狐肺炎大肠杆菌HtrA蛋白原核表达及多抗血清制备

Prokaryotic expression and polyclonal antibody preparation of HtrA protein from Escherichia coli causing pneumonia in foxes

利用PCR方法从银狐大肠杆菌基因组中克隆出htra基因,测序无误后将其构建到pET32a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SDS-PAGE和Western blot方法验证重组蛋白得到很好表达;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大肠杆菌HtrA蛋白,为研究HtrA蛋白的作用机制奠定基础。

To investigate the roles of HtrA protein in bacterial adaption to stress conditions,htra gene was cloned from Escherichia coli strain isolated from silver fox,double digested,and ligated with expression vector pET32a. The recombinattion plasmid pET32a-htra was transformed into E. coli BL21 strain for protein expression,and identified by SDS-PAGE and Western blot analysis. The collected cells were lysed by ultraphonic,purified with Ni-agrose and thereby used for mouse vaccine. The obtained polyclonal serum specifically bound to endogenous HtrA protein in E. coli as demonstrated by Western blot assay, and may help to analyze the biological roles of HtrA in E. coli.