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目的:构建多表位抗原基因、hIL-12、小分子干扰RNA(siRNA)共质粒表达的新型复合丙型肝炎DNA疫苗并在HepG2细胞中表达。方法:设计并合成复合多表位丙肝抗原基因,将其与加强型绿色荧光蛋白(EGFP)基因融合克隆进真核表达载体pVAX1的多克隆位点中;利用pVAX1载体上卡那霉素抗性基因与复制起始位点之间的非功能区域,将带CMV启动子的hIL-12表达单元克隆进该区域的BspH I位点中;同时将8组针对丙肝的siRNA表达单元分别克隆进载体复制起始位点下游非功能区域的MluI位点上,构建三重表达的HCV DNA疫苗pVAX1-HC-EGFP-hIL12-siRNA。以该重组质粒转染人肝癌细胞系HepG2,通过EGFP的荧光标记观察多表位抗原的表达;以靶向EGFP的siRNA做为对照验证siRNA的表达及其干扰效果;ELISA测定培养细胞上清中hIL-12的表达。结果:经酶切鉴定和测序证实三重表达的复合HCV DNA疫苗构建成功。在转染细胞中检测到绿色荧光,证实抗原表达;靶向EGFP的siRNA亦能显著抑制EGFP的表达;转染后48 h hIL-12的检出量为1 289 ng/L细胞上清,72 h检出量为1 712 ng/L细胞上清。结论:成功地构建多表位抗原基因、hIL-12和siRNA共质粒表达的丙肝DNA疫苗,并能在真核细胞中有效表达抗原基因、hIL-12并介导RNA干扰效应。为进一步研究该DNA疫苗抗HCV的免疫治疗和基因治疗效果打下基础。
OBJECTIVE: To construct a new recombinant hepatitis C DNA vaccine expressing multi-epitope antigens, hIL-12 and small interfering RNA (siRNA) plasmids and express in HepG2 cells. Methods: The multi-epitope Hepatitis C antigen gene was designed and synthesized, fused with enhanced green fluorescent protein (EGFP) gene into multiple cloning site of eukaryotic expression vector pVAX1. The kanamycin resistance of pVAX1 vector Cloning the hIL-12 expression unit with CMV promoter into the BspH I site of this region in the non-functional region between the gene and the replication initiation site; and simultaneously 8 groups of siRNA expression units for hepatitis C were cloned into the vector The triple-expressed HCV DNA vaccine pVAX1-HC-EGFP-hIL12-siRNA was constructed at the MluI site in the non-functional region downstream of the replication start site. The recombinant plasmids were transfected into human hepatocellular carcinoma cell line HepG2 and the expression of multi-epitope antigen was observed by fluorescent labeling of EGFP. The siRNA targeting EGFP was used as a control to verify the siRNA expression and its interference effect. hIL-12 expression. Results: The triple HCV DNA vaccine confirmed by restriction enzyme digestion and sequencing was successfully constructed. Green fluorescence was detected in the transfected cells, which confirmed the antigen expression; siRNA targeting EGFP also significantly inhibited the expression of EGFP; 48 h after transfection, the detection of IL-12 was 1 289 ng / L cell supernatant, 72 h The detection yield was 1 712 ng / L cell supernatant. CONCLUSION: The hepatitis C DNA vaccine expressing multi-epitope antigens, hIL-12 and siRNA costimulatory plasmids has been successfully constructed and can efficiently express antigen gene, hIL-12 in eukaryotic cells and mediate RNA interference. This will lay the foundation for further study on the anti-HCV immunotherapy and gene therapy effect of this DNA vaccine.