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

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.