Preparation and characterization of monoclonal antibodies specific for flaA protein of Campylobacter jejuni
空肠弯曲菌flaA单克隆抗体的制备与鉴定

Abstract: Objective] We xpressed and purified Campylobacter jejuni flagellin FlaA protein to develop monoclonal antibodies (mAbs) against this protein. Methods] The C. jejuni flaA gene was amplified and inserted into the expression plasmids, pET30a (+) and pGEX-6p-1. The purified rHis-FlaA protein was used as an immunogen in 8-week-old BALB/c mice, and injected subcutaneously. The purified rGST-FlaA protein used as a detecting antigen for screening mAbs against FlaA was prepared by using a denaturation and renaturation technique. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays. Results] The recombinant expression plasmids, pET30a(+)-flaA and pGEX-6p-1-flaA were obtained. The sizes of the recombinant proteins, rHis-FlaA and rGST-FlaA, were consistent with their predicted size. Specific reaction was found between flaA positive serum and expressed protein by Western-blot assay, confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines, designated 2D12, 5A12 and 6A9, secreting mAbs against FlaA were obtained. Their immunoglobulin subclasses were IgG2a, IgG1 and IgG1, respectively. The ELISA titers of the ascites fluid were 1:102 400, 1:102 400 and 1:51 200, respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-FlaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with FlaA and not the tags for the expression vectors. Conclusion] The successful preparation of three mAbs specific for the FlaA protein lays the foundation for further study regarding the biological characteristics of FlaA and the pathogenesis of C. jejuni.