分泌型重组mPSMA腺病毒的构建及表达

Construction and expression of secretary recombinant adenovis vectors carrying mPSMA

目的构建分泌型重组小鼠前列腺特异膜抗原腺病毒(Ad-mPSMA),并检测其免疫效果。方法利用AdEasy重组腺病毒载体系统,通过PCR扩增小鼠PSMA全长序列,并在基因的5′端加上人IL-2的信号肽序列,亚克隆至pAdenoVator CMV5穿梭质粒,与pAdenoVatorΔE1/E3进行同源重组获得pAd-mPSMA重组质粒,经HEK293细胞包装扩增获得重组病毒颗粒。通过RT-PCR及Western blot检测小鼠PSMA基因在HeLa细胞中的转录及表达。以该重组腺病毒免疫小鼠,对mPSMA的特异性抗体进行检测。结果成功将mPSMA基因克隆至pAdenoVatorΔE1/E3载体上,病毒滴度为1.32×1011IU/mL,RT-PCR证实mPSMA的转录,Western blot证实mPSMA的分泌表达,并在小鼠血清中检测到mPSMA的特异性抗体。结论成功构建分泌型重组mPSMA腺病毒,为前列腺癌特别是激素难治性前列腺癌的预防和治疗提供了更广阔的前景。

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 ΔE1/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 1.32×10^11IU/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.