溶藻弧菌外膜蛋白OmpK基因表达和间接ELISA检测方法的初步建立

Expression of OmpK gene of Vibrio alginolyticus and development of a detection method of indirect ELISA

溶藻弧菌是中国南部水产养殖业中弧菌病的最主要病原菌,数量居海洋类弧菌之首,其快速检测具有重要意义。以溶藻弧菌国内标准株ATCC17749基因组DNA为模板,PCR扩增出外膜蛋白OmpK基因,将其克隆入表达载体pET-28a中,获得重组质粒pET-28a-OmpK,限制性内切酶结合琼脂糖凝胶电泳分析和序列测定结果表明,该序列开放读码框正确且与已发表的OmpK结构编码序列完全一致。将重组质粒pET-28a-OmpK转化大肠杆菌BL21pLysE,高效表达重组蛋白,将其纯化后免疫小鼠所得的高免血清能外膜蛋白OmpK特异性结合,表明体外表达的重组蛋白OmpK具有良好的免疫原性和反应原性。利用该抗溶藻弧菌外膜蛋白OmpK的抗血清建立了间接ELISA检测方法,检测灵敏度为104CFU/mL,能特异性检测出溶藻弧菌,与其他菌株无交叉反应。

Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China,the number of which is the largest in marine Vibrio class.In the present study,the outer membrane protein K gene（OmpK）of V.alginolyticus strain ATCC17749 was amplified by PCR and cloned into high efficient expression vector pET28a.The results of sequencing and restriction enzyme analysis combined with agarose gel electrophoresis showed that the open reading frame sequence was correct and fully consistent with what had reported.The recombinant plasmid of pET-28a-OmpK was successfully constructed and transformed into E.coli BL21 pLys E.The fusion protein was expressed under the IPTG inducing condition,and the expression product was purified by an affinity chromatographic method.Mouse anti-OmpK poly-clonal antibodies（PAbs）were obtained via applying protein OmpK as antigen to immune mice.Western-blotting analysis proved the recombinant protein has a good reactive ability against OmpK positive serum.Then,an indirect Enzyme-Linked Immunosorbent Assay（ELISA）for the rapid diagnosis of V.alginolyticus has been developed using the PAbs.The lowest V.alginolyticus suspension was 104 CFU/mL.Cross reactions of antisera with other bacteria were detected,and all results were negative.The results indicated the suitability and simplicity of the test as a rapid,field diagnostic tool for V.alginolyticus and it can be used for the rapid detection of V.alginolyticus.Further investigation will be focused on the development of an indirect ELISA Kit for the detection of V.alginolyticus.