人热休克蛋白90AB1基因克隆、原核表达、中试发酵及纯化  

Cloning,Prokaryotic Expression,Pilot Fermentation and Purification of Human Heat Shock Protein 90AB1

作　　者：刘凯胜[1] 王绍祥[1] 刘忠[1] 张嘉萱[1] 张庶民[2] 王一飞[1] 

机构地区：[1]暨南大学生物医药研究开发基地广东省生物工程药物重点实验室基因工程药物国家工程研究中心,广州510632 [2]中国药品生物制品检定所,北京100050

出　　处：《生物技术通报》2011年第10期120-125,134,共7页Biotechnology Bulletin

基　　金：国家高技术研究发展计划"863"计划(2007AA02Z142)

摘　　要：通过克隆人热休克蛋白90AB1基因(Hsp90AB1),构建其原核表达载体pET28a(+)-hHsp90AB1,表达纯化获得目的蛋白,为hHsp90靶向药物筛选奠定基础。提取宫颈癌HeLa细胞总RNA,并经RT-PCR获得hHsp90AB1-cDNA。以hH-sp90AB1-cDNA为模板进行PCR体外扩增,胶回收纯化hHsp90AB1。将hHsp90AB1及pET-28a(+)质粒经NdeⅠ和SalⅠ双酶切、胶回收纯化酶切片段后,体外连接酶切片段,使其定向重组,再将重组DNA转化DH5α,经复苏后,在含卡那霉素的LB固体养基上筛选出阳性克隆。挑取LB固体培养基上的单菌落经酶切及测序鉴定,证实为阳性克隆,即pET28a-hHsp90AB1体外重组成功,命名为pET28a(+)-hHsp90AB1。重组质粒转化Rosetta(DE3)菌株,IPTG诱导表达,并对表达条件优化,以及通过SDSA-PAGE和Western blotting分析均表明Hsp90AB1蛋白得到了正确的表达,且表达产物以可溶性形式存在,优化条件下蛋白表达量约占菌体总蛋白13%。接种到18 L发酵罐中试发酵,表达产物经Ni-NTA凝胶亲和层析,蛋白纯度达到98%。Through cloning human Hsp90AB1 and constructing its prokaryotic expression vector,the purified protein laid the foundation for vitro screening of Hsp90 inhibitors.The total RNA was isolated from HeLa cells and the cDNA was gained by RT-PCR.hHsp90AB1-cDNA as a template for PCR,gel extraction and purification of the hHsp90AB1,hHsp90AB1 and pET-28a（＋） DNA were digested by NdeⅠ and SalⅠ,respectively.After purification,the two fragments obtained were ligased using T4DNA ligase.This recombinant DNA was then transformed into E.coli competent cells DH5α and positive clones were selected on the LB agarose plate containing Kanamycin.Single clones were identified by double digestion with NdeⅠ and SalⅠ,and two fragments with the size 5.4 kb and 2.1 kb were produced as expected.After gene sequencing,the hHsp90AB1 gene was successfully inserted into the prokaryotic expression vector pET-28a（＋） by recombination technique in vitro.The recombinant plasmid was transformed into Rosetta（DE3） strain,and then induced with IPTG.The target fusion protein was successfully expressed and identified in a soluble form by SDS-PAGE and Western blotting analysis,and the fusion protein was about up to 13% of the total protein.During the pilot fermentation with optimal condition in the 18 L culture medium,the purity of the protein was 98% after purification by Ni-NTA chromatography.

关 键 词：热休克蛋白90AB1(Hsp90β) 克隆 原核表达 中试发酵 纯化 

分 类 号：Q51[生物学—生物化学]