sequence with 1 conservative amino acid substitution compared with the sequence shown as SEQ ID NO. 2;

The amino acid sequence of the heavy chain CDR2 is a sequence shown as SEQ ID NO.3 or an amino acid sequence with 1 conservative amino acid substitution compared with the sequence shown as SEQ ID NO. 3;

the amino acid sequence of the heavy chain CDR3 is a sequence shown as SEQ ID NO.4 or an amino acid sequence with 1 conservative amino acid substitution compared with the sequence shown as SEQ ID NO. 4;

the amino acid sequence of the light chain CDR1 is a sequence shown as SEQ ID NO.6 or an amino acid sequence with 1 conservative amino acid substitution compared with the sequence shown as SEQ ID NO. 6;

The amino acid sequence of the light chain CDR2 is a sequence shown as SEQ ID NO.7 or an amino acid sequence with 1 conservative amino acid substitution compared with the sequence shown as SEQ ID NO. 7;

the amino acid sequence of the light chain CDR3 is a sequence shown as SEQ ID NO.8 or an amino acid sequence with 1 conservative amino acid substitution compared with the sequence shown as SEQ ID NO. 8.

In a further aspect, the invention also relates to a monoclonal antibody or antigen binding fragment thereof, the heavy chain variable region amino acid sequence is shown as SEQ ID NO.1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 5.

The present invention also relates to the above monoclonal antibodies or antigen-binding fragments thereof, which are Fab fragments, fab 'fragments, F (ab') ₂ fragments, single chain antibodies or humanized antibodies, which are capable of recognizing and binding campylobacter because they retain the variable regions of the light and heavy chains, or only the variable regions of the heavy chains.

Furthermore, the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the above-described antibody or antigen binding fragment thereof, and an expression vector comprising the above-described nucleic acid molecule, said expression vector being capable of expressing the above-described antibody or antigen binding fragment thereof. The invention also relates to a recombinant comprising the above nucleic acid molecule or the above expression vector, which can produce the above antibody or antigen-binding fragment thereof. In another aspect, the invention relates to an anti-campylobacter monoclonal antibody hybridoma cell line which secretes the monoclonal antibody described above. Further, the invention relates to a monoclonal antibody hybridoma cell strain resisting campylobacter, which is a mouse hybridoma cell strain CJ116 and has a preservation number of CGMCC No.46326.

In a further aspect, the invention relates to the use of a monoclonal antibody as described above, or an antigen binding fragment thereof, for the preparation of a product for detecting campylobacter. Further, the present invention relates to a kit for detecting campylobacter, which comprises the monoclonal antibody or the antigen-binding fragment thereof for recognizing and binding to campylobacter. Further, the invention relates to a kit for detecting campylobacter, which is a colloidal gold immunochromatography kit, and comprises the monoclonal antibody or the antigen binding fragment thereof, wherein the monoclonal antibody or the antigen binding fragment thereof is used as a capture antibody or is used as a detection antibody.

Description of biological Material preservation

The monoclonal antibody hybridoma cell strain is a mouse hybridoma cell strain CJ116 which is preserved in China general microbiological culture Collection center (CGMCC), the registration number of the preservation center is CGMCC No.46326, and the preservation date is 2025, 2 months and 13 days. The China general microbiological culture Collection center (China general microbiological culture Collection center) addresses are 1 to 3 in the West way of North Star in the Korean area of Beijing, and the postal code is 100101.

Drawings

FIG. 1 is a B cell epitope profile of Campylobacter jejuni (Campylobacter jejuni) RacR protein.

FIG. 2 is an alignment chart of amino acid sequence identity analysis of 5 major pathogenic campylobacter RacR proteins.

FIG. 3 is a SDS-PAGE electrophoresis showing prokaryotic expression of the antigen RacR of Campylobacter jejuni, wherein each mark is M,1 is whole fungus supernatant before loading, 2 is Ni column penetrating fluid, 3 is 25mmol/L imidazole washing fluid for eluting the antigen, and 4 is 250mmol/L imidazole washing fluid for eluting the antigen.

FIG. 4 is a graph showing the judgment of the results of detecting Campylobacter by colloidal gold immunochromatography, wherein the C line is a control line, the T line is a detection line, and the S is a sample well, wherein the positive is that a red band appears at the C line and a red band of unequal intensity appears at the T line at the same time, the negative is that a red band appears only at the C line and no band appears at the T line, and the ineffective is that no band appears at the C line, regardless of whether a red band appears at the T line.

FIG. 5 is a graph showing the results of detection of Campylobacter by colloidal gold immunochromatography, wherein line C is a control line, line T is a detection line, line S is a sample well, the detection sensitivity for Campylobacter jejuni and Campylobacter coli reaches 10 ² CFU/mL, and the detection sensitivity for Campylobacter fetus, campylobacter Uppsalanus and Campylobacter gull reaches 10 ³ CFU/mL.

Fig. 6 is a diagram showing a specific detection result of detecting campylobacter by a colloidal gold immunochromatography, wherein line C is a control line, line T is a detection line, and line S is a sample well, wherein 1 is pathogenic escherichia coli, 2 is salmonella enteritidis, 3 is yersinia enterocolitica, 4 is shigella flexneri, 5 is shigella pallidum, and 6 is staphylococcus aureus.

FIG. 7 is a graph showing the results of subtype identification of monoclonal antibody CJ116 of high immunogenicity consistent sequence against campylobacter RacR.

Detailed Description

The invention aims to provide an anti-campylobacter monoclonal antibody prepared by using a fused hybridoma cell strain, wherein the strain of the anti-campylobacter monoclonal antibody can identify 5 main pathogenic campylobacteria. The specific preparation process comprises the steps of firstly determining the bacterial proteins which are all present in 5 main pathogenic campylobacteria and have high sequence homology through bioinformatics analysis, then analyzing the common sequence with high immunogenicity antigen epitope, preparing monoclonal antibodies by taking the monoclonal antibodies as immunogens to immunize mice, and obtaining a mouse hybridoma cell strain which expresses the monoclonal antibodies with high affinity and high specificity after screening, wherein the detection sensitivity of the mouse hybridoma cell strain to campylobacter and campylobacter coli can reach 10 ² CFU/mL, and the detection sensitivity of the mouse hybridoma cell strain to fetal campylobacter, uplasia and campylobacter seaguli can reach 10 ³ CFU/mL, can identify all 5 main pathogenic campylobacter, has no cross reaction with other common intestinal pathogens such as pathogenic escherichia coli, salmonella, enterocolitis yersinia, shigella flexneri and staphylococcus aureus, and shows that the high-coverage anti-campylobacter monoclonal antibody 116 can be used for detecting campylobacter infection, and has high sensitivity and high specificity. The inventor reserves the cell strain in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.46326 in the year 2025, 2 and 13.

The inventors then sequenced the monoclonal antibody secreted by mouse hybridoma cell line CGMCC No.46326 and analyzed the immunoglobulin domain sequence, and found that the heavy chain variable region amino acid sequence was ：EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWFRQTPEKRLEWVAYISSGGDR MYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCGRIDNWGQGTTLTVS(SEQ ID NO.1), in which the CDR1 amino acid sequence was GFAFSSYD (SEQ ID NO. 2), the CDR2 amino acid sequence was ISSGGDRM (SEQ ID NO. 3), and the CDR3 amino acid sequence was GRIDN (SEQ ID NO. 4). Light chain variable region amino acid sequence ：DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEEGPSWKS(SEQ ID NO.5), wherein the CDR1 amino acid sequence is RASKSVSTSGYSYMH (SEQ ID No. 6), CDR2 amino acid sequence is LVSNLES (SEQ ID No. 7), and CDR3 amino acid sequence is QHIRELTR (SEQ ID No. 8).

The high-coverage anti-campylobacter monoclonal antibody CJ116 can be used as a capture antibody or a detection antibody, and the result shows that the detection sensitivity of campylobacter jejuni and campylobacter coli can reach 10 ² CFU/mL, and the detection sensitivity of campylobacter jejuni, uprasugrel campylobacter jejuni and seagull campylobacter is 10 ³ CFU/mL, so that all 5 main pathogenic campylobacter bacteria can be identified, and the high-coverage anti-campylobacter monoclonal antibody CJ116 has no cross reaction with other common intestinal pathogens such as pathogenic escherichia coli, enteritis salmonella, enterocolitis yersinia, shigella flexneri, shigella pallidus and staphylococcus aureus.

It is well known in the art that the heavy and light chain CDR regions of antibodies are important amino acid sequence regions that recognize and bind to the corresponding antigen, and that 1 conservative amino acid substitution in the amino acid sequence of the CDR regions generally does not alter the structure of the protein, so that a single conservative amino acid substitution in the region may still possess the property of binding to the corresponding antigen. Thus, monoclonal antibodies or antigen-binding fragments thereof obtained after 1 conservative amino acid substitution of heavy chain CDR1 and/or heavy chain CDR2 and/or heavy chain CDR3 and/or light chain CDR1 and/or light chain CDR2 and/or light chain CDR3 are still capable of recognizing and binding to campylobacter. In the present patent application, conservative amino acid substitutions refer to the replacement of one amino acid in a protein with another, chemically similar amino acid, such as the replacement of aromatic amino acids Phe, trp, tyr, the replacement of aliphatic amino acids Ala, gly, leu, ile, val, the replacement of polar amino acids Gln, asn, the replacement of basic amino acids Lys, arg, his, the replacement of acidic amino acids Asp, glu, and the replacement of hydroxy amino acids Ser, thr, etc.

Those skilled in the art can also prepare various antibody fragments, i.e., antigen-binding fragments, such as, but not limited to, fab ', F (ab') ₂, capable of binding to campylobacter from the monoclonal antibodies of the invention by techniques known in the art. Fab fragments are regions of an antibody structure that bind antigen and consist of a complete variable region VH and constant region CH1 domain (Fd segment) of the light and heavy chains, with a constant region and a variable region both present, and disulfide linkages between the light and heavy chains. Antigen binding fragments can be prepared, for example, by enzymatic cleavage using papain, whereby the antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, the antibody IgG is degraded into a F (ab ') ₂ fragment and a pFc' fragment, and the F (ab ') ₂ fragment is further reduced to form two Fab' fragments. Because the antigen binding fragments are still capable of binding to the corresponding antigens, they can be used to prepare products for detection of campylobacter.

The skilled artisan can also prepare single chain antibodies (scFv) from the monoclonal antibodies of the invention by techniques known in the art. The single chain antibody is formed by connecting a heavy chain variable region and a light chain variable region of the antibody through a short peptide linker of a plurality of amino acids, and only one chain is an artificially synthesized antibody. The single chain antibody may contain only the antibody heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used for the monoclonal antibody of the present invention can be determined by simple repeated experiments. The single chain antibody may be expressed in, for example, E.coli by genetic engineering techniques. The single-chain antibody of the invention prepared in this way has the characteristic of combining with campylobacter and can be applied to the detection of campylobacter.

The skilled person can design and synthesize a nucleic acid molecule encoding the same based on the amino acid sequence of the variable region of the anti-campylobacter monoclonal antibody, or insert the synthesized nucleic acid molecule into a nucleic acid vector, so as to construct an expression vector capable of expressing the anti-campylobacter monoclonal antibody or antigen-binding fragment thereof. The person skilled in the art is also able to introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, yeasts and the like to obtain recombinants, and express the antibodies or antigen-binding fragments thereof of the present invention via the recombinant bodies as described above, the antibodies or antigen-binding fragments thereof thus expressed being able to bind and recognize campylobacteria, and thus the nucleic acid molecules, expression vectors and recombinants as described above are within the scope of the claims of the present invention. And all of the above techniques are well known in the art and can be carried out by those skilled in the art without the need for creative effort.

As described above, the antibody or antigen-binding fragment thereof of the present invention is capable of specifically recognizing and binding to campylobacter, and thus can be used for preparing a kit for detecting campylobacter, which can be any kit using a binding reaction of the antibody or antigen-binding fragment thereof of the present invention with campylobacter, such as, but not limited to, a kit for colloidal gold immunochromatography, fluorescence immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunoblotting, immunohistochemical methods, wherein the high coverage anti-campylobacter monoclonal antibody CJ116 of the present invention can be used as a capture antibody or as a detection antibody.

In order to describe the technical contents of the technical solution in detail, the achieved objects and effects, the following description will be made with reference to specific embodiments.

Example 1 screening for Curvularia common antigen

According to literature studies 9 antigens with sequence conservation and useful for detection of campylobacter were selected, flagellin FliD, temperature response regulatory protein RacR, adhesion related proteins PebA and CadF, invasion related proteins CiaB and FlaC, outer membrane protein OMP18, and cell lethal tumefaction toxins CdtA and CdtC, respectively. First, NCBI reference sequences (NCBI Reference Sequence) of amino acid sequences of 9 detection antigens, namely Campylobacter jejuni (Campylobacter jejuni), campylobacter coli (Campylobacter coli), campylobacter fetus (Campylobacter fetus), campylobacter Uppsalanus (Campylobacter upsaliensis) and Campylobacter gull (Campylobacter lari), are listed in Table 1 from NCBI's GenBank database (https:// www.ncbi.nlm.nih.gov /), respectively, and specific sequences thereof are listed in Table 2 below. Next, the homology of the corresponding Protein sequences of the other 4 species of Campylobacter strains was analyzed and aligned using the "Align two or more sequences" module in NCBI website Protein BLAST (https:// BLAST. NCBI. Lm. Nih. Gov/BLAST. Cgi) with the corresponding Protein sequence of Campylobacter jejuni (Campylobacter jejuni) having the highest infection rate as a target sequence, and the results are shown in Table 3. The sequence comparison analysis result shows that only RacR proteins have higher homology among 5 main pathogenic campylobacter, so that RacR proteins are selected as detection targets.

TABLE 1.5 NCBI reference sequences for detection of related proteins by primary pathogenic campylobacter

Note that "-" indicates that the corresponding sequence is not found in NCBI

TABLE 2 amino acid sequences of 9 detection antigens of 5 major pathogenic campylobacteria

TABLE 3 amino acid sequence homology analysis with campylobacter jejuni protein

Proteins	Campylobacter coli	Campylobacter foetidus	Uppsala campylobacter	Acremodella gull
					FliD	98(628/642)	36(237/666)	63%(411/653)	41%(210/517)
RacR	93%(207/223)	76%(169/223)	78%(175/223)	79%(177/223)
					PebA	84%(218/259)	61%(159/260)	81%(211/259)	—
CadF	80%(266/331)	40%(138/344)	75%(237/318)	57%(186/329)
					CiaB	79%(483/608)	52%(317/609)	70%(429/610)	64%(389/607)
FlaC	95%(237/249)	49%(123/249)	84%(209/248)	66%(165/249)
					Omp18	94%(155/165)	54%(93/173)	89%(147/165)	—
CdtA	99%(266/268)	36%(103/290)	57%(151/266)	52%(136/264)
					CdtC	46%(85/183)	35%(64/183)	58%(111/190)	64%(109/169)

Note that "-" indicates that the corresponding sequence was not found in NCBI and no sequence alignment was possible

Example 2 high immunogenicity consistent sequence analysis of Curvularia common antigen RacR

B cell epitope distribution of Campylobacter jejuni RacR protein was analyzed by BIOSUN software, the amino acid sequence was first entered, then B cell epitopes were analyzed, the epitope distribution map was shown in FIG. 1, and the corresponding epitope sequence was shown in Table 4.

TABLE 4B cell epitope sequences and scores of Campylobacter jejuni RacR protein

Then, the amino acid sequences of the RacR main pathogenic campylobacter proteins were aligned and analyzed by DNAMAN6.0 software, and the result shows that the amino acid sequence homology of the RacR proteins of the 5 main pathogenic campylobacter bacteria is 87.09%. Combining epitope analysis and sequence identity analysis results, epitope 6 (101-PYDPKEM-107, score 3.31852), epitope 10 (169-SVSREQL-175, score 2.51627), and epitope 11 (182-LKDKDSK-188, score 3.85282) were determined to be highly immunogenic consensus sequences.

EXAMPLE 3 preparation of RacR highly immunogenic consensus synthetic peptides

In order to be able to detect all 5 major pathogenic campylobacteria, monoclonal antibodies were prepared using the RacR high immunogenic consensus sequences determined by screening as immunogens. The high-immunogenicity consistent sequence epitope 6, epitope 10 and epitope 11 are firstly synthesized into a synthetic peptide, one amino acid is covered before and after each epitope in order to ensure the integrity of the epitope (because the amino acid behind the epitope 6 and the amino acid in front of the epitope 10 are both Y, only one amino acid Y is added between the epitopes 6 and 10), and one cysteine (C) is added at the N-terminal of the synthetic peptide in order to facilitate coupling, namely the high-immunogenicity consistent sequence synthetic peptide has a sequence CKPYDPKEMYSVSREQLVNLKDKDSKS (SEQ ID NO. 52). The immunogen synthetic peptide is coupled to hemocyanin KLH to enhance immunogenicity. In order to screen and determine the epitope recognized by the monoclonal antibody, synthetic peptides of epitope 6, epitope 10 and epitope 11 were synthesized, respectively, and a cysteine (C) was added to the N-terminal of the synthetic peptide for ease of coupling, i.e., epitope 6 synthetic peptide sequence CKPYDPKEMY (SEQ ID NO. 53), epitope 10 synthetic peptide sequence CYSVSREQLV (SEQ ID NO. 54) and epitope 11 synthetic peptide sequence CNLKDKDSKS (SEQ ID NO. 55). The screening was performed using synthetic peptides coupled to bovine serum albumin BSA to facilitate detection by coated ELISA plates. Polypeptide synthesis and KLH or BSA coupling were carried out by Shanghai De Fin Biotechnology Co.

Example 4 preparation of monoclonal antibodies to RacR highly immunogenic consensus sequences

The RacR high immunogenicity consistent sequence synthetic peptide is used as immunogen, and BALB/c female mice with the age of 6-8 weeks are adopted, 100 mug/antigen is added with equivalent Freund complete adjuvant, and after thorough emulsification by a stirrer, the mice are immunized by subcutaneous and intraperitoneal injection on the back, and 3 mice are immunized. The second immunization was performed 4 weeks apart, the third immunization was performed 8 weeks later, 50. Mu.g/antigen was added with incomplete Freund's adjuvant, and after thorough emulsification with a stirrer, the mice were immunized by subcutaneous and intraperitoneal injection from the back. After the third immunization for one week, mice were collected blood from the tail vein and tested for immune serum titers, and mice with the highest titers were selected for intraperitoneal injection for booster immunization (50 μg/mouse), and spleen cells were taken for fusion after 3 days. The SP20 myeloma cells were resuscitated and cultured until they were in the log phase of growth. Immunized BALB/c mice were taken and spleen thereof was taken to prepare spleen cell suspensions. Mixing the spleen cells and myeloma cells according to the proportion of 9:1, centrifuging at 1500rpm for 5 minutes in a serum-free DMEM culture medium, sucking out the supernatant, gently shaking to disperse the cells, fusing in a 37 ℃ water bath, adding 1mL of preheated 50% PEG fused cells within 1 minute, gently shaking while adding the mixture, standing for 90 seconds after the addition, adding the serum-free DMEM culture medium to terminate the fusion, standing for 10 minutes at 37 ℃, centrifuging at 1500rpm for 5 minutes, suspending the precipitate by using the HAT culture medium, subpackaging into 96-well cell plates containing feeder cells, culturing in a cell culture box with 37 ℃ and 5% CO ₂ for 5 days, replacing the supernatant by using the HAT culture medium once, replacing the supernatant by using the HAT culture medium on the 10 th day, and taking the cell culture supernatant when the fused cells cover about 60% of the bottom of the hole, and respectively carrying out serial dilution with PBS (phosphate buffer solution) with dilution factors of 400, 800, 1600, 6400 and 12800. Positive clones were screened by indirect ELISA. The specific method comprises diluting epitope 6 synthetic peptide, epitope 10 synthetic peptide and epitope 11 synthetic peptide with carbonate coating buffer solution at concentration of 2.5 μg/ml, coating 150 μl per well at 4deg.C overnight, washing the plate 2 times with washing solution, adding 200 μl per well blocking solution, blocking at room temperature for 6 hr, and washing the plate 5 times with washing solution. After adding 100. Mu.l of sample dilution to each well, 10. Mu.l of different gradient dilutions of the cell culture supernatant were added, incubated at room temperature for 30min, and the solution was discarded. Washing the plate for 5 times, inversing the washed ELISA plate on absorbent paper, beating to dry, adding 100 μl/hole HRP-marked goat anti-mouse IgG antibody, and incubating at room temperature for 30min. The plate was washed 5 times. Each 50. Mu.L of TMB developing solution A and TMB developing solution B are added into each hole, and the color development is carried out at room temperature in a dark place for 15min. 50. Mu.L of 2M H ₂SO₄ stop solution was added to each well to stop the reaction. The detection wavelength of the enzyme label instrument is set to 450nm, the OD value of each hole is measured, and the reading is carried out within 10 minutes after termination.

A total of 121 positive clones were obtained, of which 47 positive clone-recognizing epitope 6,29 positive clone-recognizing epitope 10,45 positive clone-recognizing epitope 11. One clone with a titer of 1:12800 was selected from each group recognizing different epitopes for subsequent campylobacter detection studies, clone CJ116 recognizing epitope 6, clone CJ39 recognizing epitope 10 and clone CJ107 recognizing epitope 11, respectively. The hybridoma cell line was cultured in 1640 medium containing 10% fetal bovine serum. Each BALB/c male mouse was intraperitoneally injected with 0.5mL liquid paraffin. Cells were collected after 10 days, resuspended in 10mL of physiological saline at a cell density of 1X 10 ⁷ cells/mL and injected intraperitoneally with 0.5mL each. After 2 weeks, ascites was collected. Antibody purification was performed using Thermo company Melon Gel Monoclonal IgG Purification Kit kit and the purified antibodies were stored at-20 ℃ after sub-packaging.

EXAMPLE 5 preparation of Campylobacter jejuni RacR antigen

To facilitate expression in vitro, the RacR full-length nucleotide sequence for E.coli expression was deduced using E.coli genetic code preference from the amino acid sequence of the Campylobacter jejuni RacR antigen published by the GenBank database of NCBI (NCBI reference number ALK 81821.1) 5'-ATGATTAACGTGCTGATGATCGAAGATGACCCGGATTTTGCGCAGTTGCTGAGCG AGTATTTAGCCCAATTCAATATTAAAATCACCAACTTTGAAAATCCAAAGTCTGCGCTGAACGTTGGCGTGCAGGGTTACGATTGCCTTATTCTGGACCTCACGCTGCCGGGCATCGATGGTCTGGAAGTCTGTCGTGAGATTCGCCAGAAATCCAATATCCCAATTATTATCTCTAGCGCACGTGGCGACTTGAGTGATAAAGTTGTAGGTCTGCAAATTGGCGCTGATGATTATTTACCGAAGCCATACGACCCGAAAGAAATGTATGCGCGCATCATGTCTCTGATTCGTCGCACTAAACGTGTGGAACATGCCAACAATGAGAACATCAACTCGGCATTCAAGATTGATGAACGCCGTCACGAAATCACCTACGAGGATAAAGTTCTTACTCTGACGCCAGCTGAATTTGAAATTCTGGAGTATCTGATTCAGCAGCATGGTTACAGCGTCAGCCGTGAACAACTGGTGTCTCGCTGCAAAAATTTGAAGGACAAAGATTCCAAATCGCTGGACGTTATCATTGGCCGTTTACGCGTGAAGATCGGCGATAGCAGTAAATCTCCGAAACACATTTTCTCGGTCCGTGGTATCGGCTATAAGCTGATTGGT-3'

(SEQ ID NO. 56) and then the full-length nucleotide sequence, the upstream primer 5'-GCGGATCCATGATTAACGTGCTGA-3' (SEQ ID NO. 57) and the downstream primer 5'-GCGAATTCTTAACCAATCAGCTTATAG-3' (SEQ ID NO. 58) were delegated to Beijing biological science, inc. The synthesized RacR nucleotide sequence is used as a template to amplify the full-length gene under the conditions of 95 ℃ for 2min, 95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 45s, 30 cycles, and 72 ℃ for 5min. The 2% agarose electrophoresis identification shows that the relative molecular weight of the amplified fragment is about 700bp. The purified PCR product was digested at BamHI and EcoRI sites, and ligated into pCold I plasmid (purchased from Takara Co.) to obtain pCold-RacR recombinant plasmid. The recombinant expression plasmid with correct sequence is transformed into E.coli BL21 competent cells, single colony is selected and cultured overnight in LB liquid medium containing ampicillin sodium at 37 ℃ in a shaking way, the next day is inoculated in 250mL of fresh LB liquid medium, the culture is carried out to logarithmic phase, the temperature is adjusted to 15 ℃, 150 mu.L of IPTG induction liquid with the concentration of 1mol/L is added after 30 minutes, and the induction is carried out for 12-14 hours at 15 ℃. The induced cells were collected by centrifugation, resuspended in 25mmol/L Tris-HCl (pH 8.5), sonicated, and the supernatant collected by centrifugation at 20000g at high speed for 30min at 4℃was subjected to Ni column purification, first the Ni column was equilibrated with equilibration buffer (25 mmol/L TE, 1% beta-mercaptoethanol, 6mol/L urea, pH 8.5), the supernatant was added to the Ni column, after the sample had completely entered, the target protein was collected by eluting with 25mmol/L and 250mmol/L imidazole containing washes, respectively, and subjected to SDS-PAGE gel electrophoresis, racR antigen was expressed as soluble, and most of the protein was eluted with 250mmol/L imidazole washes, with a molecular weight of about 27.5kDa, as shown in FIG. 3.

Example 6 preparation of rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody

Selecting healthy male white rabbits, mixing 1.0mg of campylobacter jejuni RacR antigen expressed by escherichia coli prokaryotic in the embodiment 5 with 1.0ml of Freund's complete adjuvant, thoroughly emulsifying by a stirrer, injecting subcutaneously at two sides of the spine of the white rabbits at each point by 0.2ml, taking 1.0mg RacR antigen and 1.0ml of Freund's incomplete adjuvant after 4 weeks, performing secondary immunization at different points after thoroughly emulsifying by the stirrer, and performing third booster immunization after 4 weeks to prepare polyclonal antibody serum. After 1 week heart blood is taken, after blood coagulation and blood clot contraction, centrifugation is carried out at 5000rpm for 15 minutes, and serum is separated and stored in a refrigerator at-20 ℃ for standby. The use of RacR antigen as detection antigen coated enzyme-linked plate, the indirect ELISA method is also adopted to detect the titer of the purified rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody, and the result shows that the titer of the prepared rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody reaches 1:1024000.

Example 7 detection of 5 major pathogenic campylobacteria by anti-RacR highly immunogenic consensus monoclonal antibodies

The rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody prepared in example 6 is used as a coating antibody, the three mouse anti-RacR high immunogenicity consistent sequence monoclonal antibodies prepared in example 4, namely CJ116, CJ39 or CJ107 are used as detection antibodies, a double antibody sandwich method is established for detecting 5 main pathogenic campylobacter, and the detection capability of the monoclonal antibodies is evaluated. Firstly, 5 main pathogenic campylobacter strains, namely campylobacter jejuni (Campylobacter jejuni ATCC 33560), campylobacter coli (Campylobacter coli DSM 100395), campylobacter fetus (Campylobacter fetus DSM 105764), campylobacter Upsoas (Campylobacter upsaliensis ATCC 43954) and campylobacter seagull (Campylobacter lari ATCC 35221), are subjected to gradient dilution with PBS to be 10⁶CFU/mL、10⁵CFU/mL、10⁴CFU/mL、10³CFU/mL、10²CFU/mL、10¹CFU/mL., then the 5 main pathogenic campylobacter strains are detected by adopting a double-antibody sandwich method, specifically, an enzyme-linked plate is coated with rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody, the concentration is 2.0 mu g/mL, each hole is coated with 100 mu L and is subjected to 4 ℃ overnight, the plate is washed with washing liquid for 2 times, 120 mu L/hole sealing liquid is added for 6 hours at room temperature, the plate is washed with washing liquid for 5 times, 50 mu L serial diluted bacterial liquid and 50 mu L sample treatment liquid are added into each hole, incubation is carried out for 60 minutes at 37 ℃, and liquid discarding is carried out. The plates were washed 5 times with wash solution, 100. Mu.L of horseradish peroxidase-labeled mouse anti-RacR high-immunogenicity consensus monoclonal antibody was added to each well and incubated at 37℃for 60min. Washing the plate for 5 times, beating, adding 50 mu L of TMB developing solution A and B solution into each hole, and developing for 15min at room temperature in dark place. The reaction was terminated by adding 2M H ₂SO₄ stop solution at 50. Mu.L/well. The enzyme label instrument detects the wavelength of 450nm, and the OD value of each hole is measured, and the reading is carried out within 10 minutes after termination. The critical value is 0.15, the OD value is more than or equal to 0.15, positive is judged, and the OD value is less than 0.15, negative is judged.

As shown in Table 5, the results showed that the detection sensitivity of the double-antibody sandwich test campylobacter was highest, and the detection sensitivity of campylobacter jejuni and campylobacter coli was 10 ² CFU/mL, and the detection sensitivity of campylobacter jejuni, campylobacter Uppsalanus and campylobacter gull was 10 ³ CFU/mL, and the detection sensitivity of campylobacter jejuni and campylobacter sea was 10 ² CFU/mL, and the detection sensitivity of campylobacter jejuni was 10 ³ CFU/mL, and the detection sensitivity of campylobacter foetidus and campylobacter sea was 10 ⁴ CFU/mL, and the detection sensitivity of campylobacter jejuni and campylobacter sea was 10 ² CFU/mL, and the detection sensitivity of campylobacter jejuni and campylobacter sea was 10 ³ CFU/mL, but campylobacter jeppsalanus could not be detected, respectively, when the monoclonal antibody CJ107 was used as the detection antibody. Thus, the CJ116 monoclonal antibody can detect all 5 main pathogenic campylobacteria, and has highest detection sensitivity.

TABLE 5 detection of 5 major pathogenic campylobacteria by anti RacR highly immunogenic consensus monoclonal antibodies

EXAMPLE 8 detection of Curvularia by colloidal gold immunochromatography

The colloidal gold immunochromatography detection method for detecting campylobacter is established by using the mouse anti-RacR high-immunogenicity consistent sequence CJ116 monoclonal antibody prepared by the invention and the rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody prepared by the embodiment 6 by adopting a colloidal gold immunochromatography technology. The rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody prepared by the invention is coated on a nitrocellulose membrane at a T line, the CJ116 monoclonal antibody of the invention marked by colloidal gold is fixed on a gold mark pad, and a sheep anti-mouse IgG polyclonal antibody (purchased from the Zhuhai Bome Biotechnology Co., ltd.) is coated on a C line.

The 5 main pathogenic campylobacteria were also tested for gradient dilution to 10⁶CFU/mL、10⁵CFU/mL、10⁴CFU/mL、10³CFU/mL、10²CFU/mL、10¹CFU/mL. In addition, pathogenic Escherichia coli, salmonella enteritidis, yersinia enterocolitica, shigella flexneri, shigella pallidum and Staphylococcus aureus at a concentration of 1X 10 ⁷ CFU/mL were detected, and the specificity of campylobacter was evaluated by colloidal gold immunochromatography.

The specific operation is that the test card is horizontally placed on a drying plane, and 100 mu L of the sample is respectively vertically and slowly dripped into the sample adding holes of the test card. And judging the result for 5-15 minutes, and judging the result to be invalid after 15 minutes. When a sample to be measured is dropped into the sample well of the test card, the sample will move forward along the test card by capillary action. When migrating to the gold-labeled pad, if the sample contains the main pathogenic campylobacter, the monoclonal antibody of CJ116, which is the sequence consistent with the high immunogenicity of the mouse antibody RacR marked by colloidal gold, is combined to form an immune complex. The immune complex formed continues to migrate forward and is captured by a rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody (T line) immobilized on a nitrocellulose membrane, so as to form a 'mouse anti-RacR high immunogenicity consistent sequence CJ116 monoclonal antibody-main pathogenic campylobacter antigen-rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody' immune complex, and a red T line is generated. Regardless of whether the sample contains campylobacter antigen or not, the goat anti-mouse IgG coated in the C line region can be combined with an excessive mouse anti-RacR high-immunogenicity consistent sequence CJ116 monoclonal antibody to form a red band. The result judgment is as shown in fig. 4, wherein the red stripe appears at the C line, the red stripe with unequal intensity appears at the T line at the same time is judged as positive, the red stripe appears only at the C line, the no stripe appears at the T line as negative, the no stripe appears at the C line, and the red stripe appears at the T line or not as invalid.

As shown in the experimental results in FIG. 5 and FIG. 6, the colloidal gold immunochromatographic assay method established by using the mouse anti-RacR high-immunogenicity consistent sequence CJ116 monoclonal antibody prepared by the invention and the rabbit anti-campylobacter jejuni RacR antigen polyclonal antibody prepared by the example 6 can also completely detect 5 main pathogenic campylobacter, has high detection sensitivity, reaches 10 ² CFU/mL for campylobacter jejuni and campylobacter coli, has 10 ³ CFU/mL for campylobacter foetidus, campylobacter jejuni and campylobacter favui (FIG. 5), has high detection specificity, and does not have non-specific cross reaction with pathogenic escherichia coli, salmonella enteritidis, yersinia enterocolitica, shigella flexneri, shigella baumannii and staphylococcus aureus with the concentration of 1×10 ⁷ CFU/mL (FIG. 6).

Example 9 analysis of the highly immunogenic consensus monoclonal antibody CJ116 subtype against Curvularia RacR

The heavy and light chain subtypes of the mouse antibodies were identified using a rapid detection card (cat# THJ-ISO-M8 a-10/20) from Antaiji (Beijing) Biotechnology Co., ltd. The antibody was first diluted to 1. Mu.g/mL with PBS, then 100. Mu.l of diluted antibody was added to each well, and the result was observed and recorded after standing for 5-10 min. As a result, FIG. 7 shows that the anti-campylobacter RacR highly immunogenic consensus monoclonal antibody CJ116 was of the mouse IgG1 subtype and the antibody light chain was of the Ig kappa subtype.

EXAMPLE 10 determination of sequence of variable region of monoclonal antibody CJ116 against high immunogenicity of Campylobacter RacR

After culturing the mouse hybridoma cell strain CJ116 and extracting total RNA of the hybridoma cells by the Trizol method and reversely transcribing cDNA, PCR amplification is carried out by using a mouse monoclonal antibody Fab fragment primer synthesized by Beijing engine biotechnology Co Ltd (primer design is referred to pages 70-72 in "genetic engineering antibody technology" of chapter three of modern immunology experiment technology ", second edition), preheating is carried out for 2min at 95 ℃,30 cycles are carried out at 95 ℃ for 30 seconds, 58 ℃ for 30 seconds and 72 seconds, finally, 72 ℃ is extended for 5min, a pMD18-T vector (purchased from TaKaRa Co.) is connected, E.coli JM109 is transformed, and positive clones are selected for sequencing. IgBLAST (https:// www.ncbi.nlm.nih.gov/IgBLAST /) of the assay sequences in the BLAST module of the NCBI website was aligned to the mouse-derived monoclonal antibody CDR region sequences.

The sequence analysis revealed that the heavy chain variable region has an amino acid sequence of 111 amino acids, whose sequence is ：EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWFRQTPEKRLEWVAYISSGGDRMYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCGRIDNWGQGTTLTVS(SEQ ID NO.1),, wherein the underlined sequence is CDR series, CDR1 is located at 26-33aa, the amino acid sequence is GFAFSSYD (SEQ ID NO. 2), CDR2 is located at 51-58aa, the amino acid sequence is ISSGGDRM (SEQ ID NO. 3), CDR3 is located at 97-101aa, and the amino acid sequence is GRIDN (SEQ ID NO. 4). The light chain variable region has an amino acid sequence of 109 amino acids, whose sequence is ：DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEEGPSWKS(SEQ ID NO.5), wherein the underlined sequence is the CDR series, CDR1 is at 24-38aa, amino acid sequence is RASKSVSTSGYSYMH (SEQ ID NO. 6), CDR2 is at 54-60aa, amino acid sequence is LVSNLES (SEQ ID NO. 7), CDR3 is at 93-100aa, amino acid sequence is QHIRELTR (SEQ ID NO. 8).

Claims

1. An anti-campylobacter monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 and a light chain variable region comprising CDR1, CDR2 and CDR3, characterized in that,

The amino acid sequence of the heavy chain CDR1 is a sequence shown as SEQ ID NO. 2;

The amino acid sequence of the heavy chain CDR2 is a sequence shown as SEQ ID NO. 3;

The amino acid sequence of the heavy chain CDR3 is a sequence shown in SEQ ID NO. 4;

the amino acid sequence of the light chain CDR1 is a sequence shown in SEQ ID NO. 6;

the amino acid sequence of the light chain CDR2 is a sequence shown in SEQ ID NO. 7;

the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 8.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region amino acid sequence is set forth in SEQ ID No.1 and the light chain variable region amino acid sequence is set forth in SEQ ID No. 5.

3. The monoclonal antibody according to claim 2, which is secreted by a mouse hybridoma cell line CJ116 having a accession number of CGMCC No. 46326.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab 'fragment, a F (ab') ₂ fragment, a single chain antibody or a humanized antibody.

5. A nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. Bacterial or yeast recombinant, characterized in that it comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The hybridoma cell strain secreting the anti-campylobacter monoclonal antibody is characterized by being a mouse hybridoma cell strain CJ116 with a preservation number of CGMCC No. 46326.

9. Use of the anti-campylobacter monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for the preparation of a product for detecting campylobacter.

10. A kit for detecting campylobacter, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

11. The kit according to claim 10, characterized in that it is a colloidal gold immunochromatographic kit, and the monoclonal antibody or antigen-binding fragment thereof is used as a capture antibody or as a detection antibody.