Liu Tao, Zhao Jumei, Tian Ling, Wei Yuquan, Liang Chuanyu
Department of Otorhinolargngology, West China Hospital, Sichuan University, Chengdu 610041, China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2006 Dec;23(6):1289-93.
The purpose of this study was to clone human HSP90beta 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-hHSP90beta and pcDNA3.1(+) DNA were digested by AflII 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 (100 microg/ml). Single clones were identified by double digestion with AflII and Xbal, and two fragments with the size 5.4 kb and 2.1 kb were produced as expected. The hHSP90beta gene was successfully inserted into the eukaryote expression vector pcDNA3.1(+) by the recombination technique in vitro.
本研究的目的是克隆人HSP90β cDNA并构建其真核表达载体。采用TRIzol试剂（Invitrogen公司）从人鼻咽癌组织中提取总RNA，通过RT-PCR获得其cDNA。将纯化的RT-PCR产物与PGEM-T Easy载体连接，转化至XL1-blue大肠杆菌中。挑选白色菌落，提取质粒，通过双酶切和测序进一步鉴定。分别用AflII和Xbal酶切PGEM-hHSP90β和pcDNA3.1(+) DNA。纯化后，用T4 DNA连接酶（Fermentas公司）将获得的两个片段连接起来。然后将该重组DNA转化至大肠杆菌感受态细胞XL1-blue中，在含有氨苄青霉素（100μg/ml）的LB琼脂平板上筛选阳性克隆。通过用AflII和Xbal双酶切鉴定单克隆，预期产生大小为5.4 kb和2.1 kb的两个片段。通过体外重组技术成功地将hHSP90β基因插入真核表达载体pcDNA3.1(+)中。
