丙型肝炎治疗性DNA疫苗的实验研究

发布时间：2018-06-12 12:12

本文选题：丙型肝炎 + 治疗性疫苗　；参考：《吉林大学》2006年博士论文

【摘要】：为了进行丙型肝炎(HCV)治疗性DNA疫苗的研制,本实验成功建造了HCV感染的细胞模型。以红色荧光蛋白(RFP)和绿色荧光蛋白(GFP)为报告基因,将其分别插入乙肝核心抗原基因(HBc)的e1 loop和e2 loop处,构建质粒pcDNA 3.1-HBc-GFP和pcDNA 3.1-HBc-GFP并在Hela细胞内表达。构建了含有HCV T细胞表位的真核表达质粒pcDNA3.1-HBc-T1T2,通过RFP和GFP表达情况的观察、质粒pcDNA 3.1-HBc-T1T2表达产物HBcAg-T1T2电镜下成颗粒性观察及融合蛋白HBcAg-T1T2三维构象展示,证实了HBc作为疫苗免疫载体的可行性。真核表达质粒pcDNA 3.1-HBc-T1T2肌肉注射免疫小鼠,适时进行了抑瘤实验、CTL细胞毒活性、HBcAb、脾淋巴细胞胞内细胞因子IL-4和IL-γ、血清中细胞因子IL-5、IL-10和IL-12、脾淋巴细胞刺激指数(SI)、血液和脾淋巴细胞亚群、IgG2α/IgG1及脾淋巴细胞培养上清中IL-2水平的检测,以对真核表达质粒pcDNA 3.1-HBc-T1T2的免疫原性进行分析。本研究结果揭示了,真核表达质粒pcDNA3.1-HBc-T1T2能够诱导出特异性的细胞免疫应答,可作为HCV治疗性DNA疫苗的候选,本实验为HCV的治疗奠定了基础。
[Abstract]:In order to develop a therapeutic DNA vaccine for hepatitis C virus (HCV), a cell model of HCV infection was successfully constructed. Using red fluorescent protein (RFP) and green fluorescent protein (GFP) as reporter genes, the recombinant plasmids pcDNA3.1-HBc-GFP and pcDNA3.1-HBc-GFP were constructed and expressed in Hela cells by inserting them into e1 loop and e2 loop, respectively. The eukaryotic expression plasmid pcDNA3.1-HBc-T1T2 containing HCV T cell epitopes was constructed. By observing the expression of RFP and GFP, the plasmid pcDNA3.1-HBc-T1T2 was granulated under electron microscope and the fusion protein HBcAg-T1T2 was displayed in 3D conformation. The feasibility of HBc as vaccine immune vector was confirmed. Eukaryotic expression plasmid pcDNA3.1-HBc-T1T2 was injected intramuscularly into mice. The cytotoxic activity of CTL, IL-4 and IL- 纬, the cytokines IL-5, IL-10 and IL-12 in serum, the spleen lymphocyte stimulation index (SI), IgG2 伪 / IgG1 in blood and splenic lymphocyte subsets, and the culture of splenic lymphocytes were carried out in a timely manner. The detection of IL-2 level in the serum, The immunogenicity of eukaryotic expression plasmid pcDNA3.1-HBc-T1T2 was analyzed. The results showed that the eukaryotic expression plasmid pcDNA3.1-HBc-T1T2 could induce a specific cellular immune response and could be used as a candidate for HCV therapeutic DNA vaccine. This study laid a foundation for the treatment of HCV.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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