重组质粒pGEM-HERV-K gag的构建与鉴定  

Construction and identification of recombinant plasmid pGEM-HERV-K gag

作　　者：张宗彦 张雯 刘妙龄[2] 贲亚琍 ZHANG Zongyan;ZHANG Wen;LIU Miaoling;BEN Yali(Immune Disease Institute of Medical School;Department of Student Work,Jianghan University,Wuhan 430000,Hubei Province,China)

机构地区：[1]江汉大学医学院免疫疾病研究所,湖北武汉430000 [2]江汉大学学生工作部,湖北武汉430000

出　　处：《西南医科大学学报》2018年第5期398-400,448,共4页Journal of Southwest Medical University

基　　金：中央引导地方科技发展专项资金项目(财教[2016]150号)

摘　　要：目的:构建pGEM-HERV-K gag重组质粒。方法:分离人外周血单核细胞,提取总RNA,逆转录得到cDNA,PCR扩增目的基因后回收并纯化目的基因,TA基因克隆方法构建重组质粒,并采用酶切和PCR进行鉴定。结果:以PBMC的cDNA为模板,PCR扩增获得446 bp的HERV-K gag目的基因,克隆到pGEM-T载体构建pGEM-HERV-K gag重组质粒,酶切及PCR结果证实重组质粒构建成功。结论:成功构建pGEM-HERV-K gag质粒,此方法可用于比较病人与健康人的gag基因的序列。Objective To construct the recombinant plasmid pGEM-HERV-K gag. Methods： The recombi-nant plasmid was conducted through the following steps： collection of human blood, isolation of human peripheralblood mononuclear cells （PBMCs）, total RNA extraction, reverse transcription, PCR amplification, gel DNA extrac-tion, and TA cloning. And then the recombinant plasmid was identified by enzyme digestion and PCR. Results： Thetarget gene （446bp HERV-K gap） which was produced by PCR amplification with the cDNA of PBMC as the tem-plate was successfully cloned into pGEM-T vector to construct the recombinant plasmid （pGEM-HERV-K gag）.And the recombinant plasmid was confirmed by the results of enzyme digestion and PCR amplification. Conclusion：The pGEM-HERV-K gag plasmid has been successfully constructed. The method can be used to compare the gaggene sequences between patients and healthy controls.

关 键 词：HERV-Kgag基因 TA克隆 外周血单核细胞 

分 类 号：Q812[生物学—生物工程]