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 line.Methods PD-1 gene was amplified by polymerase chain reaction from pGEX-5X-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.FCMresultshowed that the expression of PD-1 molecules in L929 transfection cell lines was 97.6%.Conclusion HumanPD-1 gene has been cloned,recombinant retrovirus vector containing PD-1 gene constructed and L929 transfection cell line expressing PD-1 molecules selected.