大鼠前列腺特异膜抗原基因重组腺病毒诱导小鼠特异性抗体的研究

Immunological Response of Mice Induced by Recombinant Adenovirus Carrying Rat Prostate-specific Membrane Antigen Gene

目的构建含大鼠前列腺特异膜抗原基因(PSMA)的重组腺病毒Ad-rPSMA,并研究其在小鼠体内的特异性体液免疫反应。方法提取大鼠脑组织总RNA,经RT-PCR扩增PSMA基因片段,利用Adeasy腺病毒载体系统构建pAd-rPSMA重组腺病毒质粒,包装复制缺陷型重组腺病毒Ad-rPSMA,制备病毒颗粒。用制备的病毒感染HeLa细胞,Western blot检测PSMA基因的表达。以Ad-rPSMA重组腺病毒免疫C57小鼠,检测血清中PSMA特异性抗体。结果成功构建了含有大鼠PSMA基因的重组腺病毒,制备的病毒颗粒数为1.97×1012VP/mL,A260/A280值为1.32,病毒滴度为5×1010IU/mL;Western blot检测可见大鼠PSMA基因在HeLa细胞中的表达;ELISA法检测出免疫小鼠血清中存在PSMA特异性抗体。结论成功构建了大鼠前列腺特异膜抗原基因重组腺病毒Ad-rPSMA,并诱导小鼠产生了针对PSMA的特异性抗体。

Objective To construct a recombinant adenovirus carrying rat prostate-specific membrane antigen（PSMA） gene and to investigate the immune response of mice to it.Methods PSMA cDNA fragment was amplified by RT-PCR using total RNA extracted from rat brain tissue.Recombinant plasmid pAd-rPSMA was constructed by using adeasy adenovirus vector system.Packaged recombinant adenovirus Ad-rPSMA were obtained.The expression of rat PSMA gene in HeLa cells was confirmed by Western blot.The recombinant adenovirus were applied to C57 mice and the resulting anti sera against PSMA were detected with ELISA.Results Recombinant adenovirus carrying rat PSMA gene was constructed successfully.The virus particle count,A260/A280 value and titer were 1.97×1012 VP/mL,1.32,5×10^10 IU/mL respectively.Western blot analysis proved the expression of rat PSMA gene in HeLa cells.PSMA-specific sera antibody was produced in mice.ConclusionRecombinant adenovirus Ad-rPSMA was constructed and it could induce the mice to produce PSMA-specific antibody.