人CCR5基因真核表达质粒的构建及其鉴定  

Construction and characterization of plasmid expressing human CCR5 gene in eukaryotes

作　　者：程林[1] 宋红勇[1] 吴喜林[1] 吴稚伟[1] 

机构地区：[1]南京大学医学院公共健康医学中心,江苏南京210093

出　　处：《东南大学学报（医学版）》2012年第1期40-43,共4页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(30870124);科技部国际合作项目(2009DFA31260)

摘　　要：目的:构建人CCR5基因的真核表达质粒并对其进行功能鉴定。方法:PCR扩增人CCR5基因,将其克隆入真核表达载体pcDNA3.1内,构建含人CCR5基因的真核表达质粒pcDNA3.1-CCR5。使用RT-PCR、流式细胞术和HIV假病毒感染实验的方法,鉴定CCR5在真核细胞中的表达和功能。结果:克隆的人CCR5基因与GenBank中已登记的基因序列100%同源。瞬时转染真核细胞后,RT-PCR在预期的位置检测出目的条带,流式细胞术检测到约25.6%的细胞表达CCR5蛋白,且该蛋白能介导HIV假病毒的感染。结论:成功构建了含人CCR5基因的真核表达质粒。Objective： To construct and characterize a eukaryotic system for expressing human CCR5 gene.Methods： Human CCR5 gene was amplified by PCR,and subcloned into pcDNA3.1 vector to construct a recombinant plasmid pcDNA3.1-CCR5.The expression of human CCR5 gene in eukaryotic cells was verified by RT-PCR and flow cytometry.HIV-1 env pseudotyped virus infection assay was used to detect the function of CCR5 gene in eukaryotic cells.Results： The sequence of inserted CCR5 gene fragment was 100% homology compared to human CCR5 gene registered in GenBank.After transfection of eukaryotic cells with pcDNA3.1-CCR5,the target band was identified by RT-PCR and about 25.6% of the CCR5 protein was detected by flow cytometry.Furthermore,the protein could mediate HIV pseudotype virus infection.Conclusion： A functional eukaryotic expression plasmid pcDNA3.1-CCR5 has been established successfully.

关 键 词：CCR5基因 质粒 真核表达 人类免疫缺陷病毒 

分 类 号：R394.3[医药卫生—医学遗传学]