论文部分内容阅读
目的构建重组原核表达载体pGEX-5X-1-IKKγ,诱导GST-IKKγ融合蛋白的表达并以GST-pulldown方法得到纯化的融合蛋白。方法应用PCR技术,以M6P8载体为模板扩增得到IKKγ全长序列,并亚克隆至带有GST标签的pGEX-5X-1载体中。经酶切、测序鉴定后,重组载体转化至原核细胞中并进行诱导表达,以SDS-PAGE凝胶电泳染色鉴定融合蛋白的表达。融合蛋白经GSTbeads沉降后,Western blot分析、鉴定融合蛋白的表达及纯化情况。结果将人IKKγ亚克隆至原核表达载体pGEX-5X-1,酶切、测序鉴定无误。经诱导得到高表达的融合蛋白,经SDS-PAGE电泳染色后可见高表达条带。Westernblot鉴定经GSTbeads沉降后的蛋白,在74kD鉴定出融合蛋白的特异性条带。结论成功构建了原核表达载体pGEX-5X-1-,并在原核细胞内表达,融合蛋白可以经GSTbeads沉降用于GST-pulldown实验。
Objective To construct the recombinant prokaryotic expression vector pGEX-5X-1-IKKγ and induce the expression of GST-IKKγ fusion protein and obtain the purified fusion protein by GST-pulldown method. Methods The full-length IKKγ sequence was amplified by PCR using M6P8 vector and subcloned into pGEX-5X-1 vector with GST tag. After digestion and sequencing, the recombinant vector was transformed into prokaryotic cells and induced to express, and the fusion protein was identified by SDS-PAGE gel electrophoresis. The fusion protein was precipitated with GSTbeads and analyzed by Western blot to identify the expression and purification of the fusion protein. Results Human IKKγ was subcloned into the prokaryotic expression vector pGEX-5X-1, digested and sequenced. High expression of the fusion protein was induced by SDS-PAGE electrophoresis showed high expression bands. Western blot analysis of proteins precipitated by GSTbeads identified a specific band of fusion protein at 74 kD. Conclusion The prokaryotic expression vector pGEX-5X-1- was successfully constructed and expressed in prokaryotic cells. The fusion protein could be precipitated by GSTbeads for GST-pulldown experiments.