Development Of An Indirect ELISA Based On CjaA Protein For The Detection Of Avian Campylobacter Jejuni Antibodies And Preparation Of Its Specific Monoclonal Antibodies

Campylobacter jejuni is one of the most important food-borne pathogens associated with human diarrhea worldwide. These involve acute and self-limiting enteritis and may be complicated by severe systemic sequelae, such as GBS which is considered a sequela by infection of C. jejuni with specifically serotype. Such infections are frequently associated with the consumption of contaminated chicken meat, it is a great significance to investigate the prevalence of C. jejuni among the chickens. Although conventional investigations of pathogen epidemiology has a high specificity, the false negative results often occur due to the stringent culture condition needed by C. jejuni. The seroepidemiological survey, which is simple and sensitive, can serve as a auxiliary mean to detect C. jejuni infection rapidly to improve the epidemiological data. The objectives of this study were: (1) cloning and expressing the cjaA gene, and purifying recombinant protein His-CjaA,GST-CjaA; (2) preparation of monoclonal antibodies against CjaA, using GST-CjaA as immunogen, His-CjaA as the detecting antigen; (3) developing an indirect ELISA based on CjaA protein for the detection of C. jejuni antibodies in chicken serum.1. Prokaryotic expression of Campylobacter jejuni cjaA gene in recombinant E.coli and preparation of monoclonal antibodies against expression productCjaA protein is a membrane transporting protein, which acts as a protective antigen of C. jejuni. According to the sequences published in GenBank, primers of C. jejuni NCTC11168 were designed to amplify gene cjaA by PCR and inserted in pUCm-T vector. After sequencing confirmation, cjaA gene was then inserted into prokaryotic expression vector and recombinant bacteria BL21(pGEX-6p-1-cjaA),BL21(DE3)(pET-cjaA) were developed. After IPTG induction, the fusion proteins were expressed and purified, and their sizes were consistent with the prediction, their immunobiological character was verified by Western-blot assay.The 8-week-old BALB/c mice were immunized subcutaneously with the purified fusion protein GST-CjaA fully emulsified with equal volume of FCA. The immune dose was 100μg. Two weeks later, the purified protein GST-CjaA was administrated subcutaneously fully emulsified with equal volume of FIA. The interval between two immunizations was two weeks. After three immunizations, the purified protein GST-CjaA was administrated intravenously. Using the lymphocyte hybridoma technique, three positive hybridoma clones named 2B6,3C2,4F11 were obtained. The antibody titers of their ascitic fluid were 1×105,2×105,4×105, respectively. The immunoglobulin subclasses of the 3 McAb were IgG1. In Dot-ELISA,three McAb only reacted with bacteria expressing CjaA, they did not react with other bacteria without CjaA. All the results suggested that three McAbs were specific for CjaA. In Western-blot assay, they can react with fusion proteins His-CjaA and appear specific bands. The CjaA monoclonal antibodies could be applied in the study of biological characteristics of CjaA protein, the mechanism of C. jejuni infection and development of methods in the detection of C. jejuni.2. Establishment and application of an indirect ELISA method for the detection of avian Campylobacter jejuni antibodies based-on CjaA proteinThe BLAST results in NCBI show that C. jejuni cjaA gene is a highly conserved one. Primers were designed to amplify cjaA gene by PCR from 200 C. jejuni isolates and the positive rate of cjaA gene among them was 100%.The purified protein His-CjaA was used as coating antigen in indirect ELISA to detect specific antibodies. The optimal reaction conditions of ELISA were determined. The optimal coating buffer was TBS (0.05M pH7.6), and the optimal concentration of protein for coating microplate was 18.76μg per milliliter. 10% calf serum was added as the optimal blocking reagent and incubated for 2 hour at 37℃. The best serum dilution buffer was 5% calf serum, and the dilution of serum sample was 1:200. The working concentration of HRP-labeled goat-anti-chicken IgG antibodies was 1:10000, coloration time was 3min. The cut off value for ELISA was defined as absorbance values among negative serum samples(n=65) from field. If the OD490 value is high than 0.46, the test sample is positive. After artificial infection of C. jejuni, chicken sera were detected using the indirect ELISA method three weeks later. All forty test samples tested were positive, as well as traditional bacterial isolation method. In eight flocks, a total of 450 chicken serum samples were tested. Only one flock's positive rate was 51.7% (31/60), others were 100%. The established indirect ELISA method lays the foundation to carry out seroepidemiological studies of C. jejuni infections and provides a convenient means for further study of biological function and the pathogenesis mechanism of CjaA protein.