稳定表达细胞毒性T淋巴细胞相关抗原CTLA-4的293T细胞株的建立

Construction of 293T Cell Line Stably Expressing CTLA-4

目的：构建稳定表达细胞毒性T淋巴细胞相关抗原-4（cytotoxicTlymphocyteantigen-4，CTLA-4）的293T细胞株。方法首先从人外周血获取PBMC加以T细胞活化；PCR扩增CTLA-4转录本，连接pUCm-T质粒引入HindⅢ和EcoRⅠ双酶切位点后转入真核细胞表达质粒pcDNA3．1；重组质粒pcDNA3．1-CTLA-4经脂质体转染293T细胞，以抗生素G418筛选表达CTLA-4的293T细胞；定量PCR、免疫荧光分别检测293T细胞CTLA-4表达与细胞定位。结果血样来源T细胞经活化后表达CTLA-4转录本。HindⅢ和EcoRⅠ双酶切证实重组质粒内CTLA-4插入序列正确；RT-PCR及免疫荧光检测证实293T细胞能够稳定表达目的蛋白CTLA-4，并定位于细胞膜表面。结论成功构建的pcDNA3．1-CTLA-4重组真核表达载体，在293T细胞膜表面实现了稳定表达，为进行相关生物学理论与应用研究打下基础。

Objective To construct human embryonic kidney 293T cell line that can stably ex-press cytotoxic T lymphocyte antigen 4 （CTLA-4） .Methods Peripheral blood mononuclear cells （PBMCs） were harvested for T cell activation.CTLA-4 transcripts were amplificated by PCR and then connected with pUCm-T plasmid, which was transduced into pcDNA3.1 after introduction of HindⅢ and EcoRⅠ sites.The recombinant plasmid pcDNA3.1-CTLA-4 was transfected into 293T cells by using liposome.Antibiotic G418 was used to select the 293T cells that stably expressed CT-LA-4.The expression of CTLA-4 and its location were determined by quantitative PCR and immun -ofluoresence assay.Results Once activated, the blood-derived T cells expressed CTLA-4 tran-scripts.Double enzyme digestions with HindⅢ and EcoR-I demonstrated that the sequence of CTLA-4 in the recombinant plasmid was correct.RT-PCR and immuno-fluoresence assay revealed that 293T cells could stably express CTLA-4 protein on the cell membrane.Conclusions The recombinant eukaryotic expression vector pcDNA3.1-CTLA-4 was successfully constructed.CTLA-4 was stably expressed on the cell membrane of 293T cells.The present study lays a foundation for CTLA -4-relat-ed studies.