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 mem-brane 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 pAdeno Vator 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 pAdeno Vator △E1/E3 through homologous recombination. The recombinant adenoviruses were packaged, amplified and purified in HEK293 cells. HeLa cell was infected by recombinant ade-novirus 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 immu-nized 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 × 10~(11)IU/mL and expres-sion of mPSMA was confirmed by RT-PCR and Western blot. The specific antibody against mPSMA had been found in 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.