丙型肝炎病毒多表位蛋白的抗原性及免疫应答研究

发布时间：2018-01-03 13:38

本文关键词：丙型肝炎病毒多表位蛋白的抗原性及免疫应答研究　出处：《第二军医大学》2005年硕士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(hepatitis C virus,HCV)主要通过血液途径传播,能引起急、慢性肝炎并可导致肝硬化和肝细胞癌。全世界现有HCV感染者约1.7亿,严重影响人类健康,其治疗常用干扰素和利巴韦林,应答率也仅约为40%,因此,研制HCV疫苗具有重要意义。HCV基因组全长一般在9379～9481nt之间,编码一个约含3000个氨基酸的多聚蛋白前体,在宿主和病毒自身蛋白酶的加工下产生结构蛋白(C、E1和E2)和非结构蛋白(NS2、NS3、NS4和NS5等)。结构蛋白E2中的第384-410位氨基酸区段是个高度变异区,称为HVR1。众多实验表明,HVR1中存在中和表位,其相应的中和抗体不仅可以阻止病毒对靶细胞的粘附,而且在感染痊愈过程中起着重要作用,但该区段高度变异,诱生的抗体具有株特异性,往往对不同HCV变异株的感染缺乏交叉保护,给丙型肝炎疫苗的研制造成困难。本实验室在前期工作中,曾针对HCV的免疫学特性,优选了数条能模拟大多数HVR1免疫原性的HVR1模拟表位,同时联合应用多条HCVT细胞表位,设计合成出了一段包含9条HCVE2蛋白HVR1(384～410aa)模拟B细胞表位,2条C区保守CTL表位(35～44aa,132～140aa)、1条NS3区保守CTL表位(1073～1081aa)及1条NS3区保守Th表位(1251～1259aa)的多表位抗原融合基因(multi-epitope fragment combination,mfc),并对其免疫原性进行了分析,发现该多表位抗原基因能够诱生针对HCV的特异性免疫应答。在此基础上,为进一步提高mfc基因的免疫原性,本研究将HAsAg基因融合在mfc基因的N端,构建了以HBsAg为佐剂的HCV DNA疫苗,在小鼠中检测了其免疫效果;同时,本研究从mfc基因中优选出5条HVR1模拟B细胞表位基因(hcvme),与gst基因融合后经大肠杆菌表达并纯化出重组蛋白GST-HCVME,通过小鼠免疫实验对其免疫原性进行了分析;此外,本研究还检测了mfc基因在大肠杆菌中的重组表达产物—GST-MFC蛋白对丙肝患者外周血淋巴细胞的增殖作用情况。一、以乙型肝炎表面抗原基因为佐剂的丙型肝炎病毒多表位DNA疫苗的构建及在小鼠中的免疫应答研究为增强HCV多表位DNA疫苗的免疫效果,我们将HBsAg基因融合在mfc基因的N末端,构建了HBsAg+MFC的重组HCV DNA疫苗,体外转染HEK 293T细胞检测了其瞬时表达情况,体内实验采用肌肉注射法接种BALB/c小鼠对其免疫效果进行了评价,检测了其诱导的抗体产生情况、特异性抗体同HVR1合成肽的交叉反应率、MFC蛋白特异性的免疫小鼠脾细胞增殖反应及细胞因子IFN-γ
[Abstract]:Hepatitis C virus hepatitis C virus (HCV) is mainly transmitted through blood channels, can cause acute. Chronic hepatitis can also lead to cirrhosis and hepatocellular carcinoma. There are about 170 million HCV infected people in the world, seriously affecting human health, its commonly used interferon and ribavirin treatment, the response rate is only about 40%. Therefore, the development of HCV vaccine is of great significance. The length of HCV genome is generally between 9379 and 9481 NT, encoding a polyprotein precursor containing about 3000 amino acids. Under the processing of host and virus autoproteinases, structural proteins (Con E1 and E2) and nonstructural proteins (NS2 + NS3) were produced. NS4 and NS5 et al. The 384-410 amino acid region of structural protein E2 is a highly variable region called HVR1.Many experiments indicate that neutralization epitopes exist in HPVR1. The corresponding neutralizing antibody can not only prevent the virus adhesion to target cells, but also play an important role in the healing process of infection. It is difficult to develop hepatitis C vaccine because of the lack of cross protection against infection of different HCV variants. In our previous work, we selected several HVR1 mimic epitopes which can mimic the immunogenicity of HVR1 in view of the immunological characteristics of HCV. At the same time, using multiple HCVT cell epitopes, we designed and synthesized a segment containing 9 HCVE2 protein HVR1O384 (410aa) to mimic B cell epitopes. There were 2 conserved CTL epitopes in region C (35 ~ 44aA ~ (132140aa)). A polyepitope antigen fusion gene of a conserved CTL epitope of NS3 region 1073, 1081aa, and a conserved Th epitope of NS3 region, 1251a (1259aa). Multi-epitope fragment combination. It was found that the multiepitope antigen gene could induce the specific immune response to HCV. On this basis, the immunogenicity of mfc gene could be further improved. In this study, the HAsAg gene was fused into the N-terminal of mfc gene, and the HCV DNA vaccine with HBsAg as adjuvant was constructed, and its immune effect was tested in mice. At the same time, five HVR1 mimic B cell epitope genes (hcvme) were selected from mfc gene. The recombinant protein GST-HCVMEwas expressed and purified by E. coli after fusion with gst gene. The immunogenicity of GST-HCVMEwas analyzed by immunological assay in mice. In addition, GST-MFC protein, a recombinant expression product of mfc gene in Escherichia coli, was used to detect the proliferation of peripheral blood lymphocytes in patients with hepatitis C. 1. Construction of hepatitis C virus multiepitope DNA vaccine with hepatitis B surface antigen as adjuvant and its immune response in mice In order to enhance the immune effect of HCV multiepitope DNA vaccine, we fused HBsAg gene into the N-terminal of mfc gene. The recombinant HCV DNA vaccine of HBsAg MFC was constructed and its transient expression was detected by transfection of HEK 293T cells in vitro. In vivo, the immune effect of BALB/c mice was evaluated by intramuscular injection, and the production of specific antibody and the cross-reaction rate between specific antibody and HVR1 synthetic peptide were detected. Proliferative response of spleen cells and cytokine IFN- 纬 in immunized mice with specific MFC protein
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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