摘要
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 induction expression of enteropathogenic E.coli （EPEC） EspB gene in vitro, and obtain purified EspB protein. Methods The recombinant plasmid pET-28a-EspB was constructed in vitro, then transformed into the BL21 competent bacterium being cultured. The recombinant plasmid DNA was extracted and subjected to agarose gel electrophoresis, then the DNA sequencing was carded out. The recombinant plasmid pET-28a-EspB expression was induced by isopropyl-13-D-galactoside （IPTG）, and analyzed by SDS-polyacrylamide gel electrophoresis （SDS-PAGE） and protein blotting of pET-28a-EspB were purified and validated again by SDS-PAGE assay （Western blot）. The expression products and Western blot. Results The restriction enzyme digestion and agarose gel eleclrophoresis results showed two bright bands and the sizes matched the theoretical design. It verified that thepET-28a-EspB recombinant plasmid was successfully constructed. After the pET-28a-EspB recombinant plasmid induced by IPTG, the proteins were obviously detected in the culture broth, precipitation and supematant at various time points of induction expression. The single stable protein product, EspB protein, was obtained after the expressed protein of pET-28a-EspB was purified. Conclusion The EPEC EspB gene can be successfully induced to express EspB protein stably and efficiently in vitro.
出处
《深圳中西医结合杂志》
2013年第4期193-196,共4页
Shenzhen Journal of Integrated Traditional Chinese and Western Medicine
基金
国家自然科学基金资助项目(81170370)
深圳市科技局资助项目(201203211)
深圳市南山区科技局重点课题资助项目(2012001)
