【作者】 郭泰林；

【导师】 叶林柏； 郜金荣；

【作者基本信息】 武汉大学 ， 微生物学， 2003， 硕士

【摘要】 HCV是引起丙型肝炎的病原体。70％以上患者可转变成慢性肝炎，其中约25％的慢性丙肝病人可转变成肝硬化或肝癌。目前还没有理想的治疗药物，研究和发展有效的疫苗用以预防HCV感染是一个迫切需要解决的问题。HCV的E2包膜糖蛋白一直是国际上HCV疫苗研究的重点目标。我们以前的研究已证实E2 DNA疫苗能够诱导动物的体液免疫。能否有效地诱导产生中和性保护抗体和诱导细胞免疫，产生特异性CTL反应以清除体内病毒，是评价疫苗的重要指标。本论文研究HCV E2工程蛋白诱导的CTL反应。 为了获得E2工程蛋白，我们应用PCR技术从HCV基因组cDNA克隆质粒中扩增获得不同长度的E2基因序列，构建了两个重组真核表达质粒：pCE2-1(422-661)，pCE2-2(383-661)和两个重组原核表达质粒：pQE2(417-661)，pET-HIS-E2(417-750)，分别在猴肾细胞COS-7和工程菌株JM109、BL21中表达，我们发现，以上的所有质粒，在对应宿主中，都表达产生了E2蛋白，Western-blot结果充分地证明表达出的E2蛋白能与E2单克隆抗体特异性反应。但表达水平不高，如何提高E2的表达水平有待进一步解决。 JM109 pQ-E2菌株诱导培养5-6小时后，收集细胞用超声波振荡裂解，用Ni-NTA-Superflow亲和层析柱从裂解液中提取和纯化E2蛋白作为抗原。抗原用脂质体包被，采用腹腔、皮下、肌肉注射的方式，在盐酸普鲁卡因及淀粉的辅助作用下，免疫BALB/c鼠后取鼠脾脏制备淋巴细胞，用IL一2、Con一A、PHA及肤段定向刺激扩增后做为效应细胞，与经过重组真核表达质粒pCEZ转染的PS巧细胞(靶细胞)发生作用，利用LDH释放试验检测作用效果。在效应细胞与靶细胞的细胞数之比为200:1的情况下，三种免疫方式免疫小鼠的脾细胞对靶细胞的杀伤率都超过30%。这些结果表明工程菌株表达的HCV EZ蛋白可以诱发免疫实验动物机体产生特异性CTL应答。由此我们认为EZ蛋白是发展HCV预防工程蛋白疫苗的合适候选者。更多还原

【Abstract】 Hepatitis C Virus(HCV) is a single-stranded positive-sense RNA virus that account for most cases of post-transfection non-A non-B hepatitis. In more than 70% of the cases, HCV causes a prolonged, persistent infection, in which about 25% of the patients can eventually lead to cirrhosis and hepatocellular carcinoma. Currently, the lack of efficient antiviral treatment against HCV makes the development of a vaccine highly difficult. The envelope glycoprotein of HCV is one of the important targets in the study of efficient vaccine in the world. It has been proved that the DNA vaccine of E2 could induce humoral and cellular immunity in mice. Whether induce strong specific antibody and T lymphocytes, causing specific CTL response to eliminate virus in vivo or not is the main index for the vaccine evaluation . In this study, we focused on specific CTL inducing by E2 vaccination.The four recombinant plasmids has been constructed, pCE2-1(422-661), pCE2-2(383-661), pQE2(417-661) and pET-HIS-E2(417-750), which were transformed into COS-7, JM109 and BL21.The expression of E2 protein has been identified by Western-blot analysis using the E2 monoclonal antibody. However,the express efficiency of E2 protein in all of the four vectors are low, more efforts should focus on how to improve it.In the study of the induction of specific CTL, the recombinant E2 that its contain 6 His at N terminal end was purified with Ni-NTA-Superflow column after expression in E.coli JM109. To study the immunological properties of E2 protein, the BALB/c mice were vaccinated with purified E2 protein. Five weeks later, the vaccinated mice had been killed and extracted spleens used to prepared the effecter cells. After simulated and augment in vitro, they were used to kill the target cells P815, which were transfected by recombinant expression vector pCE2. By LDH assay, we observed that more than 30% of the target cells were killed at the ratio 200:1, and there are different results with different immunity injection routes. Altogether our data suggest that the E2 protein can provoke E2 specific CTLs in the mice, from which we conclude that the E2 protein is a good candidate for developing a vaccine to prevent HCV infection.更多还原