人载脂蛋白AV原核表达载体的构建与纯化研究  

Construction of human apolipoprotein AV prokaryotic vector and purification of the target protein

作　　者：李国平[1] 隋靖喆[1] 毕楠[2] 满永[1] 王抒[1] 陈保生[2] 黎健[1] 

机构地区：[1]卫生部北京医院卫生部-北京老年医学研究所卫生部老年医学重点实验室,100730 [2]中国医学科学院基础医学研究所-中国协和医科大学基础医学院医学分子生物学国家重点实验室

出　　处：《心肺血管病杂志》2013年第4期499-502,共4页Journal of Cardiovascular and Pulmonary Diseases

基　　金：国家重点基础研究发展计划(973计划)课题(2012CB517502);国家自然科学基金(81270948;81070634;81170381);北京医院博士启动基金(BJ-2011-30)

摘　　要：目的:构建人载脂蛋白AV(apolipoprotein AV,apoAV)的原核载体并表达纯化该蛋白。方法:从人肝癌细胞系HepG2中提取总RNA,以反转录的cDNA第一链为模板,经聚合酶链式反应(PCR)扩增得到含有编码apoAV完整成熟肽段的PCR片段。PCR产物和原核表达载体pET30b经核酸内切酶双切后连接,连接产物经转化至大肠杆菌感受态细胞Top10,挑选克隆测序。重组质粒pET30b-apoAV转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并优化诱导表达条件,重组的apoAV经镍离子螯合树脂纯化获得。结果:经测序证实人apoAV目的基因成功克隆入pET30b原核表达载体中,经过IPTG诱导有约40KD的特异性蛋白表达,与预期大小一致。该蛋白在IPTG诱导下的最佳表达时间为5h,最佳浓度在31.3μmol/L。纯化的apoAV纯度在95%以上,产率约为15 mg/L。结论:成功构建人apoAV的原核表达载体,实现了高效表达和纯化,为进一步研究其结构与功能奠定了基础。Objective：To construct the apolipoprotein AV（apoAV） prokaryotic vector and purify of the target protein.Methods： Total RNA was isolated from the human hepatoma cell line HepG2,following by the first-strand cDNA synthesis.Encoding mature apoAV DNA fragment was amplified by polymerase chain reaction（PCR）.Purified digested PCR products were ligated into the pET30b and recombinant clones were sequenced.The recombinant ApoAV protein was induced by isopropyl β-D-1-thiogalactopyranoside（IPTG） in E.coli BL21（DE3）,optimized the expressed conditions and purified by Ni-chelated resin.Results： The recombinant pET30b-apoAV was confirmed by DNA sequencing,and a specific protein about 40KD was identified after IPTG induction,consistent with the reported.The optimal induced time was shown at 5 h with 31.3 μmol/L IPTG.The purity of apoAV was more than 95% after purification,with the yield of about 15 mg/L.Conclusion： The apoAV prokaryotic expression plasmid was successfully cloned with efficient expression and purification,and has laid down the foundation for further investigation on its structure and functions.

关 键 词：载脂蛋白AV 原核表达 蛋白纯化 

分 类 号：R54[医药卫生—心血管疾病]