Construction of Z2A retroviral vector with LMP2A and BZLF1 gene

AIM:When LMP2A and BZLF1 gene are mixed,LMP2A induces CTL and BZLF1 induces EBV from latent period to burst times to kill EBV positive cell.The study was aimed to construct Z2A recombinant retroviral vector and a stable virus producing cell line. METHODS:Experiments were performed at Department of Microbiology of Jining Medical College and Department of Microbiology of Medical College of Qingdao University from August 2006 to August 2007.The target gene Z2A amplified by reverse transcription-polymerase chain reaction was subcloned into retroviral vector pMSCVpuro through connecting gene by(Gly_4Ser)_3 DNA sequence by spliced overlap exetension.The recombinant plasmid pMSCVpuro-Z2A was transfected into packaging cell PT67 by lipofectamine2000.The transfectants were selected by puromycin and viral titer tested.The expression of Z2A in PT67 cell line was identified by reverse transcription-polymerase chain reaction. RESULTS:The recombinant retroviral vector pMSCVpuro-Z2A was identified by polymerase chain reaction,restrictive analysis and DNA sequencing.A stable virus producing cell line was selected and the viral titer was 7.8×10~6 CFU/L.Z2A could effectively express in packaging cell PT67. CONCLUSION:The recombinant retroviral vector pMSCVpuro-Z2A is successfully constructed.A stable viral producing cell line PT67-Z2A is selected and established.