迟缓爱德华菌菌毛蛋白原核表达及多抗血清制备

Prokaryotic expression of PILI protein of Edwardsiella tarda and preparation of polyclonal antibody

为研究迟缓爱德华菌菌毛蛋白PILI的功能和免疫原性,本研究利用PCR方法从迟缓爱德华菌基因组中克隆出pili基因,并将其构建到pET-32a原核表达载体上,重组载体转化BL21大肠杆菌诱导重组蛋白表达,通过SDSPAGE和Western blot方法验证重组蛋白表达。用亲和层析方法纯化蛋白,纯化的蛋白免疫小鼠,所得的多抗血清能够特异性识别迟缓爱德华菌PILI蛋白,为研究PILI蛋白奠定基础。

To investigate the roles of PILl protein in bacterial adhesion host cells and immunogenic- ity. pill gene was cloned from Edwardsiella tarda strain isolated from farmed turbots,double di- gested,and ligated with expression vector pET-32a. The recombination plasmid pET-32a-pili was transformed into E. coli BL21 strain for protein expression, and identified by SDS-PAGE and Western blot analysis. The collected cells were lyzed by ultraphonic, purified with Ni agrose and thereby used for mouse vaccine. The obtained polyclonal serum specifically bound to bacterial PILI protein in E. tarda as demonstrated by Western blot assay,and may help to analyze the biological roles of PILL in E. tarda.