基于Ii分子的HCV-NS3 Th1位基因疫苗对树突细胞抗原提呈功能的影响
发布时间：2019-05-19 08:30
【摘要】： 丙型病毒性肝炎是严重危害人类健康的传染病。全球丙肝病毒(HCV)的感染人数约为一亿七千万，占世界人口总数的1—3％。在感染的人群中，约80％转为慢性，20％发展为肝硬化和肝细胞癌；我国的HCV感染者占人口总数的3.2％，估计感染者总数约为4000万。 自从1989年HCV cDNA首次被克隆成功至今，对HCV的分子生物学研究取得了突飞猛进的发展。目前已明确，HCV为单股正链RNA病毒，属黄病毒科，全长约9600个核苷酸，ORF区编码3010个氨基酸的多聚蛋白前体，经过宿主和病毒本身基因编码的蛋白酶裂解为十个功能性片段，其中四个为结构蛋白，分别是核心蛋白C、包膜蛋白E1、E2以及P7；六个为非结构蛋白，分别是NS2、NS3、NS4A、NS4B、NS5A和NS5B。尽管HCV的基本结构已经清楚，，但是HCV的防治仍然是世界性的难题，α干扰素和病毒唑联合治疗是唯一的治疗方法，但这种治疗方法只对不到50％的患者显效，且具有费用高、易复发和副作用多等缺陷；尤其是我国流行的HCV-Ⅱ／1b型，对α干扰素的治疗应答最低，因此发展治疗和预防性丙肝疫苗，对于预防HCV感染和控制感染者的病情发展，都具有重要的意义。 HCV NS3蛋白具有丝氨酸蛋白酶和解旋酶活性，参与病毒蛋白翻译后加工，为病毒复制所必须，针对NS3蛋白的T细胞应答与自限性感染关系密切。目前公认位于NS3区的1248—1261位氨基酸残基为CD4~+Th1细胞表位，而且该表位对于MHC限制性具有广谱特异性。在此之前的研究已经以BALB／c小鼠为实验动物模型，以MHCⅡ抗原提呈通路中的分子伴侣恒定链(invariant chain，Ii)为载体，用HCV-NS3的1248-1261表位替换Ii链的CLIP编码片段构建了内源性靶向基因疫苗，并在细胞水平研究了所构建的基因疫苗与抗原提呈分子之间的相互作用，用所构建的疫苗对荷瘤小鼠进行了免疫治疗并取得了好的效果。 本研究构建了内源性靶向基因疫苗的体外转录载体，在体外转录成mRNA，同时用小鼠的骨髓细胞体外诱导培养，成功的培养出小鼠骨髓来源的树突细胞，通过电转的方法将内源性靶向基因疫苗mRNA导入小鼠树突细胞(dendritic cell，DC)内，研究内源性靶向基因疫苗对DC表面分子的表达及细胞因子的分泌的影响。并用混合淋巴细胞实验来研究内源性靶向基因疫苗是否影响DC对T细胞诱导分化功能。研究结果如下： 1．内源性基因疫苗可有效地刺激树突细胞成熟，增加表面协同刺激分子CD80、CD86、I-A~d的表达。 2．内源性基因疫苗可有效地诱导DC分泌细胞因子IL-12、IFN-γ。 3．经内源性基因疫苗负载的DC可有效的刺激CD4~+T细胞增殖，并诱导其向Th1方向分化。 综上所述，用以Ii链作为载体针对HCV-NS3的Th1表位内源性靶向基因疫苗修饰DC后，可增强DC的抗原提呈功能，能有效地激活CD4~+T细胞。这种方法有望为HCV的防治提供一种可行的途径，为下一步构建靶向树突细胞的内源性基因疫苗奠定基础，在治疗性HCV基因疫苗的开发研究中具有良好的应用前景。
[Abstract]:Type C viral hepatitis is an infectious disease that is seriously harmful to human health. The worldwide prevalence of hepatitis C virus (HCV) is about 100 million, accounting for 1-3 per cent of the world's total population. In the infected population, about 80% to chronic and 20% developed into liver cirrhosis and hepatocellular carcinoma; HCV-infected people in China account for 3.2% of the total population, and the total number of people infected is estimated to be about 40 million. The molecular biology of HCV has been studied since the first cloning of the HCV cDNA in 1989. The present invention has shown that HCV is a single-stranded positive-chain RNA virus, which is a single-stranded positive-chain RNA virus, which is a polyprotein precursor of the total length of 9600 nucleotides and the ORF region encoding 3010 amino acids, and the protease which is encoded by the host and the virus itself is cracked into ten functional fragments, Four of which are structural proteins, respectively, are core protein C, envelope protein E1, E2 and P7, and six are non-structural proteins, and are NS2, NS3, NS4A and NS4B, respectively. NS5A and NS5B. Although the basic structure of HCV is clear, the prevention and treatment of HCV is still a worldwide problem. The combination therapy of interferon and virotherapy is the only treatment method, but the treatment method is effective only for less than 50% of patients, and has high cost and easy recovery. There are many defects such as hair and side effects, especially the prevalence of HCV-II/ 1b in China, and the treatment response of interferon is the lowest. Therefore, the development of treatment and preventive hepatitis C vaccine can be used to prevent HCV infection and to control the development of the infected people. The HCV NS3 protein has the activity of serine protease and helicase, participates in the post-translational processing of the viral protein, The T cell response of the 3-protein is closely related to the self-infection. It is recognized that the 1248-1261-position amino acid residue in the NS3 region is the CD4 ~ + Th1 cell epitope. And the epitope has broad-spectrum specificity for MHC restriction. The prior research has been used as an experimental animal model in BALB/ c mice, and the molecular chaperone constant chain (Ii) in the path of the MHC II antigen is taken as a carrier, and the C of the Ii chain is replaced by the 1248-1261 epitope of the HCV-NS3. The LIP coding fragment constructs the endogenous targeting gene vaccine and studies the interaction between the constructed gene vaccine and the antigen-presenting molecule at the cell level, using the constructed vaccine pair In this study, the in vitro transcription vector of the endogenous target gene vaccine was constructed and the mRNA was transcribed in vitro, and the mice were used in the same time. The bone marrow cells were cultured in vitro, and the dendritic cells from the bone marrow of the mouse were successfully cultured. The endogenous target gene vaccine mRNA was introduced into the mouse dendritic cell (DC) by the method of electrospinning. The effect of the target gene vaccine on the expression of DC surface and the secretion of cytokines. to study endogenous Whether the target gene vaccine affects the DC-to-T cell-induced differentiation function. The results are as follows:1. The endogenous gene vaccine can effectively stimulating the maturation of the dendritic cells, increasing the surface synergistic stimulation molecules CD80, CD86, I-A- 2. The endogenous gene vaccine can effectively induce the secretion of the cytokines IL-12, I, The DC of the endogenous gene vaccine load can stimulate the proliferation of CD4 ~ + T cells and induce it to differentiate into the Th1 direction. The method provided by the invention has the functions of enhancing the antigen extraction function of the DC and effectively activating the CD4 + T cell by using the Ii chain as a vector to modify the DC of the endogenous target gene of the Th1 epitope of the HCV-NS3,
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2480568
[Abstract]:Type C viral hepatitis is an infectious disease that is seriously harmful to human health. The worldwide prevalence of hepatitis C virus (HCV) is about 100 million, accounting for 1-3 per cent of the world's total population. In the infected population, about 80% to chronic and 20% developed into liver cirrhosis and hepatocellular carcinoma; HCV-infected people in China account for 3.2% of the total population, and the total number of people infected is estimated to be about 40 million. The molecular biology of HCV has been studied since the first cloning of the HCV cDNA in 1989. The present invention has shown that HCV is a single-stranded positive-chain RNA virus, which is a single-stranded positive-chain RNA virus, which is a polyprotein precursor of the total length of 9600 nucleotides and the ORF region encoding 3010 amino acids, and the protease which is encoded by the host and the virus itself is cracked into ten functional fragments, Four of which are structural proteins, respectively, are core protein C, envelope protein E1, E2 and P7, and six are non-structural proteins, and are NS2, NS3, NS4A and NS4B, respectively. NS5A and NS5B. Although the basic structure of HCV is clear, the prevention and treatment of HCV is still a worldwide problem. The combination therapy of interferon and virotherapy is the only treatment method, but the treatment method is effective only for less than 50% of patients, and has high cost and easy recovery. There are many defects such as hair and side effects, especially the prevalence of HCV-II/ 1b in China, and the treatment response of interferon is the lowest. Therefore, the development of treatment and preventive hepatitis C vaccine can be used to prevent HCV infection and to control the development of the infected people. The HCV NS3 protein has the activity of serine protease and helicase, participates in the post-translational processing of the viral protein, The T cell response of the 3-protein is closely related to the self-infection. It is recognized that the 1248-1261-position amino acid residue in the NS3 region is the CD4 ~ + Th1 cell epitope. And the epitope has broad-spectrum specificity for MHC restriction. The prior research has been used as an experimental animal model in BALB/ c mice, and the molecular chaperone constant chain (Ii) in the path of the MHC II antigen is taken as a carrier, and the C of the Ii chain is replaced by the 1248-1261 epitope of the HCV-NS3. The LIP coding fragment constructs the endogenous targeting gene vaccine and studies the interaction between the constructed gene vaccine and the antigen-presenting molecule at the cell level, using the constructed vaccine pair In this study, the in vitro transcription vector of the endogenous target gene vaccine was constructed and the mRNA was transcribed in vitro, and the mice were used in the same time. The bone marrow cells were cultured in vitro, and the dendritic cells from the bone marrow of the mouse were successfully cultured. The endogenous target gene vaccine mRNA was introduced into the mouse dendritic cell (DC) by the method of electrospinning. The effect of the target gene vaccine on the expression of DC surface and the secretion of cytokines. to study endogenous Whether the target gene vaccine affects the DC-to-T cell-induced differentiation function. The results are as follows:1. The endogenous gene vaccine can effectively stimulating the maturation of the dendritic cells, increasing the surface synergistic stimulation molecules CD80, CD86, I-A- 2. The endogenous gene vaccine can effectively induce the secretion of the cytokines IL-12, I, The DC of the endogenous gene vaccine load can stimulate the proliferation of CD4 ~ + T cells and induce it to differentiate into the Th1 direction. The method provided by the invention has the functions of enhancing the antigen extraction function of the DC and effectively activating the CD4 + T cell by using the Ii chain as a vector to modify the DC of the endogenous target gene of the Th1 epitope of the HCV-NS3,
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
【引证文献】
相关博士学位论文 前1条
1 徐全壹;“寒淫”致病及转归的转录组与代谢组实验研究[D];成都中医药大学;2012年
本文编号：2480568
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