肝癌相关抗原SMP30重组基因的构建、表达及分子伴侣共表达系统的探索  

Recombination and Expression of Hepatocellular Carcinoma Associated Antigen SMP30 and Exploration of Co-expressed System with Molecular Chaperones

作　　者：黄朋[1] 范升龙[1] 凌敏[1] 贺菽嘉[1] 周素芳[1] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,南宁530021

出　　处：《生物技术通报》2012年第4期170-175,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30760233);广西自然科学基金资助项目(2011GXNSFA018261)

摘　　要：旨在通过应用基因工程的方法构建、表达和纯化肝癌相关抗原SMP30,研究共表达分子伴侣提高基因工程蛋白表达的可溶性及效率。PCR扩增SMP30 cDNA序列,用基因工程技术构建重组表达质粒,转化E.coliBL21(DE3)pLysS宿主菌。表达蛋白经Ni-NTA亲和柱纯化获得HIS-SMP30融合蛋白;分别将4种表达不同分子伴侣的质粒(pG-KJE8、pGro7、pKJE7、pTf16)转入E.coliBL21(DE3)中;然后再将重组质粒转入含有分子伴侣质粒的细胞中,进行分子伴侣与重组质粒的共表达,SDS-PAGE检测目的蛋白的表达量与可溶性分析。经优化表达条件后,目的蛋白以包涵体形式表达,目的蛋白占总蛋白的60%以上;纯化后纯度高达95%以上;诱导共表达后,目的蛋白在上清含量极少,不到总表达目的蛋白的10%。成功构建出高效表达的SMP30重组质粒;加入到诱导表达体系中的4种分子伴侣质粒不能有效的促进可溶性蛋白的表达,pTf16共表达系统能增加目的蛋白表达量。It was to obtain and purify hepatocellular carcinoma-associated antigen SMP-30 fusion protein with a 6×HIS tag,and explore the role of molecular chaperones promoting protein’s solubility.The cDNA of SMP30 was amplified and recombined in expressing plasmid（pET30a）.The optimal expression condition was explored,and HIS-SMP30 fusion protein was expressed in E.coli BL21（DE3）pLysS and purified by Ni-NTA resin.Four molecular chaperone plasmids（pG-KJE8,pGro7,pKJE7,pTf16）was transferred into E.coli BL21（DE3） respectively,then the recombined plasmid pET30a-SMP30 was transferred.The interest protein’s expression level and its solubility was assayed.Expression level of interest protein（HIS-SMP30）was as high as 60 % when the culture conditions were optimal,and inclusion body protein was up to 95% after purification.Molecular chaperones improved solubility protein expression was rare,but tig（a chaperones protein expressed by pTf16）improved the HIS-SMP30 protein’s expression level tremendously compared to the other molecular chaperones.The high expression level of SMP30 recombined plasmid was successfully constructed.Molecular chaperones could not promote solubility protein expressed efficiently,but the pTf16 co-expressed system could enhance the interest protein’s expression.

关 键 词：SMP30 基因工程 表达与纯化 分子伴侣质粒 共表达 

分 类 号：R735.7[医药卫生—肿瘤]