【作者】 许信刚；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 博士

【摘要】 丙型肝炎病毒(HCV)是输血后非甲非乙型肝炎以及社区获得性肝炎的主要病原因子,目前全世界约有2 亿HCV感染者。HCV感染后缺乏有效的保护性免疫。目前对HCV感染尚未找到有效的治疗手段,因此需要提高治疗手段和发展有效的疫苗以控制HCV感染。HCV 为单股正链RNA 病毒,属于黄病毒科的成员。其基因组全长9.4kb,编码约3 000 氨基酸的多聚蛋白前体,在宿主及病毒蛋白酶的作用下,将其切割成3 个结构蛋白和7 个非结构蛋白,其中有2 个糖蛋白E1 和E2 为HCV的包膜蛋白。由于其位于病毒颗粒的最外侧,是宿主免疫系统攻击的主要目标,也是HCV 疫苗研究的首选抗原。近年来各国学者运用基因重组技术对HCV包膜糖蛋白的结构和功能进行了深入研究,以期进一步理解HCV感染后慢性化机制及病毒与宿主相互作用的关系,并寻找有效的保护性疫苗及治疗药物。Chiron公司的研究人员在1994 年首先报道了用哺乳动物细胞表达的E1 和E2 蛋白共免疫大猩猩,可诱生出中和抗体能抵抗同源病毒攻击的。他们认为在大猩猩免疫试验中,E2 区尤其是E2 的高变区之一HVR1(386～411 位氨基酸)对于诱导中和抗体的保护性免疫具有更为重要的作用。但是由于E2 区的高度变异性,针对某种型或株的抗体往往对其他型或株的HCV 无中和作用。而且有的学者认为抗E2 抗体对病毒感染的预防作用仍难以肯定。由于尚未建立HCV 体外复制系统,病人血清中的HCV 病毒粒子又极难纯化,迄今为止的HCV被膜蛋白研究几乎完全以各种系统中表达的重组蛋白为对象。有证据表明,只有哺乳动物细胞中表达的E1、E2 糖蛋白才能较好地反映天然HCV病毒粒子上被膜蛋白的构象和性质。但就目前而言,E1 及E2 糖蛋白在哺乳动物细胞中的高效表达,以及表达蛋白的纯化,仍然是世界范围内都未攻克的难题,严重影响了被膜蛋白相关研究及其临床应用的进展,说明HCV 中和抗体的研究还面临许多困难。本研究由三部分构成,第一部分是用逆转录病毒载体将HCV囊膜蛋白基因整合到SP2/0 细胞基因组中,在SP2/0 细胞的细胞膜上成功表达了HCV 囊膜蛋更多还原

【Abstract】 Hepatitis C virus (HCV) is a major causative agent of post-transfusion and community-acquired hepatitis in the world. The majority of HCV-infected individuals develop chronic hepatitis progressing eventually to liver cirrhosis and hepatocellular carcinoma. Neither an effective treatment for chronic HCV infection nor a vaccine to prevent HCV infection is available at the present time. The only available treatment, a combination of alpha interferon and ribavirin, is efficacious in only a minority of patients. The development of such therapies will be aided greatly by a more complete picture of the structure-function features of HCV proteins. HCV is an enveloped plus-strand RNA virus of the Flaviviridae family. Its genome is 9.4 kb in length with one open reading frame that is translated as a single polyprotein, which is processed by host and virus proteases into at least three structural and seven non-structual proteins with various enzymatic activities. Two glycoproteins are probably virion envelope proteins, which are obvious candidates for inclusion in a subunit vaccine because of its potential role in HCV attachment. Recently, scholars from all over the world studied structure and function of HCV deeply by means of gene recombination technique .They want to understand infectious chronic mechanism and reciprocity of HCV and host and search effective vaccine and curative medicine. In 1994, researchers of Chiron company report experiment result of HCV vaccine using gorillas. They found gorillas immuned by mammiferous cells express HCV E1E2 protein could induce neutralizing antibody and resist HCV attack. They thinked HCV E2 region especially E2 hypervariable region (HVR, aa386~411) have important action for inducing neutralizing antibody. On the other side, E2 region has high variance and different types and strains have not neutralization. Some scholars considered E2 antibody have no neutralization. Because we have no HCV in vitro reproducing system and concentrated patients serum HCV difficultly, we studied E2 recombinant protein expressed by all kinds of expression systems. Evidences showed that mammiferous cells expressing HCV E1E2 protein can embody structure and character of natural HCV membrane protein. But recently, it is very difficult problem in the world to express and concentrate E1E2 protein highly in mammiferous cells. This effect seriously study and application of E1E2 membrane protein. All of these show study of HCV neutralizing antibody faces a lot of difficulties. The paper has three parts. The first part includes HCV E1E2 protein was expressed in SP2/0 cells’envelope successfully using retroviral express system. Balb/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells and induced specific anti-E1E2 antibody in mouse serum. Virus neutralization antibody was detected .These established bases for studying HCV neutralizing antibody .The second part includes HCV E1E2 gene vaccine was constructed and Balb/c mice were injected intramuscularly with the nucleic acid vaccine. Anti-HCV E1E2 neutralization antibody was screened .The third part is expression of the major antigen region of E2 gene of HCV in E.coli , the fused E2 protein was highly expressed in E.coli prokaryotic expression system in the experiments and provided the basic materials for the HCV diagnose. Main research contents include: 1. The recombinant retroviral vector pBABE-puro-E1E2 was constructed by inserting full-length HCV E1E2 gene of H77 strain into pBABE-puro. Both the recombinant retroviral vector and pVSVg plasmid were transfected into eukaryotic cells 293GP by calcium phosphate transfection method. And then, the pseudovirus was produced. The pseudovirus infected eukaryotic cells SP2/0 and E1E2 protein was expressed. E1E2 protein was detected by puromycin-resistant and FACS analysis. Balb/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells. Anti-HCV E1E2 antibody was screened by FACS. The results showed that HCV E1E2 protein was expressed in SP2/0 cells’envelope successfully. FACS could detect specific anti-E1E2 antibody in SP2/0 cells immune mouse serum. Virus neutralization antibody was detected by HCV pseudovirus .The result showed anti-HCV E1E2 antibody has no neutralization activity. Mouse spleen cells were fused with myeloma SP2/0. Culture supernatants of hybridoms were screened by FACS and four cell lines which could secrete against E1E2 protein of HCV McAbs stably were obtained. The McAbs could react with HCV E1E2 protein specially. The McAbs was detected by HCV pseudovirus .The result showed McAbs has no neutralization activity too. 2. Hepatitis C Virus(HCV) H77 strain E1E2 gene vaccine was constructed by inserting full-length cDNA of HCV E1E2 into an eukaryotic expression vector pcDNA4.0. The recombinant plasmid was transfected into eukaryotic cells 293T bycalcium phosphate transfection method and transient expressive E1E2 envelope protein was analyzed by FACS. Balb/c mice were injected intramuscularly with the recombinant plasmid. Anti-HCV E1E2 antibody was screened by FACS and antigen was SP2/0 cells which expressed HCV E1E2 protein. The results showed that HCV E1E2 protein was expressed transiently in 293T cells. Specific anti-E1E2 antibody could be detected in DNA immune mouse serum by FACS and the antibody could react specially to SP2/0 cells which expressed HCV E1E2 protein. Virus neutralization antibody was detected by HCV pseudovirus .The result showed anti-HCV E1E2 antibody has no neutralization activity. 3. The major antigen region E1and E2 gene of HCV H77 strain were cloned into the expression vector pET32a, the recombinant plasmids pET32a-E1 and pET32a-E2 were obtained. The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis. SDS-PAGE indicated that both of the recipient germs translated and induced by the recombinant plasmid pET32a-E2 could express the major antigen region of E2 gene. Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of HCV.更多还原