治疗性乙型肝炎病毒DNA疫苗的基础研究

发布时间：2018-05-16 16:03

  本文选题：乙型肝炎 + DNA疫苗　； 参考：《中国人民解放军军医进修学院》2005年博士论文

【摘要】：慢性乙型肝炎(CHB)严重危害人类健康,目前尚无特效治疗手段。乙型肝炎慢性化的主要原因之一是被感染者缺乏有效的特异性细胞免疫应答,不能清除其体内的乙型肝炎病毒(HBV)。DNA疫苗将编码外源蛋白基因的质粒DNA直接导入机体组织,外源基因在体细胞中表达后,表达产物被递呈,与主要组织相容性复合物结合,可刺激机体产生相应的细胞毒性T淋巴细胞(CTL)和抗体,介导细胞和体液免疫应答。 乙肝DNA疫苗可诱导机体产生抗原特异性细胞及体液免疫,接种后可诱导多种实验动物产生特异性CTL及保护性抗体,清除HBV转基因鼠血清HBsAg及HBV DNA,接种于对重组亚单位疫苗无应答的健康志愿者可产生保护性抗体。由于可诱导特异性细胞免疫,乙肝DNA疫苗有可能抑制或清除CHB患者体内的HBV,但要作为临床治疗CHB的工具尚需解决许多难题,主要是确保安全性并增强免疫效果。既往的研究大多通过联合使用免疫佐剂、细胞因子及采用不同的接种方法增强疫苗的免疫原性。本研究在乙肝DNA疫苗中插入HBV自身复制调控元件—增强子(ENH)及前S2(PreS2)、前C(PreC)抗原基因片段,通过基因调控的方法增强乙肝DNA疫苗目的基因的表达以增强DNA疫苗的免疫原性,并采用FDA已批准用于临床试验的载体VR1012构建疫苗,所构建的疫苗确保安全性,可进入临床试验进行深入研究。 本研究采用常规PCR法从adr亚型HBV全基因DNA序列中分别扩增HBsAg,PreS2-HBsAg,HBsAg—ENH Ⅰ,PreS2-HBsAg-ENH Ⅰ,HBcAg和ENH Ⅱ-PreC-HBcAg基因片段,重组到载体VR1012中,构建6种HBV重组质粒(重组质粒是乙肝DNA疫苗的主体),转染HepG2细胞及COS-7细胞,免疫BALB/C小鼠及HBV转基因鼠。采用细胞内酶免疫染色、ELISA、ELISPOT等方法检测其在HepG2细胞、COS-7细胞内的表达,BALB/C小鼠的体液、细胞免疫及HBV转基因鼠血清HBsAg、HBV DNA及肝组织病理改变。发现转染的HepG2细胞、COS-7细胞均表达相应的目的蛋白—
[Abstract]:Chronic hepatitis B (CHB) is a serious hazard to human health. One of the main causes of chronic hepatitis B infection is the lack of specific cellular immune response in infected patients, and the hepatitis B virus (HBV) virus DNA vaccine can not be removed directly into the tissues of the body by introducing the plasmid DNA encoding foreign protein gene into the body tissue. After the expression of exogenous gene in somatic cells, the expressed product was presented and combined with the major histocompatibility complex, which could stimulate the production of cytotoxic T lymphocyte (CTL) and antibodies, and mediate cellular and humoral immune responses. Hepatitis B DNA vaccine can induce antigen-specific cells and humoral immunity, and induce many experimental animals to produce specific CTL and protective antibodies after inoculation. The serum HBsAg and HBV DNA of HBV transgenic mice were cleared, and protective antibodies were produced in healthy volunteers who did not respond to the recombinant subunit vaccine. Hepatitis B DNA vaccine may inhibit or eliminate HBV in patients with CHB due to its ability to induce specific cellular immunity. However, as a tool for clinical treatment of CHB, many problems need to be solved, mainly to ensure safety and enhance the immune response. Previous studies have mostly enhanced the immunogenicity of vaccines by combined use of immune adjuvants, cytokines and different inoculation methods. In order to enhance the immunogenicity of hepatitis B DNA vaccine, we inserted HBV self-replicating regulatory element (Enh) and pre-S _ 2 PreS _ (2) and pre-C _ (2) prec _ (2) gene fragments into hepatitis B DNA vaccine, and enhanced the expression of target gene of DNA vaccine by means of gene regulation, so as to enhance the immunogenicity of DNA vaccine. The vaccine was constructed with VR1012, which has been approved by FDA for clinical trials. The constructed vaccine ensures safety and can be further studied in clinical trials. In this study, HBcAg and ENH 鈪，

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