Construction of Human IL-17F-recombinant Retrovirus Vector and Its Stable Expression in 293T Cells

Objective To construct human IL-17F-recombinant retrovirus vector and express it stably in 293T cells.Methods The recombinant retrovirus vector pSIV-1/hIL-17F(including the 20aa signal peptide sequence,moreover,hIL-17F gene inside GAATTC→GAACTC synonymous mutation)was constructed by PCR according to pUCm-T/hIL-17F and identified by PCR,double endonuclease digestion and DNA sequencing.The pSIV-1/hIL-17F together with its two helper virus vectors pHIT456 and pHIT60 was cotransfected into the package cell 293T by lipofectin to produce mature recombinant retrovirus,then which was used to infect 293T cell.The transgenic 293T cell line stably expressing rhIL-17F protein was selected in the presence of G418 and the integration,transcription,expression of hIL-17F target gene in 293T cell were identified by PCR, RT-PCR and dot-ELISA,respectively.Results The recombinant retrovirus vector pSIV-1/hIL-17F and its transgenic 293T cell expressing stably rhIL-17F protein were obtained successfully.Conclusion This is the first time for us to obtain the transgenic 293T cells expressing stably rhIL-17F protein in China,which paves the way for future study on biological functions and mechanism of hIL-17F.