CONSTRUCTION OF A RETROVIRAL VECTOR CARRYING LMP2A AND ESTABLISHMENT OF RECOMBINANT VIRUS PRODUCING CELL LINE

Objective To establish a retroviral vector encoding Epstein-Barr virus(EBV) latent membrane protein 2A(LMP2A) and a stable virus producing cell line. Methods The target gene LMP2A amplified by RT-PCR was subcloned into retroviral vector pMSCVpuro.The recombinant plasmid pMSCVpuro-2A was transfected into packaging cell PT67 by lipofectamine 2000.The transfectants were selected by puromycin and viral titer tested.The expression of LMP2A in PT67 cell line was identified by RT-PCR. Results The recombinant retroviral vector pMSCVpuro-2A was identified by polymerase chain reaction(PCR),restrictive analysis,and DNA sequencing.A stable virus producing cell line was selected and the viral titer was 3.2×107 CFU/L.The expected 1 500 bp fragment was amplified from PT67-LMP2A cells,this result indicated that LMP2A could effectively express in packaging cell PT67. Conclusion The recombinant retroviral vector pMSCVpuro-2A was constructed successfully.A stable viral producing cell line PT67-LMP2A was selected and established.This study established a foundation for further study of LMP2A.