Cloning,Expression and Purification of APOBEC3G,and Its Polyclonal Antibodies Production

To prepare human APOBEC3G and to produce its antibodies,the total RNA was extracted from H9 cells,and APOBEC3G gene was achieved by RT-PCR.The resulting DNA construct was cloned into a prokaryotic expression vector(pET-32a)and recombinant pET-APOBEC3G was expressed in Eserichia coli BL21(DE3)as an insoluble protein.The vector also contained a six-histidine(His6)tag at the C-terminus for convenient purification and detection.To express and purify the APOBEC3G in E.coli cells,the accuracy of inserted genes and specificity of APOBEC3G proteins were detected by two enzymes digestion technology, SDS-PAGE,and Western Blot(WB).Rabbits were immuzied by purified APOBEC3G proteins.Serum samples were tested by indirect enzyme-linked immunosorbent assays(ELISAs)to determine the level of antibodies.The purity of APOBEC3G was above 80%.The titer of the antibodies was 1:102 400