人热休克蛋白90β-cDNA基因克隆及其真核表达载体的构建

Cloning Human Heat Shock Protein 90β-cDNA and Constructing its Eukaryon Vector

本研究旨在克隆人热休克蛋白90β(HSP90β)基因,构建其真核表达载体pcDNA3.1(+)-hHSP90β,为研究hHSP90β的基因治疗奠定实验基础。按Invitrogen公司操作说明用TRIzolReagent提取鼻咽癌总RNA,并经RT-PCR获得hHSP90βcDNA。用纯化的hHSP90βcDNA与PGEMEasyTVector连接,挑选白色菌落进行鉴定,结果为重组质粒,命名为PGEM-hHSP90β。将PGEM-hHSP90β及pcDNA3.1(+)质粒经AflII和Xbal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XL1-blue,经复苏后,在含Ampr的LB固体培养基上筛选出阳性克隆。挑取的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hHSP90β与pcDNA3.1(+)体外重组成功,命名为pcDNA3.1(+)-hHSP90β。采用体外重组技术,成功地将人HSP90β插入了真核表达载体pcDNA3.1(+)中。

The purpose of this study was to clone human HSP90β cDNA and construct its eukaryote expression vector . The total RNA was isolated by TRIzol Reagent （Invitrogen） from human NPC and its cDNA was gained by RT-PCR. The purified RT-PCR products and PGEM-T Easy Vector were ligated and transformed into XL1-blue E. coli bacteria. The white clones were selected and the plasmid was purified, which was further identified by double enzyme digestion and sequenced. PGEM-hHSP90β and pcDNA3.1 （＋） DNA were digested by AfllI and Xbal respectively, After purification, the two fragments obtained were ligased by using T4 DNA ligase （Fermentas）. This recombinant DNA was then transformed into E. coli Competent Cells XL1-blue and positive clones were selected on the LB agarose plate containing Ampr （100μg/ml）. Single clones were identified by double digestion with AfllI and Xbal, and two fragments with the size 5.4 kb and 2.1 kb were produced as expected. The hHSP90β gene was successfully inserted into the eukaryote expression vector pcDNA3.1 （＋） by the recombination technique in vitro.