空肠弯曲菌表面蛋白特异性多表位抗原的设计、表达与鉴定

Design,Expression and Identification of Specific Multi-Epitope Antigen of Campylobacter Jejuni Surface Proteins

构建空肠弯曲菌表面蛋白特异性多表位抗原原核表达载体,并在大肠杆菌中诱导表达,免疫动物后检测其抗血清与空肠弯曲菌菌体的反应性。从空肠弯曲菌的6个表面蛋白中分析到特异性抗原表位,人工合成其串联基因片段,将其插入原核表达载体,获得重组质粒pET-32a(+)-CJMEA-A,经IPTG诱导后表达出25k的目标蛋白,纯化后免疫新西兰大白兔,Western blot和间接ELISA检测重组蛋白抗原性和抗血清反应性。结果表明目标蛋白具有良好的反应原和免疫原性,抗血清能与菌体发生反应。其抗体可用于C.jejuni免疫磁珠和免疫胶体金等检测方法的建立。

To construct and to express the gene of specific multi-epitope antigen from Campylobacterjejuni and to detect the reactivity of its antibody to this bacteria. Firstly ,some specific antigenic determinants were obtained after 6 surface proteins of Campylobacter jejurri were analyzed. Secondly these epitopes were linked in series and its gene was synthesized. Thirdly, the recombinant plasmid pET-32a（ ＋ ）-CJMEA-A was obtained when the gene was inserted into pET-32a（ ＋ ） vector. Then, after being induced by IPTG ,the 25ku of fusion protein was expressed, purified and used for the immunization of rabbits. Finally,SDS-PAGE and indirect ELISA were used to detect the antigenicity of the protein and the reactivity of the antiserum. The results showed that this protein was expressed successfully;it possesses good immunogenieity and reactogenicity; its antiserum can react to thalli of C. jejuni . A conclusion can be made that the antibody of this protein could be used to detect C. jejuni by immunomagnetic beads or immune colloidal gold techtuque.