靶向抗原递呈细胞激活gB的重组质粒的构建及其对树突状细胞凋亡的影响

Construction of recombinat plasmid expressing gB gene targeting antigen-presenting dendritic cells activation and its preliminary investigation of reducing apoptosis of dendritic cells

目的:观察靶向抗原递呈细胞激活表达gB的重组DNA疫苗对树突状细胞凋亡的抑制作用。方法:构建含gB与靶向BAK siRNA共表达的重组质粒载体pgB-U6BAK,将重组质粒pgB-U6BAK及pgB分别转染经体外培养的树突状细胞;Western blotting法检测转染后树突状细胞BAK和gB基因表达变化,MTT法检测转染后树突状细胞凋亡发生率。结果:成功构建含gB和靶向BAK siRNA共表达的重组质粒载体pgB-U6BAK。重组质粒pgB-U6BAK转染树突状细胞后在gB稳定表达的同时,其BAK蛋白表达水平显著降低;重组质粒pgB-U6BAK转染后树突状细胞凋亡明显减少。结论:重组DNA疫苗pgB-U6BA能够在稳定表达gB的同时通过抑制凋亡基因BAK而抑制树突状细胞发生凋亡,有望成为较为有效的抗巨细胞病毒疫苗。

Objectives To investigate construct of targeted dendritic cells activation of vaccine strain expressing gB gene and its function of reduced apoptosis of dendritic cells. Methods A plasmid DNA vector encoding the gB and target BAK siRNA was constructed. The recombinant plasmid was used to infect DC in vitro and the expressed products were detected by Western blot. The activity of DC2.4 was assessed by MTT. Results Plasmid pgB-U6BAK was constructed. Murine DC infected with plasmid pgB-U6BAK could express gB protein stably, and reduce BAK. The apoptosis of DC2.4 was apparently reduced after transfection with plasmid pgB-U6BAK. Conclusion The results showed that pgB-U6BAK could express gB protein stably, and reduce apoptosis of DC2.4 by inhibiting BAK, would be potentially used as effective anti-CMV vaccine.