Construction and expression of secretary recombinant adenovis vectors carrying mPSMA / 基础医学与临床

ABSTRACT

Objective To construct secretary recombinant adenoviral vector carrying the mouse prostate-specific membrane antigen gene through AdEasy vector system and the immunological outcome was assessed.Methods mPSMA was amplified from plasmid pCR-BluntII-TOPO by PCR and subcloned into transfer vector pAdenoVator CMV5,The signal peptide DNA sequence of hIL-2 was fused to 5′terminal of mPSMA gene to construct a secretary Ad-mPSMA.pAdv-mPSMA was co-transformed with pAdenoVator ?E1/E3 through homologous recombination.The recombinant adenoviruses were packaged,amplified and purified in HEK293 cells.HeLa cell was infected by recombinant adenovirus Ad-mPSMA and the expression of mouse prostate-specific membrane antigen gene was detected by RT-PCR and Western blot.The recombinant adenovirus had been immuned mice,sera antibody against mPSMA from immunized mice was detected by ELISA.Results The secretary pAd-mPSMA was constructed successfully and typical cytopathic effect(CPE) was observed.The titer of the recombinant adenovirus was 1.32?1011IU/mL and expression of mPSMA was confirmed by RT-PCR and Western blot.The specific antibody against mPSMA had been foundin serum of the immunized mice.Conclusion mPSMA gene recombinant adenovirus was constructed successfully,which provide a basis for further study on the anti-tumor immunotherapy role of PSMA.