人IKKγ基因重组载体的构建及在原核体内的表达纯化

Construction and expression of recombinant vector of hIKKγ gene in prokaryotic cells

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 expression plasmid of pGEX-SX-I-IKKγ and study its fusion protein expression in prokaryotic cells. Methods The IKK coding sequence was amplified by polymerase chain reaction and subcloned into pGEX-5X-I vector. After identification by enzymes digesting and sequencing, the vector was induced to express the GST-IKKγ protein. BL21 cell lysate was analyzed by SDS-PAGE gel electrophoresis and the fusion protein purified by GST-pull down was identified by Western blot assay. Results The pGEX-SX-l-IKKγ vector was constructed successfully, the segments digested by enzymes and sequencing were coincident with expected. The expression of fusion protein induced by IPTG was observed after SDS-PAGE gel electrophoresis, the fusion protein showed a specificity band at 74kD detected by western-blot after purified by GST-pull down. Conclusion The human IKKγ, gene was subcloned into pGEX-SX-1 successfully and expressed in prokaryotic cells, the GST-fused protein could be prepared for GST-pull down analysis.