中文摘要：

目的：构建含有靶向乙肝表面抗原（HBsAg）基因的siRNA、乙肝复合多表位抗原基因和hIL-12共质粒表达的新型DNA疫苗，并在HepG2细胞中检测siRNA的效果以及各基因的表达。方法：设计并合成复合多表位HBV抗原基因，将其与增强型绿色荧光蛋白（EGFP）基因融合克隆进真核表达载体pVAX1的多克隆位点中，同时将带CMV启动子的完整hIL-12表达单元克隆进载体的BspnⅠ位点之间，再设计并合成乙肝siRNA表达单元，将其克隆进载体的MluⅠ位点之间，得到真核三元共表达重组质粒pVAX1-siHB—HB—EGFP—hIL12。以该重组质粒瞬时转染人肝癌细胞系HepG2，通过EGFP的荧光标记观察多表位抗原的表达，以ELISA测定培养细胞上清中hIL-12的表达，以rtPCR检测siRNA对HBsAg基因的沉默效果。结果：经酶切鉴定和测序证实共表达siRNA、hIL-12的HBV多表位DNA疫苗构建成功。转染细胞中检测到绿色荧光，证实抗原表达；转染后48h hIL-12的检出量为1289pg／ml细胞上清，72h检出量为1712pg／ml细胞上清；转染后HBsAg表达量明显降低，证实siRNA效果良好。结论：成功构建乙肝复合多表位抗原基因与siRNA、hIL-12共质粒表达的DNA疫苗，并能在真核细胞中有效表达抗原与hIL-12基因，而且siRNA对HBsAg显示出明显的沉默效果。为进一步研究该复合型DNA疫苗抗HBV的治疗效果打下基础。

英文摘要：

Objective：To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods： The HBV muhi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein（EGFP） gene, and cloned into the multi-clone site（MCS） of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH Ⅰ and Mlu Ⅰ site of pVAX1 respectively. Then the recombinant plasmid pVAXⅠ-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results ： The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion： The DNA vaccine coexpressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.