迟缓爱德华菌溶血活化蛋白EHA的原核表达及生物学活性

目的研究迟缓爱德华菌(Et)溶血活化基因(eha)原核表达蛋白EHA的生物学特性。方法构建重组载体pET28a+-eha,转入大肠埃希菌BL21,得到克隆子pEHA1。克隆子pEHA1超声破碎后离心,上清用SDS-PAGE电泳分析。在不同条件下,IPTG诱导pEHA1菌表达EHA蛋白,该蛋白经Ni-NTA亲和层析柱纯化后,检测其溶血活性及Vero细胞毒性。结果测序结果表明,pET28a+-eha重组质粒的eha序列完全正确。SDS-PAGE电泳显示表达出一条17kDa左右的条带。在30℃,IPTG浓度为0.5mmol/L的情况下,克隆子pEHA1被诱导6h产生的蛋白量最多。纯化的EHA蛋白无溶血活性及Vero细胞毒性。结论eha基因不是溶血素结构基因,可能是一个调控基因。

Prokaryotic expression and biologic characterization of Edwardsiella tarda hemolysin activator protein

To investigate the prokaryotic expression and biologic characterization of Edwardsiella tarda hemolysin activator(EHA)protein,the recombinant plasmid pET28a(+)-eha was constructed and transformed to E coli BL21 to obtain clone pEHA1. The clone pEHA1 was broken by supersound and centrifuged and then the supernatant was analyzed by SDA electrphoresis. IPTG at different concentrations was used to induce the clone pEHA1 to express EHA protein.The protein was then purified through Ni-NTA affinity chromatograph. The hemolytic activity of the purified EHA protein and its cytotoxici effect to Vero cells were Determined It was demonstrated that,as demonstrated by the results in sequencing,the eha gene in recombinant plasmid pET28(+)-eha was completely correct. A band of approximate 17 kDa of protein was found in SDS electrophoresis. Under 0.5 mmol/L concentration of IPTG clone pEHA1 was induced to express large amount of EHA protein. No hemolytic activity or the cytotoxicity effect to Vero cells of the purified protein could be demonstrated . From these observations,it is clear that the eha is not the structural gene of hemolysin,but it may serve as a regulatory gene to regulate the activity of hemolysin.