人甲胎蛋白AFP在大肠杆菌中表达和鉴定

Expression and Identification of Human Alpha Fetoprotein in E. coli

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21(DE3)中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21(DE3)中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。

In this study, through the application of genetic engineering techniques, the prokaryotic expression vector pET22b-AFP of full-length human alpha-fetoprotein AFP gene was recombined, constructed, encoded, and then transformed into E. coli host strain BL21 （DE3） to induce the expression of the target protein AFP. AFP coding sequence was amplified by polymerase chain reaction （PCR）, and then cloned into the prokaryotic expression vector pET-22b containing His-tag. The constructed vector, verified by restriction endonuclease digestion and DNA sequencing, was then transformed into E. coli host strain BL21 （DE3） for expression by using IPTG-inducible promoters. Different concentrations of IPTG and different temperatures were optimized to induce the expression of target protein. Expression products were identified by SDS-PAGE electrophoresis and Western Blot. The results showed that AFP gene fragments were obtained by PCR amplification. By double enzyme digestion and gene sequencing methods, it was confirmed that the recombinant plasmid was constructed successfully. Expression product was revealed in the vicinity of the 66 kDa band by using SDS-PAGE and Western Blot., which was consistent with the expected value indicating that the recombinant AFP protein succeeded in expressing.