SERPINA3K原核表达系统的构建及其表达  

Construction and expression of prokaryotic expression vector of SERPINA3K

作　　者：郭妍伶[1] 成姝婷[1] 李世平[1] 后望[1] 杨淑红[1] 江舟[1] 汪宇辉[1] 肖静[1] 郭慧玲[1] 刘延友[1] 王正荣[1] 

机构地区：[1]四川大学华西基础医学与法医学院,时间生物学卫生部重点实验室,四川成都610041

出　　处：《四川生理科学杂志》2013年第4期145-148,共4页Sichuan Journal of Physiological Sciences

基　　金：国家自然科学资金资助(编号:41074131);四川大学青年科学基金(编号:2013SCU1107)

摘　　要：目的:通过构建丝氨酸蛋白酶抑制因子SERPINA3K的原核表达载体,并将其转化到Rosetta-gami2感受态细胞中,表达重组SERPINA3K,从而为对其功能的深入研究奠定了基础。方法:以小鼠MS-1细胞cDNA为模板通过PCR的方法扩增Serpina3k基因的表达全片段,再将其插入到原核表达载体pET-41a中,酶切并测序鉴定重组体。将构建好的重组质粒转化大肠杆菌Rosetta-gami2感受态细胞,表达产物用Western blot鉴定。结果:原核表达载体pET-41a-Serpina3k成功构建,可在大肠杆菌Rosetta-gami2中高效表达,得到重组蛋白SERPINA3K,经Western blot鉴定正确。结论:成功构建SERPINA3K原核表达载体且获得表达,为研究SERPINA3K生物学活性及产品开发提供了实验基础。Objective： To construct those which the prokaryotic expression vector of Serpina3k is transfected into the Rosetta- gami2 competent cells to secret recombinant SERPINA3K, the access was to deep seek the protein function. Methods： As the template gene, all length sequence of mouse MS-1 cell cDNA was amplified by PCR. Serpina3k were cloned into the prokaryotic expression vector pET-41a through restriction enzyme digestion and sequencing recombinant. The constructed recombinant plasmid was transfected into the E. coli Rosetta- gami2 competent cells, in which product of cell expression was tested by Western blotting. Results： The experimental results illuminated that the prokaryotic expression vector pET-41a- Serpina 3k succeeded in transfecting in- to the E. coli Rosetta- gami2. Mean while, the recombinant protein SERPINA3K was correctly identified with Western blotting, largely in line with expectations. Conclusion： The establishment in prokaryotic expression vector of Serpina3k made an fundamental contribution to seek the biology activity and product development on SERPINA3K.

关 键 词：丝氨酸蛋白酶抑制因子 原核表达系统 SERPINA3K重组蛋白 

分 类 号：Q78[生物学—分子生物学]