目的：克隆人PD-L1(B7-H1)基因，并构建含有该目的基因的重组逆转录病毒载体，获得稳定表达PD-L1基因的L929细胞。方法：从人心脏cDNA文库中扩增出PD-L1基因，通过双酶切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，72 h后，加入Zeocin进行筛选，挑选出能稳定表达PD-L1蛋白的L929细胞株。结果：构建了用于表达的含PD-L1基因的重组逆转录病毒载体；经转染包装细胞293T后，包装出具有感染能力的重组PD-L1逆转录病毒；经RT-PCR、流式细胞仪表型检测表明，筛选出的L929转基因细胞能稳定表达人PD-L1蛋白。结论：构建了含人PD-L1基因重组逆转录病毒载体和稳定表达人PD-L1蛋白的细胞株，为该基因功能的后续研究和单克隆抗体的研制奠定了基础。

Objective:To clone human PD-L1(B7-H1) gene and construct recombinant retrovirus vector carrying the target gene which can be expressed stably in mammal cell line L929. Methods: PD-L1 gene was amplified by PCR (polymerase chain reaction) from the human heart cDNA library and confirmed by DNA-sequence analysis. Digested with the restriction endonucleases PstI and EcoRI, the PD-L1 gene was inserted into retrovirus vector pGEZ-Term. The recombinant retrovirus vector together with its two helper virus vectors cotransfected into the package cell 293T in the context of LipfectAMINE. Then the supernatant of 293T was used to infect L929 cells. L929 cell line stably expressing PD-L1 protein was selected in the presence of Zeocin(500 μg/ml). Results: The full-length PD-L1(B7-H1) gene was successfully cloned; and the recombinant retrovirus vector carrying PD-L1 gene for expression was constructed; by transfecting package cell line 293T, recombinant PD-L1 retrovirus with infective capability was packaged and after 2 weeks of selection, the infected L929 cells formed monoclonal colonies in selective medium. Results of RT-PCR and flow cytometry indicated that L929 transgenetic cells could stably express human PD-L1 protein on the membrance of cells. Conclusion: Cloning of human PD-L1(B7-H1) gene and construction of the recombinant retrovirus vector and L929 cell line stably expressing PD-L1 protein could contribute to further biological function research and monoclonal antibody preparation.

国家重大基础研究项目（2001CB51003）资助