Monoclonal Antibodies Against Pathogenic Campylobacter and Their Applications

Abstract

The present invention discloses a monoclonal antibody CJ116 against pathogenic Campylobacter and its application. In the monoclonal antibody CJ116, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.5. This monoclonal antibody CJ116 has high affinity and high specificity, and the detection sensitivity for Campylobacter jejuni and Campylobacter coli can reach 10<supgt;2< / supgt> CFU / mL, and the detection sensitivity for Campylobacter fetus, Campylobacter upsaliensis and Campylobacter lari is 10<supgt;3< / supgt> CFU / mL, but there is 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.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a monoclonal antibody against pathogenic Campylobacter and an application thereof. Background Art

[0002] Infectious diarrhea is a common symptom caused by gastrointestinal infections caused by bacteria, viruses, or parasites. It is the second leading cause of morbidity and mortality worldwide. In developing countries, acute infectious diarrhea is a major cause of morbidity and mortality in children aged one to three years and the most common cause of malnutrition in children.

[0003] Campylobacter spp. are common foodborne zoonotic pathogens and are important global causes of infectious diarrhea. Species pathogenic to humans include Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter lari, and Campylobacter upsaliensis. Campylobacter jejuni and Campylobacter coli are the primary pathogens of human infection, accounting for 95% of all reported human Campylobacter infections, with Campylobacter jejuni accounting for over 90% of all cases. Campylobacteriosis is a self-limited gastroenteritis lasting approximately 5-7 days. Typical symptoms range from mild diarrhea to inflammatory diarrhea, including diarrhea, abdominal cramps, 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.

[0004] In recent years, the incidence of campylobacteriosis has been steadily increasing. Globally, Campylobacter jejuni causes approximately 166 million cases of diarrhea annually, accounting for approximately 5-14% of all diarrheal illnesses. Currently, in Europe and the United States, Campylobacter jejuni infection ranks first among pathogenic bacterial infections causing diarrhea, and in some countries, it ranks second only to Salmonella or Shigella. In my country, infants and young children are at high risk of Campylobacter enteritis, particularly in the developed eastern coastal areas during the summer and autumn months, when they are most susceptible to Campylobacter infection and diarrhea. Recent surveillance results in China have shown that the detection rate of Campylobacter jejuni in children with diarrhea ranges from 4.0% to 17.7%, and in adults from 3.85% to 9.61%. Furthermore, studies have shown that Campylobacter jejuni can be carried in healthy individuals, with a carriage rate ranging from 0.6% to 9.4%. Therefore, rapid and accurate detection of Campylobacter is crucial for preventing and controlling Campylobacter outbreaks, providing early treatment for infected patients, and preventing and controlling serious complications.

[0005] Currently, detection methods for Campylobacter mainly include isolation and culture, conventional biochemical methods, nucleic acid detection, immunoassays, and mass spectrometry. Isolation and culture of the pathogen is the gold standard for the clinical diagnosis of Campylobacter infection. However, due to Campylobacter's high nutritional requirements and specialized culture conditions, traditional isolation and culture methods are time-consuming and labor-intensive, with low sensitivity, and thus fall short of clinical needs. The gold standard for species-level identification of Campylobacter is to isolate and culture the bacteria using enrichment and selective culture media, followed by biochemical differentiation. However, some Campylobacter species are resistant to biochemical reactions, potentially misdiagnosing Campylobacter jejuni as Campylobacter coli, thus limiting the clinical application of biochemical methods. In recent years, molecular biological detection methods such as nucleic acid hybridization and PCR have provided more reliable methods for Campylobacter detection. However, due to their complex operation and the need for specialized equipment, they remain unsuitable for large-scale, rapid testing.

[0006] Immunological tests include antibody testing and antigen testing. There have been many studies on the production of antibodies after people are infected with Campylobacter. 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, anti-Campylobacter antibodies in the serum can last for a long time, with IgG dropping to baseline levels after 4-5 months and IgM dropping rapidly to baseline levels after 30-50 days. However, some studies have found that there is no significant change in antibodies in patients with diarrhea caused by Campylobacter infection. Therefore, serological antibody testing for Campylobacter is not currently used as a clinical diagnostic standard for Campylobacter infection.

[0007] Campylobacter antigen structure is relatively complex and has a wide range of antigenic diversity or differences. Currently, there is a lack of monoclonal antibodies that can recognize all major pathogenic Campylobacter species. 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, which can easily lead to missed detection, and existing test kits are unable to detect other pathogenic Campylobacter species such as Campylobacter fetus. Therefore, there is an urgent need in the field to develop high-coverage monoclonal antibodies that can recognize all major pathogenic Campylobacter species, as well as Campylobacter infection detection products, to enable rapid and effective diagnosis of Campylobacter infection. Summary of the Invention

[0008] To this end, the present invention aims to provide an anti-Campylobacter monoclonal antibody prepared using a fused hybridoma cell line. The monoclonal antibody obtained experimentally can recognize five major pathogenic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter lari, and Campylobacter upsaliensis), does not cross-react with other non-Campylobacter pathogens, and has a detection sensitivity of 10 for Campylobacter when used together with a polyclonal antibody. 2 ~10 3 CFU / mL is significantly higher than existing reagents and can be used to detect Campylobacter in stool samples or samples after stool culture.

[0009] Therefore, one aspect of the present invention relates to 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:

[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 one 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 one 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 one 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 one 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 one 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 one 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 antigen-binding fragment thereof, 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. These antibodies or antigen-binding fragments can recognize and bind to Campylobacter because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain.

[0018] In addition, the present invention also relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment, and an expression vector comprising the above-mentioned nucleic acid molecule, wherein the expression vector is capable of expressing the above-mentioned antibody or its antigen-binding fragment. 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 its antigen-binding fragment. On the other hand, the present invention relates to an anti-Campylobacter monoclonal antibody hybridoma cell line, wherein the monoclonal antibody hybridoma cell line secretes the above-mentioned monoclonal antibody. Furthermore, the present invention relates to an anti-Campylobacter monoclonal antibody hybridoma cell line, wherein the monoclonal antibody hybridoma cell line is the mouse hybridoma cell line CJ116, with a deposit number of CGMCC No. 46326.

[0019] In another aspect, the present invention relates to the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof in the preparation of a product for detecting Campylobacter . Furthermore, the present invention relates to a kit for detecting Campylobacter , comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof, for recognizing and binding Campylobacter . Furthermore, the present invention relates to a kit for detecting Campylobacter , which is a colloidal gold immunochromatography kit comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof serves as a capture antibody or a detection antibody.

[0020] Description of biological material deposit

[0021] The monoclonal antibody hybridoma cell line of the present invention, mouse hybridoma cell line CJ116, is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC), with the registration number of the CGMCC No. 46326 and the deposit date of February 13, 2025. The address of the General Microbiology Center of the China Culture Collection Administration of Microorganisms is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a map of the B cell epitope distribution of Campylobacter jejuni RacR protein.

[0023] Figure 2 This is a comparison chart of the amino acid sequence consistency analysis of RacR proteins of five major pathogenic Campylobacter species.

[0024] Figure 3 The figure shows the SDS-PAGE electrophoresis diagram of prokaryotic expression of Campylobacter jejuni RacR antigen, wherein the labels are: M is Marker; 1 is the supernatant of whole bacteria 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 This is a judgment chart showing the results of colloidal gold immunochromatography detection of Campylobacter. Line C is the control line, line T is the test line, and S is the sample well. A positive result is when a red band appears at line C and red bands of varying intensities appear at line T. A negative result is when a red band appears only at line C and no band appears at line T. An invalid result is when no band appears at line C, regardless of whether a red band appears at line T.

[0026] Figure 5 This is a graph showing the sensitivity of colloidal gold immunochromatography for detecting Campylobacter. Line C is the control line, line T is the test line, and line S is the sample well. The sensitivity of detecting Campylobacter jejuni and Campylobacter coli is 10 2 CFU / mL, the detection sensitivity for Campylobacter fetus, Campylobacter Uppsala and Campylobacter gull is 10 3 CFU / mL.

[0027] Figure 6 This is a graph showing the specific detection results of colloidal gold immunochromatography for detecting Campylobacter. Line C is the control line, line T is the test line, and S is the sample well. 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 Figure 1 shows the results of subtype identification of the highly immunogenic consensus sequence monoclonal antibody CJ116 against Campylobacter RacR. DETAILED DESCRIPTION

[0029] The present invention aims to provide an anti-Campylobacter monoclonal antibody prepared using a fused hybridoma cell line. This anti-Campylobacter monoclonal antibody can recognize five major pathogenic Campylobacter species. The specific preparation process is as follows: first, through bioinformatics analysis, bacterial proteins that are present in all five major pathogenic Campylobacter species and have high sequence homology are identified; then, common consensus sequences with highly immunogenic antigenic epitopes are analyzed and used as immunogens to immunize mice to prepare monoclonal antibodies. After screening, a mouse hybridoma cell line expressing high-affinity and high-specificity monoclonal antibodies was obtained, named CJ116, which has a detection sensitivity of 10 for Campylobacter jejuni and Campylobacter coli. 2 CFU / mL, the detection sensitivity for Campylobacter fetus, Campylobacter Uppsala and Campylobacter gull is 10 3 CFU / mL, it can recognize all five major pathogenic Campylobacter species, and has no cross-reaction with other common intestinal pathogens such as pathogenic Escherichia coli, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii, and Staphylococcus aureus, indicating that the high-coverage anti-Campylobacter monoclonal antibody CJ116 of the present invention can be used for the detection of Campylobacter infection with very high sensitivity and specificity. The inventors deposited this cell line with the General Microbiology Center of the China National Culture Collection Administration on February 13, 2025, with the deposit number CGMCC No. 46326.

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

[0031] The inventors used enzyme-linked immunosorbent assay and colloidal gold immunochromatography to determine the affinity and specificity of the monoclonal antibody for Campylobacter detection. The high-coverage anti-Campylobacter monoclonal antibody CJ116 of the present invention can be used as both a capture antibody and a detection antibody. The results showed that its detection sensitivity for Campylobacter jejuni and Campylobacter coli can reach 10 2 CFU / mL, the detection sensitivity for Campylobacter fetus, Campylobacter Uppsala and Campylobacter gull is 10 3 It can identify all five major pathogenic Campylobacter species and has no cross-reaction with other common intestinal pathogens such as pathogenic Escherichia coli, Salmonella Enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii and Staphylococcus aureus.

[0032] It is well known in the art that the heavy and light chain CDR regions of antibodies are important amino acid sequence regions for recognizing and binding to corresponding antigens. Furthermore, a single conservative amino acid substitution in the amino acid sequence of these CDR regions generally does not alter the protein structure. Therefore, a single conservative amino acid substitution in these regions may still confer binding properties to the corresponding antigen. Therefore, monoclonal antibodies or antigen-binding fragments thereof obtained by making a single conservative amino acid substitution in the 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 may still be able to recognize and bind to Campylobacter. In the present patent application, conservative amino acid substitution refers to the replacement of a certain amino acid in a protein by another chemically similar amino acid, such as the mutual replacement between aromatic amino acids Phe, Trp, and Tyr, the mutual replacement between aliphatic amino acids Ala, Gly, Leu, Ile, and Val, the mutual replacement between polar amino acids Gln and Asn, the mutual replacement between basic amino acids Lys, Arg, and His, the mutual replacement between acidic amino acids Asp and Glu, and the mutual replacement between hydroxy amino acids Ser and Thr, etc.

[0033] Those skilled in the art can also use existing techniques to prepare various antibody fragments capable of binding to Campylobacter, i.e., antigen-binding fragments, such as, but not limited to, Fab, Fab', and F(ab')2, from the monoclonal antibodies of the present invention. The Fab fragment is the region of the antibody structure capable of binding to an antigen. It consists of a complete light chain, the variable region VH of the heavy chain, and the constant region CH1 domain (Fd segment). Both the light and heavy chains have a constant region and a variable region, and the light and heavy chains are linked by disulfide bonds. Antigen-binding fragments can be prepared, for example, by enzymatically degrading IgG with papain into two Fab fragments and an Fc fragment. Under the action of pepsin, the IgG is degraded into an F(ab')2 fragment and a pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because these antigen-binding fragments can still bind to the corresponding antigen, they can be used to prepare 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 by using existing technologies in the art. A single-chain antibody is an antibody formed by connecting the heavy chain variable region and the light chain variable region of an antibody through a short peptide linker of several amino acids. It has only one chain and is an artificially synthesized antibody. A single-chain antibody may also contain only the heavy chain variable region of an antibody. 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 can be expressed in, for example, Escherichia coli by genetic engineering technology. The single-chain antibody of the present invention prepared in this way has the property of binding to Campylobacter and can be used in the detection of Campylobacter.

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

[0036] As described above, the antibodies or antigen-binding fragments thereof of the present invention can specifically recognize and bind to Campylobacter and can therefore be used to prepare a kit for detecting Campylobacter. The kit can be any kit that utilizes the binding reaction between the antibodies or antigen-binding fragments thereof of the present invention and Campylobacter, such as, but not limited to, kits for colloidal gold immunochromatography, fluorescent immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunoblotting, and immunohistochemistry. The high-coverage anti-Campylobacter monoclonal antibody CJ116 of the present invention can be used as both a capture antibody and a detection antibody.

[0037] In order to explain the technical content, achieved objectives and effects of the technical solution in detail, the following is an explanation in conjunction with specific embodiments.

[0038] Example 1: Screening of common Campylobacter antigens

[0039] Based on a literature review, nine antigens with conserved sequences that could be used for Campylobacter detection were screened. These antigens include the flagellar capping protein FliD, the temperature-responsive regulatory protein RacR, the adhesion-associated proteins PebA and CadF, the invasion-associated proteins CiaB and FlaC, the outer membrane protein OMP18, and the cytolethal distending toxins CdtA and CdtC. First, the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / ) was searched for five major pathogenic Campylobacter species: Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter upsaliensis, and Campylobacter lari. The NCBI Reference Sequences for the amino acid sequences of the nine detection antigens are listed in Table 1, and their specific sequences are listed in Table 2 below. Next, using the "Align two or more sequences" module in the NCBI Protein BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), we used the protein sequence of Campylobacter jejuni, the strain with the highest infection rate, as the target sequence to analyze and align the corresponding protein sequences of four other Campylobacter species. The results are shown in Table 3. Sequence alignment analysis revealed that only the RacR protein shared high homology among the five major pathogenic Campylobacter species, and therefore, RacR was selected as the detection target in this study.

[0040] Table 1. NCBI reference sequences of proteins related to 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 Uppsala Campylobacter seagulls 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) CdC 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 was not possible.

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

[0057] BIOSUN software was used to analyze the B cell epitope distribution of Campylobacter jejuni RacR protein. First, its amino acid sequence was input, and then the B cell epitopes were analyzed. The resulting 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, the amino acid sequences of RacR proteins of five major pathogenic Campylobacter species were compared and analyzed using DNAMAN6.0 software. Figure 2 As shown, the amino acid sequence homology of the RacR proteins of the five major pathogenic Campylobacter species was 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) were identified as highly immunogenic consensus sequences.

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

[0063] In order to detect all five major pathogenic Campylobacter species, the RacR highly immunogenic consensus sequence identified by screening was used as an immunogen to prepare monoclonal antibodies. First, the highly immunogenic consensus sequence epitopes 6, 10, and 11 were synthesized into a synthetic peptide. To ensure the integrity of the epitopes, one more amino acid was included before and after each epitope (because the amino acid after epitope 6 and the amino acid before epitope 10 were both Y, only one amino acid Y was added between epitopes 6 and 10). At the same time, for the convenience of coupling, a cysteine ​​(C) was added to the N-terminus of the synthetic peptide, that is, the highly immunogenic consensus sequence synthetic peptide sequence was CK. PYDPKEM Y SVSREQL VN LKDKDSK S (SEQ ID NO. 52). The immunogenic synthetic peptide was coupled to hemocyanin KLH to enhance immunogenicity. In order to screen and determine the epitopes recognized by the monoclonal antibodies, synthetic peptides for epitope 6, epitope 10, and epitope 11 were synthesized. To facilitate coupling, a cysteine ​​(C) was added to the N-terminus of the synthetic peptide, i.e., the sequence of the synthetic peptide for epitope 6 was CK. PYDPKEM Y (SEQ ID NO.53), the synthetic peptide sequence of epitope 10 is CY SVSREQL V (SEQ ID NO.54), the synthetic peptide sequence of epitope 11 is CN LKDKDSK S (SEQ ID NO. 55). The synthetic peptides used for screening were coupled to bovine serum albumin (BSA) to facilitate detection on an ELISA plate. Peptide synthesis and KLH or BSA coupling were performed by Shanghai Dechi Biotechnology Co., Ltd.

[0064] Example 4: Preparation of highly immunogenic anti-RacR consensus sequence monoclonal antibodies

[0065] A synthetic peptide with a highly immunogenic consensus sequence of RacR was used as the immunogen. 6-8 week old BALB / c female mice were immunized with 100 μg / mouse antigen plus an equal amount of Freund's complete adjuvant. After thorough emulsification with a blender, the mice were immunized subcutaneously and intraperitoneally. Three mice were immunized. A second immunization was performed 4 weeks apart, and a third immunization was performed 8 weeks later. 50 μg / mouse antigen was added to incomplete Freund's adjuvant, thoroughly emulsified with a blender, and then injected subcutaneously and intraperitoneally. One week after the third immunization, blood was collected from the tail vein of the mice to test the titer of the immune serum. The mice with the highest titer were selected for intraperitoneal booster immunization (50 μg / mouse). Three days later, spleen cells were harvested for fusion. SP20 myeloma cells were resuscitated and cultured until they were in the logarithmic growth phase. The spleens of the immunized BALB / c mice were removed and spleen cell suspensions were prepared. Spleen cells and myeloma cells were mixed at a 9:1 ratio in serum-free DMEM medium. The mixture was centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, and the cells were gently shaken to disperse. The cells were then fused in a 37°C water bath. Within 1 minute, 1 mL of prewarmed 50% PEG was added to the cells, gently shaking them. After addition, the cells were allowed to stand for 90 seconds. Serum-free DMEM medium was added to terminate fusion. The cells were allowed to stand at 37°C for 10 minutes and centrifuged at 1500 rpm for 5 minutes. The pellet was suspended in HAT medium and aliquoted into 96-well plates containing feeder cells. After 5 days of incubation at 37°C and 5% CO2, the medium was replaced with HAT medium. On the 10th day, the medium was replaced with HAT medium. When the fused cells covered approximately 60% of the well bottom, the cell culture supernatant was removed and serially diluted with PBS at dilution factors of 400, 800, 1600, 3200, 6400, and 12800. Positive clones were screened by indirect ELISA. The specific method is as follows: Synthetic peptides for epitope 6, epitope 10, and epitope 11 were diluted in carbonate coating buffer to a concentration of 2.5 μg / ml, and 150 μl was coated per well at 4°C overnight. The plate was washed twice with washing buffer, and then blocked with 200 μl / well of blocking buffer at room temperature for 6 hours. The plate was then washed five times with washing buffer. After adding 100 μl of sample dilution buffer to each well, 10 μl of serial dilutions of cell culture supernatant were added, and the cells were incubated at room temperature for 30 minutes before discarding the solution. The plate was washed five times, inverted onto absorbent paper, and patted dry. HRP-conjugated goat anti-mouse IgG antibody (100 μl / well) was added and incubated at room temperature for 30 minutes. The plate was washed five times. TMB color development solution A and B (50 μl each) were added to each well, and the color was developed at room temperature in the dark for 15 minutes. The reaction was terminated by adding 50 μl of 2 M H2SO4 stop solution to each well. Set the detection wavelength of the microplate reader to 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, of which 47 positive clones recognized epitope 6, 29 positive clones recognized epitope 10, and 45 positive clones recognized epitope 11. From each group recognizing different epitopes, a clone with a titer of 1:12800 was selected for subsequent Campylobacter detection research, namely clone CJ116 recognizing epitope 6, clone CJ39 recognizing epitope 10, and clone CJ107 recognizing epitope 11. Hybridoma cell lines were taken and 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 and resuspended in 10 mL of normal saline at a cell density of 1×10 7 Each mouse was intraperitoneally injected with 0.5 mL of the antibody. After 2 weeks, ascites was collected. Antibodies were purified using the Thermo Fisher Scientific MelonGel Monoclonal IgG Purification Kit and aliquoted and stored at -20°C.

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

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

[0069] (SEQ ID NO. 56). Beijing Qingke Biotechnology Co., Ltd. was then commissioned to synthesize the full-length nucleotide sequence, along with the upstream primer 5'-GCGGATCCATGATTAACGTGCTGA-3' (SEQ ID NO. 57) and the downstream primer 5'-GCGAATTCTTAACCAATCAGCTTATAG-3' (SEQ ID NO. 58). The synthesized RacR nucleotide sequence was used as a template to amplify the full-length gene. Amplification conditions included 95°C for 2 minutes, 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds, for a total of 30 cycles, followed by extension at 72°C for 5 minutes. 2% agarose gel electrophoresis confirmed the amplified fragment had a molecular weight of approximately 700 bp. The purified PCR product was double-digested using the BamHI and EcoRI restriction sites and ligated into the pCold I plasmid (purchased from Takara) to generate the pCold-RacR recombinant plasmid. The correctly sequenced recombinant expression plasmid was transformed into E. coli BL21 competent cells, and a single colony was picked and placed in 5 mL of LB liquid medium containing ampicillin sodium. The culture was shaken at 37°C overnight. The next day, it was inoculated into 250 mL of fresh LB liquid medium and cultured until the logarithmic growth phase. The temperature was adjusted to 15°C. After 30 minutes, 150 μl of 1 mol / L IPTG induction solution was added and the cells were induced at 15°C for 12-14 hours. The induced bacteria were collected by centrifugation, resuspended with 25mmol / L Tris-HCl (pH8.5), and ultrasonically disrupted. The supernatant was collected by high-speed centrifugation at 20000g for 30min at 4°C for Ni column purification. First, the Ni column was equilibrated with equilibration buffer (25mmol / L TE, 1% β-mercaptoethanol, 6mol / L urea, pH8.5). The supernatant was added to the Ni column. After the sample was completely entered, the target protein was eluted with washing solutions containing 25mmol / L and 250mmol / L imidazole, respectively. SDS-PAGE gel electrophoresis was performed. RacR antigen was expressed in a soluble form. The 250mmol / L imidazole washing solution eluted most of the protein with a molecular weight of approximately 27.5kDa. The results are as follows: Figure 3 shown.

[0070] Example 6: Preparation of rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibodies

[0071] Healthy male white rabbits were selected. 1.0 mg of the Campylobacter jejuni RacR antigen expressed in Escherichia coli as described in Example 5 was mixed with 1.0 ml of complete Freund's adjuvant and thoroughly emulsified using a blender. 0.2 ml was then injected subcutaneously at points along the rabbit's spine. Four weeks later, 1.0 mg of the RacR antigen was mixed with 1.0 ml of incomplete Freund's adjuvant and thoroughly emulsified using a blender. A second immunization was performed at different locations along the same spine. A third booster immunization was performed four weeks later to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After coagulation and clot retraction, the blood was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored in a -20°C refrigerator until further use. RacR antigen was used as the test antigen to coat an enzyme-linked immunosorbent assay (ELISA) plate. The titer of the purified rabbit anti-Campylobacter jejuni RacR antigen polyclonal antibody was determined using an indirect ELISA assay. 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 five major pathogenic Campylobacter species using highly immunogenic anti-RacR consensus sequence monoclonal antibodies

[0073] The rabbit anti-C. jejuni RacR antigen polyclonal antibody of the present invention prepared in Example 6 was used as the coating antibody, and the three mouse anti-RacR highly immunogenic consensus sequence monoclonal antibodies, CJ116, CJ39, or CJ107, prepared in Example 4, were used as the detection antibodies. A double antibody sandwich assay was established to detect five major pathogenic Campylobacter species, and the detection ability of the monoclonal antibodies was evaluated. First, the five major pathogenic Campylobacter species, Campylobacter jejuni ATCC 33560, Campylobacter coli DSM 100395, Campylobacter fetus DSM 105764, Campylobacter upsaliensis ATCC 43954, and Campylobacter lari ATCC 35221, were serially diluted to 10% with PBS. 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1CFU / mL. Five major pathogenic Campylobacter species were then tested using a double-antibody sandwich assay. The following steps were performed: 100 μL of rabbit anti-C. jejuni RacR antigen polyclonal antibody was coated into an ELISA plate at a concentration of 2.0 μg / mL, and each well was coated with 100 μL of the antibody. The plate was then incubated at 4°C overnight. The plate was washed twice with wash buffer and blocked with 120 μL / well of blocking buffer at room temperature for 6 hours. The plate was washed five times with wash buffer, and 50 μL of serially diluted bacterial solution and 50 μL of sample treatment buffer were added to each well. The plate was incubated at 37°C for 60 minutes, and the solution was discarded. The plate was washed five times with wash buffer, and 100 μL of horseradish peroxidase-labeled mouse anti-RacR highly immunogenic consensus sequence monoclonal antibody was added to each well. The plate was incubated at 37°C for 60 minutes. The plate was washed five times, patted dry, and 50 μL each of TMB colorimetric reagents A and B were added to each well. The plate was developed at room temperature in the dark for 15 minutes. The reaction was terminated by adding 50 μL / well of 2M H₂SO₄ stop solution. Measure the OD value of each well using a microplate reader at a wavelength of 450 nm and read the value within 10 minutes after termination. The cutoff value is 0.15; OD values ​​≥ 0.15 are considered positive, and OD values ​​< 0.15 are considered negative.

[0074] The results are shown in Table 5. When CJ116 monoclonal antibody was used as the detection antibody, the double antibody sandwich detection of Campylobacter had the highest sensitivity, which could reach 10 for Campylobacter jejuni and Campylobacter coli. 2 CFU / mL, the detection sensitivity for Campylobacter fetus, Campylobacter Uppsala and Campylobacter gull is 10 3 CFU / mL; when CJ39 monoclonal antibody is used as the detection antibody, the detection sensitivity of Campylobacter jejuni can reach 10 2 CFU / mL, and the detection sensitivity for Campylobacter coli is 10 3 CFU / mL, and the detection sensitivity for Campylobacter fetus and Campylobacter gull is 10 4 CFU / mL, the sensitivity of detection for Campylobacter Uppsala is 10 5 CFU / mL; when CJ107 monoclonal antibody is used as the detection antibody, the detection rate for Campylobacter jejuni and Campylobacter coli can reach 10 2 CFU / mL, the detection sensitivity for Campylobacter fetus and Campylobacter gull is 10 3 CFU / mL, but could not detect Campylobacter Uppsala. This shows that the CJ116 monoclonal antibody can detect all five major pathogenic Campylobacter species with the highest detection sensitivity.

[0075] Table 5. Detection of five major pathogenic Campylobacter species by highly immunogenic anti-RacR consensus sequence monoclonal antibodies

[0076]

[0077] Example 8: Colloidal gold immunochromatographic detection of Campylobacter

[0078] A colloidal gold immunochromatographic assay for detecting Campylobacter was established using colloidal gold immunochromatographic technology, using the mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody prepared in 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 in the present invention was coated at the T line on a nitrocellulose membrane. The colloidal gold-labeled CJ116 monoclonal antibody of the present invention was immobilized on a gold label pad. The C line was coated with a goat anti-mouse IgG polyclonal antibody (purchased from Zhuhai Bomei Biotechnology Co., Ltd.).

[0079] The same test was performed with a serial dilution 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 of 5 major pathogenic Campylobacter species. In addition, the concentration was 1×10 7 The specificity of colloidal gold immunochromatographic assay for detecting Campylobacter was evaluated by testing the bacterial liquid of pathogenic Escherichia coli, Salmonella Enteritidis, Yersinia enterocolitica, Shigella flexneri, Shigella boydii and Staphylococcus aureus with a CFU / mL level.

[0080] The specific procedure is as follows: Place the test card flat on a dry surface and slowly drip 100 μL of the sample into each well vertically. Read the results after 5 to 15 minutes; read the results after 15 minutes. When the sample is added to the sample well of the test card, it migrates along the card due to capillary action. Upon migration to the gold pad, if the sample contains the major pathogenic Campylobacter, it binds to the colloidal gold-labeled mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody to form an immune complex. This immune complex continues to migrate forward and is captured by the rabbit anti-C. jejuni RacR antigen polyclonal antibody (T line) immobilized on the nitrocellulose membrane, forming an immune complex of "mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody-major pathogenic Campylobacter antigen-rabbit anti-C. jejuni RacR antigen polyclonal antibody," which produces the red T line. Regardless of whether the sample contains Campylobacter antigen, the goat anti-mouse IgG coated in the C-line area will bind to the excess mouse anti-RacR highly immunogenic consensus sequence CJ116 monoclonal antibody to form a red band. Figure 4 As shown, among them: the appearance of a red stripe at the C line and red stripes of varying intensities at the T line are judged as positive; the appearance of a red stripe only at the C line and no stripe at the T line is judged as negative; the absence of a stripe at the C line, regardless of whether a red stripe appears at the T line, is judged as invalid.

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

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

[0083] The mouse antibody subtype rapid detection card (catalog number: THJ-ISO-M8a-10 / 20) from Antaiji (Beijing) Biotechnology Co., Ltd. was used to identify the heavy chain and light chain subtypes of mouse antibodies. 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 minutes, the results were observed and recorded. Figure 7 As shown, the highly immunogenic consensus sequence monoclonal antibody CJ116 against Campylobacter RacR is of mouse IgG1 subtype, and the antibody light chain is of Igκ subtype.

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

[0085] Mouse hybridoma cell line CJ116 was cultured, and total RNA from hybridoma cells was extracted using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primers for the Fab region of a mouse monoclonal antibody synthesized by Beijing Qingke Biotechnology Co., Ltd. (primer design was based on pages 70-72 of Chapter 3, "Genetic Engineering Antibody Technology," in the second edition of Modern Immunology Experimental Techniques). The amplification was performed by preheating at 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The amplified fragments were ligated into the pMD18-T vector (purchased from TaKaRa) and transformed into Escherichia coli JM109. Positive clones were selected for sequencing. The determined sequences were compared with the mouse monoclonal antibody CDR region sequences using the IgBLAST module on the NCBI website (https: / / www.ncbi.nlm.nih.gov / igblast / ).

[0086] After sequence analysis, it was found that the amino acid sequence of the heavy chain variable region is 111 amino acids, and its sequence is as follows: EVKLVESGGGLVKPGGSLKLSCAAS GFAFSSYD MSWFRQTPEKRLEWVAY ISSGGDR M YYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYC GRID NWGQGTTLTVS (SEQ ID NO. 1), where the underlined sequence is the CDR series, CDR1 is located at 26-33aa, with the amino acid sequence of GFAFSSYD (SEQ ID NO. 2); CDR2 is located at 51-58aa, with the amino acid sequence of ISSGGDRM (SEQ ID NO. 3); CDR3 is located at 97-101aa, with the amino acid sequence of GRIDN (SEQ ID NO. 4). The amino acid sequence of the light chain variable region is 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISY RASKSVSTSGYS YMH WNQQKPGQPPRLLIY LVSNLE S GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEEGPSWKS (SEQ ID NO.5), in which the underlined sequence is 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 ID NO.7); CDR3 is located at 93-100aa, and the amino acid sequence is QHIRELTR (SEQ ID NO.8).

Claims

1. 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. It is characterized in that the amino acid sequence of the heavy-chain CDR1 is 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; the amino acid sequence of the heavy-chain CDR3 is 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; the amino acid sequence of the light-chain CDR2 is the sequence shown in SEQ ID NO.7; the amino acid sequence of the light-chain CDR3 is the sequence shown in SEQ ID NO.

8.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, 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.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by the mouse hybridoma cell line CJ116 with the preservation 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, an 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 its antigen-binding fragment 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 bacterial 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 a monoclonal antibody against Campylobacter, characterized in that, It is the mouse hybridoma cell line CJ116 with the preservation number of CGMCC No.46326.

9. Use of the monoclonal antibody against Campylobacter or an 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, It comprises the monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4.

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

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