人PD-1基因克隆及其在L929基因转染细胞上的表达

目的 克隆人PD-1基因，构建含有该目的基因的重组逆转录病毒载体，并转染入L929细胞，获得稳定高表达PD-1分子的L929细胞。方法 用PCR法从pGEX-5X-3-PD-1扩增出PD-1基因，通过双酶切（PstI BamHI）装入逆转录病毒载体pGEZ Term中，脂质体法共转染包装细胞293T，用含有完整病毒颗粒的293T细胞的培养上清感染L929细胞，72h后，加入Zeocin进行筛选，挑选出能稳定表达PD-1分子的L929细胞株。结果 成功克隆了人PD-1基因，构建了含PD-1基因的重组逆转录病毒载体，包装出具有感染能力的重组PD-1逆转录病毒，筛选出具有稳定表达人PD-1分子的L929基因转染细胞。经流式细胞仪检测：PD-1分子在L929基因转染细胞中具有高表达，达到97．6％。结论 构建了含人PD-1基因重组逆转录病毒的载体，筛选出稳定表达人PD-1分子的L929细胞株。

Cloning of human PD-1 gene and its expression in L929 transfection cell line

Objective To clone human PD-1 gene and obtain a stablly expressing PD-1 molecules in L929 transfected cell ling Methods PD-1 gene was amplified by polymerase chain reaction from pGEX-SX-3-PD-1 and confirmed by DNA sequencing. The PD-1 gene was then digested with the restriction endonucleases Pst I and BamH I and inserted into retrovirus vector pGEZ Term. The recombinant retrovirus vector was cotransfected into the package cell 293T with LipfectAMINE transfection method. The supernatant of 293T was infected into L929 cell lines. After 72 hours, L929 cell stablly expressed PD-1 molecules was selected by Zeocin. Results The full length of human PD-1 gene was cloned and the recombinant retrovirus vector was constructed. The L929 cell line expressing PD-1 molecules was successfully transfected and selected. FCM result showed that the expression of PD-1 molecules in L929 transfection cell lines was 97.6%. Conclusion Human PD-1 gene has been cloned, recombinant retrovirus vector containing PD-1 gene constructed and L929 transfection cell line expressing PD-1 molecules selected.