果蝇TCTP基因的原核表达与纯化

摘要

翻译控制肿瘤蛋白（translationally controlled tumor protein,TCTP）即在翻译水平受到抑制的肿瘤相关蛋白,是一个高度保守且和细胞生长、死亡等功能有关的蛋白。采用PCR技术从果蝇基因组DNA中扩增果蝇dTCTP基因,经酶切后插入到表达质粒pGEX-4T-2上构建原核表达载体,转化大肠杆菌,经菌落PCR和质粒酶切鉴定确认;表达载体经过IPTG诱导后、经超声破碎和亲和层析分离纯化出目的蛋白并通过SDS-PAGE进行鉴定。结果：成功构建果蝇dTCTP基因表达载体,并表达可溶的、有活性的dTCTP融合蛋白。%The translationally controlled tumor protein is a tumor-related protein which is regulated at the translational level.It is a highly conservative protein and involves in cell growth and death.Drosophila TCTP gene was amplified from the Drosophila genome in this experiment using PCR.After enzyme digestion,the gene was inserted into the pGEX-4T-2 vector and transformed into E.coli.The expression vector was identified using the colony PCR and enzyme digestion and then induced by IPTG.The expressed protein was separated,purified and identified using sonication,affinity chromatography and SDS-PAGE respectively.The results： the expression vector was successfully constructed and the soluble and active fusion proteins were obtained.