The cloning of healthy human APOBEC3G gene and the construction and identification of eukaryotic expression vectors

Objective To clone the cDNA of human apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like 3G(APOBEC3G) gene and construct the eukaryotic expression vector for the in vitro expression and identification.Methods The coding region of APOBEC3G gene was amplified by RT-PCR from peripheral blood mononuclear cells(PBMCs) of healthy humans,and then the gene fragment was inserted into the recombinant eukaryotic expression vector pcDNA3.1 to construct the eukaryotic expression plasmid of pcDNA3.1-A3G,which was transfected into HepG2 and HL7702 cells,respectively.The expressed products in the HepG2 and HL7702 cells were detected by immunocytochemistry and Western blot.Results The results of double enzyme digestion and sequencing showed that the pcDNA3.1-A3G-transfected HepG2 and HL7702 cells were both highly expressed,while the cells transfected with the blank plasmid pcDNA3.1 and those untransfected cells were low expressed or unexpressed.Conclusion The eukaryotic expression vector of human APOBEC3G,i.e.,pcDNA3.1-A3G was successfully constructed,which lays an experimental basis for the study of APOBEC3G against HBV.