nm23-H1cDNA克隆及腺病毒载体的构建

Cloning of nm23-H1 cDNA gene and construction of recombinant adenovirus vector

目的:在正常人肝组织中克隆nm23 -H1cDNA基因,构建腺病毒克隆载体。方法:应用逆转录多聚酶链式反应(RT- PCR)技术,从正常人肝组织中扩增出nm23- H1cDNA,连接到质粒pMD18- T上,经测序、鉴定,与腺病毒穿梭质粒正向连接,构建腺病毒克隆载体。结果:克隆的基因经测序鉴定,与已知nm23-H1cDNA编码区基因100%符合,以此基因成功构建正向腺病毒穿梭质粒载体。结论:利用分子克隆技术,成功克隆中国人nm23- H1cDNA基因,构建出正向重组腺病毒穿梭质粒载体。

Objective:To clone human nm23-H1 c DNA gene and to recombine it into adenovirus vector.Methods:Nm23 -H1cDNA fragment was amplified from human liver by reverse transcription-polym erase chain reaction(RT-PCR) and cloned into pMD18-T plasmid. The gene was exa mined by sequencing. Then pAd-Shuttle-CMV vector containing nm23-H1 was const ructed. Results:The results showed that the gene fragment cloned in pMD18-T was coincident with the sequence of nm23-H1.A pAd-Shuttle -CMV v ector containing nm23-H1 gene with correct order was con firmed by Kpnl digestion. Conclusion:nm23-H1 may be cloned into recombined adenovirus vector pAd-Shuttle-CMV.