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Gene Cloning of Human PD-L1(B7-H1) and the Corresponding Recombinant Retrovirus Construction and Stable Expression
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Abstract:
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.
Keywords:
PD-L1 ; retrovirus ; stable expression ; selection
