Monoclonal antibody for resisting pathogenic campylobacter and application thereof

Abstract

The invention discloses a monoclonal antibody CJ116 for resisting pathogenic campylobacter and an application of the monoclonal antibody CJ116. In the monoclonal antibody CJ116, the amino acid sequence of a heavy chain variable region is as shown in SEQ ID NO.1, and the amino acid sequence of a light chain variable region is as shown in SEQ ID NO.5. The CJ116 monoclonal antibody has high affinity and high specificity, the detection sensitivity of the CJ116 monoclonal antibody to campylobacter jejuni and campylobacter colons can reach 10 < 2 > CFU / mL, and the detection sensitivity of the CJ116 monoclonal antibody to campylobacter fetus, campylobacter upsackii and campylobacter seagull is 10 < 3 > CFU / mL. However, the enteropathogenic bacteria have no cross reaction with other common enteropathogenic bacteria such as pathogenic Escherichia coli, salmonella enteritidis, yersinia enterocolitica, Shigella flexneri, Shigella baumannii and Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a monoclonal antibody against pathogenic Campylobacter and its application. Background Art

[0002] Infectious diarrhea is a common symptom caused by bacterial, viral or parasitic infections of the gastrointestinal tract, 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 childhood malnutrition.

[0003] Campylobacter spp. are common foodborne zoonotic pathogens and important pathogens causing infectious diarrhea worldwide. The species pathogenic to humans include Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, etc. Campylobacter jejuni and Campylobacter coli are the main pathogens of human infection, accounting for 95% of all reported human Campylobacter infection cases, with Campylobacter jejuni infection exceeding 90%. Campylobacteriosis is a self - limiting gastroenteritis disease that lasts about 5 - 7 days, and typical symptoms can range from mild diarrhea to inflammatory diarrhea, including diarrhea, abdominal colic, vomiting and fever. In addition to acute gastroenteritis, Campylobacter jejuni infection can lead to serious sequelae such as Guillain - Barré syndrome, reactive arthritis and irritable bowel syndrome, etc.

[0004] In recent years, the incidence of campylobacteriosis has been gradually increasing. Approximately 166 million diarrhea cases worldwide are caused by Campylobacter jejuni each year, accounting for about 5 - 14% of diarrhea diseases. Currently, in countries such as Europe and America, diarrhea caused by Campylobacter jejuni infection ranks first among diarrhea caused by pathogenic bacterial infections, and in some countries it is second only to Salmonella or Shigella. In China, infants and young children are a high - risk population for Campylobacter jejuni enteritis, especially in the summer and autumn seasons in the developed eastern coastal areas, where they are most likely to be infected with Campylobacter jejuni and develop diarrhea. Monitoring results in China in recent years have shown that the detection rate of Campylobacter jejuni in children with diarrhea is 4.0 - 17.7%, and the detection rate in adults with diarrhea is 3.85 - 9.61%. In addition, studies have found that Campylobacter jejuni can be carried in healthy people, with a carriage rate between 0.6 - 9.4%. Therefore, rapid and accurate detection of Campylobacter is of great significance for the prevention and control of the outbreak and epidemic of Campylobacter jejuni disease, the early treatment of infected patients, and the prevention and treatment of serious complications.

[0005] Currently, the detection methods for Campylobacter mainly include isolation and culture methods, conventional biochemical methods, nucleic acid detection methods, immunoassay methods, mass spectrometry detection methods, etc. The isolation and culture of pathogenic bacteria is the gold standard for the clinical diagnosis of Campylobacter infection. However, due to the high nutritional requirements and special culture conditions of Campylobacter, the traditional isolation and culture method is time-consuming and laborious, and has low sensitivity, unable to meet the clinical needs. The gold standard for identifying Campylobacter at the species level is to first isolate and culture bacteria through enrichment and selective media, and then conduct biochemical experiments to distinguish. However, some Campylobacter species are inert to biochemical reactions, which may misdiagnose Campylobacter jejuni as Campylobacter coli, thus limiting the clinical application of biochemical methods. In recent years, molecular biology detection methods such as nucleic acid molecular hybridization technology and PCR have provided more reliable methods for the detection of Campylobacter. However, due to their complex operation and the need for special equipment, they still cannot meet the requirements of large-scale rapid detection.

[0006] Immunology-based detection includes antibody detection and antigen detection. There have been many previous studies on the production of antibodies in the population after Campylobacter infection. It is generally believed that Campylobacter infection can stimulate the body to produce specific antibodies 5 - 7 days after the onset of the disease. Usually, the anti-Campylobacter antibodies in serum can be maintained for a long time. IgG drops to the baseline level after 4 - 5 months, and IgM drops rapidly to the baseline level after 30 - 50 days. However, some studies have also found that there are no significant changes in the antibodies of patients with diarrhea caused by Campylobacter infection. Therefore, the serological antibody detection of Campylobacter is not currently used as the clinical diagnosis standard for Campylobacter infection.

[0007] The antigen structure of Campylobacter is relatively complex, with extensive antigenic diversity or differences. Currently, there is a lack of monoclonal antibodies that can recognize all major pathogenic Campylobacter species. The existing Campylobacter antigen detection reagents mainly detect Campylobacter jejuni and Campylobacter coli, with detection sensitivities of 10 4 CFU / mL and 10 5 CFU / mL respectively, which are prone to missed detections. Moreover, none of the existing kits can detect other pathogenic Campylobacter species such as Campylobacter fetus. Therefore, there is an urgent need in this field to prepare high-coverage monoclonal antibodies that can recognize all major pathogenic Campylobacter species, as well as Campylobacter infection detection products for the rapid and effective diagnosis of Campylobacter infection. Summary of the Invention

[0008] To this end, the object of the present invention is to provide a monoclonal antibody against Campylobacter prepared using a fused hybridoma cell line, and the monoclonal antibody obtained through experiments can recognize 5 main pathogenic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis), does not cross-react with other non-Campylobacter pathogenic bacteria, and has a detection sensitivity of 10 2 ~10 3 CFU / mL when used together with a polyclonal antibody, which is significantly higher than that of existing reagents, and can be used for the detection of Campylobacter in fecal samples or samples after fecal culture.

[0009] Therefore, one aspect of the present invention relates to a monoclonal antibody against Campylobacter or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3. Among them,

[0010] the amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO.2;

[0011] the amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO.3;

[0012] the amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO.4;

[0013] the amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO.6;

[0014] the amino acid sequence of the light chain CDR2 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO.7;

[0015] the amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.8 or an amino acid sequence having 1 conservative amino acid substitution compared with the sequence shown in SEQ ID NO.8.

[0016] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0017] The present invention also relates to the above-mentioned monoclonal antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab') 2 fragment, a single-chain antibody or a humanized antibody. Since these antibodies or antigen-binding fragments retain the variable regions of the light chain and the heavy chain, or only retain the heavy chain variable region, they can recognize and bind Campylobacter.

[0018] In addition, the present invention also relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned antibody or an antigen-binding fragment thereof, and an expression vector comprising the above-mentioned nucleic acid molecule, and the expression vector can express the above-mentioned antibody or an antigen-binding fragment thereof. At the same time, the present invention also relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned antibody or an antigen-binding fragment thereof. On the other hand, the present invention relates to a monoclonal antibody hybridoma cell line against Campylobacter, and the monoclonal antibody hybridoma cell line secretes the above-mentioned monoclonal antibody. Further, the present invention relates to a monoclonal antibody hybridoma cell line against Campylobacter, and the monoclonal antibody hybridoma cell line is a mouse hybridoma cell line CJ116, and the deposit number is CGMCC No.46326.

[0019] In yet another aspect, the present invention relates to the use of the above-mentioned monoclonal antibody or an antigen-binding fragment thereof in the preparation of a product for detecting Campylobacter. Further, the present invention relates to a kit for detecting Campylobacter, and the kit comprises the above-mentioned monoclonal antibody or an antigen-binding fragment thereof for recognizing and binding Campylobacter. Further, the present invention relates to a kit for detecting Campylobacter, which is a colloidal gold immunochromatography kit, and comprises the above-mentioned monoclonal antibody or an antigen-binding fragment thereof, and the monoclonal antibody or an antigen-binding fragment thereof is used as a capture antibody or a detection antibody.

[0020] Description of the deposit of biological materials

[0021] The monoclonal antibody hybridoma cell line of the present invention: mouse hybridoma cell line CJ116, was deposited with the China General Microbiological Culture Collection Center (CGMCC), and the registration number of the deposit center is CGMCC No.46326, and the deposit date is: February 13, 2025. The address of the China General Microbiological Culture Collection Center is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101. Description of the drawings

[0022] Figure 1 It is the distribution map of B-cell epitopes of the RacR protein of Campylobacter jejuni.

[0023] Figure 2 It is the alignment analysis comparison map of the amino acid sequences of the RacR proteins of 5 major pathogenic Campylobacter species.

[0024] Figure 3 It is the SDS-PAGE electrophoresis map showing the prokaryotic expression of the Campylobacter jejuni RacR antigen. The labels are as follows: M is the Marker; 1 is the whole-bacterium supernatant before loading; 2 is the Ni column flow-through; 3 is the antigen eluted with 25 mmol / L imidazole washing solution; 4 is the antigen eluted with 250 mmol / L imidazole washing solution.

[0025] Figure 4 It is the result judgment map showing the detection of Campylobacter by colloidal gold immunochromatography. The C line is the control line, the T line is the test line, and S is the sample loading well. Among them, positive shows a red band at the C line and red bands of varying intensities at the T line; negative shows a red band only at the C line and no band at the T line; invalid shows no band at the C line, regardless of whether there is a red band at the T line.

[0026] Figure 5 It is the sensitivity detection result map showing the detection of Campylobacter by colloidal gold immunochromatography. The C line is the control line, the T line is the test line, and S is the sample loading well. The detection sensitivity for Campylobacter jejuni and Campylobacter coli reaches 10 2 CFU / mL, and the detection sensitivity for Campylobacter fetus, Campylobacter upsaliensis, and Campylobacter lari is 10 3 CFU / mL.

[0027] Figure 6 It is the specificity detection result map showing the detection of Campylobacter by colloidal gold immunochromatography. The C line is the control line, the T line is the test line, and S is the sample loading well. Among them, 1 is pathogenic Escherichia coli, 2 is Salmonella enteritidis, 3 is Yersinia enterocolitica, 4 is Shigella flexneri, 5 is Shigella boydii, and 6 is Staphylococcus aureus.

[0028] Figure 7 Identification result map of the monoclonal antibody CJ116 subtype against the highly immunogenic consensus sequence of Campylobacter RacR. Detailed implementation methods

[0029] The object of the present invention is to provide a monoclonal antibody against Campylobacter prepared by using a fused hybridoma cell line, and this monoclonal antibody against Campylobacter can recognize 5 main pathogenic Campylobacter species. The specific preparation process is as follows: First, through bioinformatics analysis, the bacterial proteins that exist in all 5 main pathogenic Campylobacter species and have high sequence homology are determined, and then the consensus sequence of their highly immunogenic antigenic epitopes is analyzed, and it is used as an immunogen to immunize mice to prepare monoclonal antibodies. After screening, a mouse hybridoma cell line expressing monoclonal antibodies with high affinity and high specificity is obtained, named CJ116. Its detection sensitivity for Campylobacter jejuni and Campylobacter coli can reach 10 2 CFU / mL, and the detection sensitivity for Campylobacter fetus, Campylobacter upsaliensis and Campylobacter lari is 10 3 CFU / mL. It can recognize all 5 main pathogenic Campylobacter species and has no cross-reaction with other common intestinal pathogenic bacteria such as pathogenic Escherichia coli, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii and Staphylococcus aureus. It shows that the high-coverage monoclonal antibody CJ116 against Campylobacter of the present invention can be used for the detection of Campylobacter infection and has very high sensitivity and specificity. The inventor deposited this cell line with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 13, 2025, and the deposit number is CGMCC No. 46326.

[0030] Subsequently, the present inventors sequenced the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No. 46326 and analyzed the immunoglobulin domain sequences, and found that the amino acid sequence of its heavy chain variable region was: EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWFRQTPEKRLEWVAYISSGGDR MYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCGRIDNWGQGTTLTVS (SEQ ID NO.1), wherein the amino acid sequence of CDR1 was GFAFSSYD (SEQ ID NO.2), the amino acid sequence of CDR2 was ISSGGDRM (SEQ ID NO.3), and the amino acid sequence of CDR3 was GRIDN (SEQ ID NO.4). The amino acid sequence of the light chain variable region: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEEGPSWKS (SEQ ID NO.5), wherein the amino acid sequence of CDR1 was RASKSVSTSGYSYMH (SEQ ID NO.6), the amino acid sequence of CDR2 was LVSNLES (SEQ ID NO.7), and the amino acid sequence of CDR3 was QHIRELTR (SEQ ID NO.8).

[0031] The present inventors determined the affinity and specificity of the above monoclonal antibody for Campylobacter detection by enzyme-linked immunosorbent assay and colloidal gold immunochromatography. The high-coverage anti-Campylobacter monoclonal antibody CJ116 of the present invention can be used as a capture antibody or a detection antibody. The results showed that its detection sensitivity for Campylobacter jejuni and Campylobacter coli could reach 10 2 CFU / mL, and the detection sensitivity for Campylobacter fetus, Campylobacter upsaliensis and Campylobacter lari was 10 3 CFU / mL. It can recognize all 5 main pathogenic Campylobacter species and has no cross-reaction with other common intestinal pathogenic bacteria such as pathogenic Escherichia coli, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii and Staphylococcus aureus.

[0032] As is well known in the art, the CDR regions of the heavy chain and light chain of an antibody are important amino acid sequence regions for recognizing and binding the corresponding antigen. Moreover, generally, a single conservative amino acid substitution in the amino acid sequence of the above CDR regions does not change the protein structure. Therefore, a single conservative amino acid substitution within the above regions may still possess the property of binding the corresponding antigen. Thus, a monoclonal antibody or its antigen-binding fragment obtained by performing a single conservative amino acid substitution on 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 can still recognize and bind Campylobacter. In this invention patent application, a conservative amino acid substitution refers to the replacement of one amino acid in a protein with another chemically similar amino acid. For example, the mutual replacement among aromatic amino acids Phe, Trp, and Tyr, the mutual replacement among aliphatic amino acids Ala, Gly, Leu, Ile, and Val, the mutual replacement between polar amino acids Gln and Asn, the mutual replacement among basic amino acids Lys, Arg, and His, the mutual replacement between acidic amino acids Asp and Glu, and the mutual replacement between hydroxyl amino acids Ser and Thr, etc.

[0033] Those skilled in the art can also prepare various antibody fragments capable of binding Campylobacter, namely antigen-binding fragments, from the monoclonal antibodies of the present invention through the existing technologies in the art. For example, but not limited to, Fab, Fab', F(ab') 2 . The Fab fragment is the region in the antibody structure that can bind to the antigen and is composed of a complete light chain and the variable region VH and the constant region CH1 domain (Fd segment) of the heavy chain. There is a constant region and a variable region in both the light chain and the heavy chain, and a disulfide bond links the light and heavy chains. The antigen-binding fragment can be prepared as follows. For example, after the enzymatic digestion of papain, the antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, the antibody IgG is degraded into one F(ab') 2 fragment and one pFc' fragment, and the F(ab') 2 fragment is further reduced to form two Fab' fragments. Since the above antigen-binding fragments can still bind the corresponding antigen, they can be applied to the preparation of products for detecting Campylobacter.

[0034] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention through the existing technologies in this field. A single-chain antibody is an antibody formed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide linker of several amino acids. It has only one chain and is an artificially synthesized antibody. The single-chain antibody can also only contain the variable region of the heavy chain of the antibody. The length and amino acid composition of the short peptide linker are well known in this field, and the short peptide linker that can be used for the monoclonal antibody of the present invention can be determined through simple repeated experiments. The single-chain antibody can be expressed in, for example, Escherichia coli through genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the property of binding Campylobacter and can be applied to the detection of Campylobacter.

[0035] Those skilled in the art can design and synthesize nucleic acid molecules encoding the above-mentioned monoclonal antibody variable regions against Campylobacter based on the amino acid sequences, and can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors, which can express monoclonal antibodies against Campylobacter or their antigen-binding fragments. Those skilled in the art can also introduce the synthesized nucleic acid molecules or the constructed expression vectors into organisms such as cells, bacteria, yeast, etc. to obtain recombinants, and produce the antibodies or their antigen-binding fragments of the present invention through the expression of the above-mentioned recombinants. The antibodies or their antigen-binding fragments expressed in this way can bind and recognize Campylobacter. Therefore, the above-mentioned nucleic acid molecules, expression vectors, and recombinants are within the scope of protection of the claims of the present invention. And the above-mentioned technologies all belong to the technologies well known in this field, and those skilled in the art can carry them out without creative labor.

[0036] As described above, the antibodies or their antigen-binding fragments of the present invention can specifically recognize and bind Campylobacter, and thus can be used to prepare kits for detecting Campylobacter. The kits can be any kits that utilize the binding reaction between the antibodies or their antigen-binding fragments of the present invention and Campylobacter, such as, but not limited to, kits for colloidal gold immunochromatography, fluorescence immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunoblotting, immunohistochemistry methods. Among them, the high-coverage monoclonal antibody CJ116 against Campylobacter of the present invention can be used as a capture antibody or a detection antibody.

[0037] To illustrate the technical content, the achieved objectives, and the effects of the technical solutions in detail, the following will be described with specific examples.

[0038] Example 1: Screening of Campylobacter common antigens

[0039] Nine antigens with sequence conservation and applicable to Campylobacter detection were screened according to literature research. They are flagellar capping protein FliD, temperature-responsive regulatory protein RacR, adhesion-related proteins PebA and CadF, invasion-related proteins CiaB and FlaC, outer membrane protein OMP18, cytolethal distending toxin CdtA and CdtC. First, the NCBI reference sequences of the amino acid sequences of the nine detection antigens of five major pathogenic Campylobacter species, namely Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter upsaliensis, and Campylobacter lari, were separately queried from the GenBank database of NCBI (https: / / www.ncbi.nlm.nih.gov / ). The NCBI reference sequences are listed in Table 1, and their specific sequences are listed in Table 2 below. Then, using the "Align two or more sequences" module in Protein BLAST of the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), with the corresponding protein sequence of Campylobacter jejuni with the highest infection rate as the target sequence, the homology of the corresponding protein sequences of the other four Campylobacter species was analyzed and compared. The results are shown in Table 3. The sequence alignment analysis results show that only the RacR protein has high homology among the five major pathogenic Campylobacter species. Therefore, the RacR protein was selected as the detection target in this invention.

[0040] Table 1. NCBI reference sequences of proteins related to the detection of five major pathogenic Campylobacter species

[0041]

[0042]

[0043] Note: "-" indicates that the corresponding sequence was not found in NCBI

[0044] Table 2. Amino acid sequences of nine detection antigens of five major pathogenic Campylobacter species

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] Table 3. Amino acid sequence homology analysis with Campylobacter jejuni proteins

[0054] Protein Campylobacter coli Campylobacter fetus Campylobacter upsaliensis Campylobacter lari 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)

[0055] Note: "-" indicates that the corresponding sequence was not found in NCBI and sequence alignment could not be performed

[0056] Example 2: Analysis of highly immunogenic consensus sequences of Campylobacter common antigen RacR

[0057] Using BIOSUN software to analyze the B-cell epitope distribution of Campylobacter jejuni RacR protein. First, input its amino acid sequence, and then analyze the B-cell epitopes. The obtained epitope distribution map is shown in Figure 1 , and the corresponding epitope sequences are shown in Table 4

[0058] Table 4. B-cell epitope sequences and scores of Campylobacter jejuni RacR protein

[0059]

[0060]

[0061] Then, using DNAMAN 6.0 software to align and analyze the amino acid sequences of RacR proteins of 5 major pathogenic Campylobacter species, the results are as Figure 2 shown. The amino acid sequence homology of RacR proteins of 5 major pathogenic Campylobacter species is 87.09%. Combining the results of epitope analysis and sequence identity analysis, 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) are determined as highly immunogenic consensus sequences

[0062] Example 3: Preparation of synthetic peptides of RacR highly immunogenic consensus sequences

[0063] In order to detect all 5 major pathogenic Campylobacter species, the RacR highly immunogenic consensus sequence determined by screening was used as an immunogen to prepare monoclonal antibodies. First, epitope 6, epitope 10, and epitope 11 of the highly immunogenic consensus sequence were synthesized into a single synthetic peptide. To ensure epitope integrity, one additional amino acid was included before and after each epitope (since the amino acid following epitope 6 and the amino acid preceding epitope 10 are both Y, only one amino acid Y was added between epitope 6 and 10). At the same time, to facilitate conjugation, a cysteine (C) was added to the N-terminus of the synthetic peptide. Thus, the sequence of the highly immunogenic consensus sequence synthetic peptide is CK PYDPKEM Y SVSREQL VN LKDKDSK S (SEQ ID NO.52). The immunogenic synthetic peptide was conjugated to keyhole limpet hemocyanin (KLH) to enhance immunogenicity. To screen and determine the epitopes recognized by the monoclonal antibodies, synthetic peptides of epitope 6, epitope 10, and epitope 11 were synthesized respectively. To facilitate conjugation, a cysteine (C) was also added to the N-terminus of the synthetic peptide. Thus, the sequence of the synthetic peptide of epitope 6 is CK PYDPKEM Y (SEQ ID NO.53), the sequence of the synthetic peptide of epitope 10 is CY SVSREQL V (SEQ ID NO.54), and the sequence of the synthetic peptide of epitope 11 is CN LKDKDSK S (SEQ ID NO.55). The synthetic peptides for screening were conjugated to bovine serum albumin (BSA) to facilitate coating of the enzyme-linked immunosorbent assay (ELISA) plate for detection. Peptide synthesis and KLH or BSA conjugation were commissioned to Shanghai Dechi Biotechnology Co., Ltd. to complete.

[0064] Example 4: Preparation of Monoclonal Antibodies Against the RacR Highly Immunogenic Consensus Sequence

[0065] Using the synthetic peptide with the RacR highly immunogenic consensus sequence as the immunogen, 6-8-week-old female BALB / c mice were used. 100 μg / antigen per mouse was added with an equal amount of Freund's complete adjuvant, and after thorough emulsification with a stirrer, the mice were immunized by subcutaneous injection in the back and intraperitoneal injection. Three mice were immunized. The second immunization was carried out at an interval of 4 weeks, and the third immunization was carried out 8 weeks later. 50 μg / antigen per mouse was added with incomplete Freund's adjuvant, and after thorough emulsification with a stirrer, the mice were immunized by subcutaneous injection in the back and intraperitoneal injection. One week after the third immunization, blood was collected from the tail vein of the mice to detect the titer of the immune serum. The mouse with the highest titer was selected for booster immunization by intraperitoneal injection (50 μg / mouse). Three days later, spleen cells were taken for fusion. Resuscitate SP20 myeloma cells and culture them until they are in the logarithmic growth phase. Take the immunized BALB / c mice and prepare a spleen cell suspension. Take the above spleen cells and myeloma cells in a ratio of 9:1, mix them evenly in serum-free DMEM medium, centrifuge at 1500 rpm for 5 minutes, aspirate the supernatant, gently shake to disperse the cells, fuse them in a 37°C water bath, add 1 mL of pre-warmed 50% PEG to fuse the cells within 1 minute, gently shake while adding, and let it stand for 90 seconds after adding. Add serum-free DMEM medium to terminate the fusion, let it stand at 37°C for 10 min, centrifuge at 1500 rpm for 5 minutes, suspend the precipitate in HAT medium, and aliquot it into a 96-well cell plate containing feeder cells. Culture it in a cell incubator at 37°C and 5% CO 2 After culturing for 5 days, change the medium with HAT medium once. On the 10th day, change the medium with HAT medium. When the fused cells cover about 60% of the bottom of the well, take the cell culture supernatant and serially dilute it with PBS. The dilution factors are 400, 800, 1600, 3200, 6400, and 12800 respectively. The indirect ELISA method was used to screen for positive clones. The specific method is as follows: Dilute the synthetic peptides of epitope 6, epitope 10, and epitope 11 respectively with carbonate coating buffer at a concentration of 2.5 μg / ml, coat 150 μl per well, and incubate overnight at 4°C; Wash the plate 2 times with the washing solution; Add 200 μl / well of blocking solution and block at room temperature for 6 hours; Wash the plate 5 times with the washing solution. After adding 100 μl of sample dilution solution to each well, add 10 μl of different gradient dilution solutions of the cell culture supernatant respectively, incubate at room temperature for 30 min, and discard the liquid. Wash the plate 5 times, invert the washed enzyme-labeled plate on absorbent paper to pat dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate at room temperature for 30 min. Wash the plate 5 times. Add 50 μL of each of TMB chromogenic solution A and B to each well, and develop color at room temperature in the dark for 15 min. Add 50 μL of 2M H 2 SO 4 Termination solution 50 μL to terminate the reaction. Set the detection wavelength of the enzyme-labeled instrument at 450 nm, measure the OD value of each well, and read the value within 10 minutes after termination.

[0066] A total of 121 positive clones were obtained, among which 47 positive clones recognized epitope 6, 29 positive clones recognized epitope 10, and 45 positive clones recognized epitope 11. One clone with a titer reaching 1:12800 was selected from each group recognizing different epitopes for subsequent Campylobacter detection studies, namely clone CJ116 recognizing epitope 6, clone CJ39 recognizing epitope 10, and clone CJ107 recognizing epitope 11. The hybridoma cell lines were cultured in 1640 medium containing 10% fetal bovine serum. Each BALB / c male mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. After 10 days, the cells were collected, resuspended in 10 mL of normal saline, and the cell density was 1×10 7 cells / mL. 0.5 mL of the cell suspension was intraperitoneally injected into each mouse. Two weeks later, ascites were collected. The antibody was purified using the Thermo MelonGel Monoclonal IgG Purification Kit. The purified antibody was aliquoted and stored at -20°C.

[0067] Example 5: Preparation of Campylobacter jejuni RacR antigen

[0068] For in vitro expression, according to the amino acid sequence of Campylobacter jejuni RacR antigen (NCBI reference number ALK81821.1) published in the GenBank database of NCBI, the full-length nucleotide sequence of RacR for E. coli expression was deduced using the E. coli genetic code preference: 5'-ATGATTAACGTGCTGATGATCGAAGATGACCCGGATTTTGCGCAGTTGCTGAGCG AGTATTTAGCCCAATTCAATATTAAAATCACCAACTTTGAAAATCCAAAGTCTGCGCTGAACGTTGGCGTGCAGGGTTACGATTGCCTTATTCTGGACCTCACGCTGCCGGGCATCGATGGTCTGGAAGTCTGTCGTGAGATTCGCCAGAAATCCAATATCCCAATTATTATCTCTAGCGCACGTGGCGACTTGAGTGATAAAGTTGTAGGTCTGCAAATTGGCGCTGATGATTATTTACCGAAGCCATACGACCCGAAAGAAATGTATGCGCGCATCATGTCTCTGATTCGTCGCACTAAACGTGTGGAACATGCCAACAATGAGAACATCAACTCGGCATTCAAGATTGATGAACGCCGTCACGAAATCACCTACGAGGATAAAGTTCTTACTCTGACGCCAGCTGAATTTGAAATTCTGGAGTATCTGATTCAGCAGCATGGTTACAGCGTCAGCCGTGAACAACTGGTGTCTCGCTGCAAAAATTTGAAGGACAAAGATTCCAAATCGCTGGACGTTATCATTGGCCGTTTACGCGTGAAGATCGGCGATAGCAGTAAATCTCCGAAACACATTTTCTCGGTCCGTGGTATCGGCTATAAGCTGATTGGT-3'

[0069] (SEQ ID NO.56), and then commissioned Beijing Tsingke Biotechnology Co., Ltd. to synthesize the full-length nucleotide sequence, as well as the upstream primer 5'-GCGGATCCATGATTAACGTGCTGA-3' (SEQ ID NO.57) and the downstream primer 5'-GCGAATTCTTAACCAATCAGCTTATAG-3' (SEQ ID NO.58). Using the synthesized RacR nucleotide sequence as a template, the full-length gene was amplified under the following conditions: 95°C for 2 min; 95°C for 30 s, 58°C for 30 s, 72°C for 45 s, for a total of 30 cycles; then extended at 72°C for 5 min. Identification by 2% agarose gel electrophoresis showed that the relative molecular weight of the amplified fragment was approximately 700 bp. Using the BamHⅠ and EcoRⅠ restriction enzyme sites, the purified PCR product was double-digested and ligated into the pCold I plasmid (purchased from Takara), obtaining the pCold-RacR recombinant plasmid. The recombinant expression plasmid with correct sequencing was transformed into E. coli BL21 competent cells. Single colonies were picked and cultured overnight at 37°C with shaking in 5 mL of LB liquid medium containing sodium ampicillin. The next day, they were inoculated into 250 mL of fresh LB liquid medium and cultured until the logarithmic growth phase. The temperature was adjusted to 15°C, and 150 μl of 1 mol / L IPTG inducer was added after 30 min. Induction was carried out at 15°C for 12 - 14 h. The induced bacteria were collected by centrifugation, resuspended in 25 mmol / L Tris-HCl (pH 8.5), sonicated, and centrifuged at 20,000 g for 30 min at 4°C to collect the supernatant for Ni column purification. First, the Ni column was equilibrated with the equilibration buffer (25 mmol / L TE, 1% β-mercaptoethanol, 6 mol / L urea, pH 8.5). The supernatant was added to the Ni column. After the sample completely entered, the target protein was eluted and collected with washing solutions containing 25 mmol / L and 250 mmol / L imidazole respectively, and SDS-PAGE gel electrophoresis was performed. The RacR antigen was expressed solubly. Most of the protein was eluted with the 250 mmol / L imidazole washing solution, and the molecular weight was approximately 27.5 kDa. The results were as Figure 3 shown.

[0070] Example 6: Preparation of polyclonal antibody against Campylobacter jejuni RacR antigen

[0071] Healthy male New Zealand white rabbits were selected. 1.0 mg of the Campylobacter jejuni RacR antigen expressed in Escherichia coli in Example 5 was mixed with 1.0 ml of Freund's complete adjuvant. After thorough emulsification with a stirrer, it was subcutaneously injected at multiple points on both sides of the rabbit's spine, with 0.2 ml injected at each point. Four weeks later, 1.0 mg of the RacR antigen was mixed with 1.0 ml of Freund's incomplete adjuvant, and after thorough emulsification with a stirrer, a second immunization was performed at different points at the above-mentioned site. A third booster immunization was carried out 4 weeks later to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After the blood coagulated and the blood clot contracted, it was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored at -20 °C in a refrigerator for later use. The RacR antigen was used as the detection antigen to coat the ELISA plate, and the indirect ELISA method was also used to detect the titer of the purified rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody. The results showed that the titer of the prepared rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody reached 1:1024000.

[0072] Example 7: Detection of 5 main pathogenic Campylobacter species by anti-RacR highly immunogenic consensus sequence monoclonal antibodies

[0073] Using the rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody of the present invention prepared in Example 6 as the coating antibody, and three mouse anti-RacR highly immunogenic consensus sequence monoclonal antibodies, namely CJ116, CJ39 or CJ107, prepared in Example 4 as the detection antibodies, a sandwich ELISA method was established to detect 5 main pathogenic Campylobacter species to evaluate the detection ability of the monoclonal antibodies. First, 5 main pathogenic Campylobacter species, namely Campylobacter jejuni (ATCC 33560), Campylobacter coli (DSM 100395), Campylobacter fetus (DSM105764), Campylobacter upsaliensis (ATCC 43954) and Campylobacter lari (ATCC 35221), were serially diluted with PBS to 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1CFU / mL. Then, the double antibody sandwich method was used to detect 5 main pathogenic Campylobacter species. The specific steps were as follows: The ELISA plate was coated with rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody at a concentration of 2.0 μg / mL, 100 μL per well, and incubated overnight at 4°C; The plate was washed 2 times with the washing solution; 120 μL / well of the blocking solution was added and blocked at room temperature for 6 hours; The plate was washed 5 times with the washing solution, 50 μL of the serially diluted bacterial solution and 50 μL of the sample treatment solution were added to each well, and incubated at 37°C for 60 min, and the liquid was discarded. The plate was washed 5 times with the washing solution, 100 μL of horseradish peroxidase-labeled mouse anti-RacR highly immunogenic consensus sequence monoclonal antibody was added to each well, and incubated at 37°C for 60 min. The plate was washed 5 times, patted dry, 50 μL of TMB chromogenic solution A and 50 μL of solution B were added to each well, and developed at room temperature in the dark for 15 min. 2M H 2 SO 4 termination solution 50 μL / well was added to terminate the reaction. The detection wavelength of the microplate reader was 450 nm, the OD value of each well was measured, and the reading was taken within 10 minutes after termination. The critical value was 0.15. If the OD value ≥ 0.15, it was judged as positive; if the OD value < 0.15, it was judged as negative.

[0074] The results are shown in Table 5. When the CJ116 monoclonal antibody was used as the detection antibody, the detection sensitivity of the double antibody sandwich for detecting Campylobacter was the highest, reaching 10 2 CFU / mL for Campylobacter jejuni and Campylobacter coli, and the detection sensitivity for Campylobacter fetus, Campylobacter upsaliensis and Campylobacter lari was 10 3 CFU / mL; When the CJ39 monoclonal antibody was used as the detection antibody, the detection sensitivity for Campylobacter jejuni could reach 10 2 CFU / mL, the detection sensitivity for Campylobacter coli was 10 3 CFU / mL, the detection sensitivity for Campylobacter fetus and Campylobacter lari was 10 4 CFU / mL, and the detection sensitivity for Campylobacter upsaliensis was 10 5 CFU / mL; When the CJ107 monoclonal antibody was used as the detection antibody, it could reach 10 2 CFU / mL for Campylobacter jejuni and Campylobacter coli, and the detection sensitivity for Campylobacter fetus and Campylobacter lari was 10 3 CFU / mL, but Campylobacter upsaliensis could not be detected. Thus, it can be seen that the CJ116 monoclonal antibody can detect all 5 main pathogenic Campylobacter species and has the highest detection sensitivity.

[0075] Table 5. Detection of 5 main pathogenic Campylobacter species by monoclonal antibodies against RacR highly immunogenic consensus sequence

[0076]

[0077] Example 8: Detection of Campylobacter by colloidal gold immunochromatography

[0078] Using the colloidal gold immunochromatography technique, a colloidal gold immunochromatography detection method for Campylobacter was established using the mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody prepared by the present invention and the rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody prepared in Example 6. The rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody prepared by the present invention was coated at the T line on the nitrocellulose membrane, the colloidal gold-labeled CJ116 monoclonal antibody of the present invention was fixed on the gold conjugate pad, and the goat anti-mouse IgG polyclonal antibody (purchased from Zhuhai Bomei Biotechnology Co., Ltd.) was coated at the C line.

[0079] Similarly, 5 main pathogenic Campylobacter strains diluted at gradients of 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 CFU / mL were detected. In addition, pathogenic Escherichia coli, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii, and Staphylococcus aureus bacterial solutions with a concentration of 1×10 7 CFU / mL were taken for detection to evaluate the specificity of the colloidal gold immunochromatography method for detecting Campylobacter.

[0080] The specific operation is as follows: Place the test card flat on a dry surface, and vertically and slowly add 100 μL of the above samples to the sample addition hole of the test card respectively. The results are read within 5 - 15 minutes, and the reading is invalid after 15 minutes. When the test sample is dropped into the sample hole of the test card, the sample will move forward along the test card under capillary action. When it migrates to the gold conjugate pad, if the sample contains the main pathogenic Campylobacter, it will bind to the colloidal gold-labeled mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody to form an immune complex. The formed immune complex continues to migrate forward and is captured by the rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody (T line) fixed on the nitrocellulose membrane, forming an "mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody - main pathogenic Campylobacter antigen - rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody" immune complex, generating a red T line. Whether or not the sample contains Campylobacter antigen, the goat anti-mouse IgG coated in the C line area will bind to the excessive mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody to form a red band. The result judgment is as Figure 4As shown in the figure, where: the appearance of a red band at line C and the simultaneous appearance of red bands with different intensities at line T are judged as positive; only the appearance of a red band at line C and no band at line T are judged as negative; no band at line C, regardless of whether a red band appears at line T or not, is judged as invalid.

[0081] The experimental results are as Figure 5 and Figure 6 shown. The colloidal gold immunochromatographic detection method established using the mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody prepared by the present invention and the rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody prepared in Example 6 can also detect all 5 major pathogenic Campylobacter species; it has high detection sensitivity, with a detection sensitivity of 10 2 CFU / mL for Campylobacter jejuni and Campylobacter coli, and a detection sensitivity of 10 3 CFU / mL for Campylobacter fetus, Campylobacter upsaliensis, and Campylobacter lari ( Figure 5 ); it has high detection specificity and shows no non-specific cross-reaction with pathogenic Escherichia coli, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii, and Staphylococcus aureus at a concentration of 1×10 7 CFU / mL ( Figure 6 ).

[0082] Example 9: Subtype analysis of the monoclonal antibody CJ116 against the highly immunogenic consensus sequence of Campylobacter RacR

[0083] The heavy and light chain subtypes of the mouse antibody were identified using the Mouse Antibody Subtype Rapid Detection Card (product number: THJ-ISO-M8a-10 / 20) from Antaiji (Beijing) Biotechnology Co., Ltd. First, the antibody was diluted to 1 μg / mL with PBS, and then 100 μl of the diluted antibody was added to each well. After standing for 5 - 10 min, the results were observed and recorded. The results are as Figure 7 shown. The monoclonal antibody CJ116 against the highly immunogenic consensus sequence of Campylobacter RacR is of the mouse IgG1 subtype, and the antibody light chain is of the Igκ subtype.

[0084] Example 10: Determination of the variable region sequence of the monoclonal antibody CJ116 against the highly immunogenic consensus sequence of Campylobacter RacR

[0085] The mouse hybridoma cell line CJ116 was cultured, and total RNA of the hybridoma cells was extracted by the Trizol method. After reverse transcription into cDNA, PCR amplification was performed using the mouse monoclonal antibody Fab fragment primers synthesized by Beijing Tsingke Biotechnology Co., Ltd. (the primer design refers to pages 70-72 of Chapter 3, "Gene Engineering Antibody Technology" in the second edition of "Modern Immunology Experimental Techniques"). It was preheated at 95°C for 2 min, and 30 cycles were carried out at 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. Finally, it was extended at 72°C for 5 min, ligated to the pMD18-T vector (purchased from TaKaRa), transformed into Escherichia coli JM109, and positive clones were selected for sequencing. The determined sequence was compared and analyzed for the CDR region sequence of the mouse-derived monoclonal antibody in the IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) module on the NCBI website.

[0086] After sequence analysis, it was found that the amino acid sequence of the heavy chain variable region was 111 amino acids, and its sequence was as follows: EVKLVESGGGLVKPGGSLKLSCAAS GFAFSSYD MSWFRQTPEKRLEWVAY ISSGGDR M YYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYC GRID NWGQGTTLTVS (SEQ ID NO.1), where the underlined sequences are the CDR series. CDR1 is located at 26-33aa, and the amino acid sequence is GFAFSSYD (SEQ ID NO.2); CDR2 is located at 51-58aa, and 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 amino acid sequence of the light chain variable region was 109 amino acids, and its sequence was as follows: DIVLTQSPASLAVSLGQRATISY RASKSVSTSGYS YMH WNQQKPGQPPRLLIY LVSNLE S GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEEGPSWKS (SEQ ID NO.5), where the underlined sequences are the CDR series. CDR1 is located at 24-38aa, and the amino acid sequence is RASKSVSTSGYSYMH (SEQ ID NO.6); CDR2 is located at 54-60aa, and the amino acid sequence is LVSNLES (SEQ IDNO.7); CDR3 is located at 93-100aa, and the amino acid sequence is QHIRELTR (SEQ ID NO.8).

Claims

1. An anti-Campylobacter monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein: The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence having one conservative amino acid substitution compared to the sequence LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that It is secreted by the mouse hybridoma cell line CJ116 with the deposit number of CGMCC No.46326.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: 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, characterized in that It comprises a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression vector, characterized in that: It comprises the nucleic acid molecule according to claim 5.

7. A cell, bacterium or yeast recombinant, characterized in that: It comprises the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

8. A hybridoma cell line secreting anti-Campylobacter monoclonal antibodies, characterized in that: It is the mouse hybridoma cell line CJ116 with the deposit 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 in the preparation of a product for detecting Campylobacter.

10. A kit for detecting Campylobacter, characterized in that: 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 The invention is a colloidal gold immunochromatography kit, wherein the monoclonal antibody or the antigen-binding fragment thereof is used as a capture antibody or as a detection antibody.

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