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目的:研究肠致病性大肠埃希菌(enteropathogenic E.coli,EPEC)EspB基因的体外诱导表达情况,获得纯化的EspB蛋白。方法:体外构建重组质粒pET-28a-EspB,转化BL21感受态细菌培养,提取重组质粒并行琼脂糖凝胶电泳和测序鉴定。重组质粒pET-28a-EspB经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹试验(Western blot)分析。pET-28a-EspB表达产物再次经纯化,获得的纯化蛋白进一步经SDS-PAGE和蛋白质Western blotting分析验证。结果:经双酶切和测序验证pET-28a-EspB重组质粒构建成功。pET-28a-EspB重组质粒经IPTG诱导后,在诱导表达不同时间点,培养菌液、沉淀和上清中均有良好的蛋白表达。经SDS-PAGE和蛋白质Western blotting分析验证我们获得了纯化的EspB蛋白。结论:体外可成功诱导EPECEspB蛋白稳定高效表达并获得纯化EspB蛋白产物。
Objective: To study the induced expression of EspB gene of enteropathogenic E. coli (EPEC) in vitro and to obtain purified EspB protein. Methods: The recombinant plasmid pET-28a-EspB was constructed in vitro and transformed into BL21 competent cells. The recombinant plasmids were extracted and sequenced by agarose gel electrophoresis and sequencing. The recombinant plasmid pET-28a-EspB was induced by IPTG and analyzed by SDS-PAGE and Western blot . The pET-28a-EspB expression product was purified again and the purified protein was further confirmed by SDS-PAGE and Western blotting analysis. Results: The recombinant plasmid pET-28a-EspB was successfully constructed by double enzyme digestion and sequencing. After induced by IPTG, pET-28a-EspB recombinant plasmid showed good protein expression in culture broth, sediment and supernatant at different time points. SDS-PAGE and Western blotting analysis confirmed that we obtained purified EspB protein. Conclusion: EPECEspB protein can be successfully induced in vitro and purified, and the purified EspB protein product can be obtained.