Construction of eukaryotic expression vector of human CD34 and establishment of stably transfected cell line

AIM:To construct the eukaryotic expression vector of human hCGβ and stably transfect B16 cell line with it.METHODS:The full length of hCGβ cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo,added the restriction enzyme position and 6×His tag.After identification of restriction digestion and PCR,The recombinant plasmid pIRES-neo-hCGβ-(His)6 was obtained.Then transfected it into B16 cells by lipofectamine 2000.After screening culture by G418,a stably transfected cell line was established,the transcription and expression of the hCGβ gene was identified by RT-PCR,Western blot and immunofluorescence assay.RESULTS:The eukaryotic expression vector pIRES-neo-hCGβ-(His)6 was successfully constructed.A stably transfected cell line was established and the expression rate of hCGβ gene was higher than 90%.CONCLUSION:The established cell line can highly express hCGβ stably,the expression of the target gene provide a solid experimental foundation for further studies on the function of the hCGβ,and which will contribute to the research of hCGβ gene in the tumor immunotherapy.