Cloning human heat shock protein 90beta-cDNA and constructing its eukaryon vector / 生物医学工程学杂志

ABSTRACT

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.