【作者】 刘晶；

【导师】 李光地；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2002， 博士

【摘要】 丙型肝炎病毒(hepatitis C virus, HCV)感染可导致慢性肝炎、肝硬化以至肝癌，是威胁人类健康的重大病原体。研制抗HCV疫苗是当前重要而且紧迫的任务。多种证据表明，HCV被膜蛋白E1(aa 192-383)和E2(384-746)在HCV感染靶细胞的过程中可能发挥了重要而且关键的作用，因此成为HCV疫苗研制的主要靶分子。基于被膜蛋白的HCV疫苗研究急需解决抗原检测和疫苗接种后的免疫反应评价的问题，需要首先得到大量高纯度的被膜蛋白抗原及其相应抗体或抗血清。为此，在大肠杆菌系统中进行了1b亚型HCV被膜蛋白表达和纯化的研究。采用6xHis融合表达体系，在大肠杆菌中实现了E2 aa 385-565、aa 450-565、aa 567-730和aa 385-730四个片段的高表达，并获得了高度纯化的产物。在免疫印迹检测中，上述片段与丙型肝炎病人血清有特异性反应，表明大肠杆菌系统表达的E2蛋白携带有HCV E2特异的、不依赖于糖化和立体构象的抗原决定簇。利用aa 385-730片段，建立了病人血清中抗E2抗体的ELISA检测体系，该体系在56%的丙型肝炎病人血清内检出了抗E2抗体，并且发现抗E2抗体的存在与HCV病毒血症存在正相关。利用aa 385-565片段，建立了E2疫苗免疫后抗体反应的ELISA评价体系，并已成功用于重组痘苗病毒疫苗和DNA疫苗的研究。利用aa 450-565和aa 385-730两片段免疫家兔，获得了可识别大肠杆菌和哺乳动物细胞表达之E2蛋白的多抗血清，并且在某些亚型之间具有一定程度的交叉反应性。采用6xHis融合表达体系，在大肠杆菌中实现了E1 aa 192-340和aa 192-326两个片段的高表达，并获得了高度纯化的产物。利用aa 192-326免疫家兔，获得了可识别大肠杆菌和哺乳动物细胞表达之E1蛋白的多抗血清，表明大肠杆菌系统表达的E1蛋白携带有HCV E1特异的、不依赖于糖化和立体构象的抗原决定簇。通过以上工作，得到了大量、高纯的被膜蛋白抗原和被膜蛋白特异的抗血清，建立了被膜蛋白抗原、抗体的检测体系。同时，在表达被膜蛋白的过程中，对E1和E2在大肠杆菌中的表达规律作了一些有益的探索。更多还原

【Abstract】 Hepatitis C virus (HCV) infection could lead to chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma, constituting a major threat to human health. There is a pressing need to develop effective anti-HCV vaccines. Different lines of evidence suggest that the envelope proteins of HCV, E1 (aa 192-383) and E2 (384-746), may play important and vital roles in its infection of target cells, thus becoming the main targets of HCV vaccine research. Research on envelope protein-based HCV vaccine demands a prompt solution to the problems of detection of antigen and evaluation of post-vaccination immune responses, which requires large amounts of highly purified envelope proteins and corresponding antibody or antiserum. Therefore, the expression and purification of subtype 1b envelope proteins by using the Escherichia coli system were studied.Four E2 fragments, aa 385-565, aa 450-565, aa 567-730 and aa 385-730, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. In immuno-blotting, these fragments reacted specifically with hepatitis C patients’ sera, suggesting that E. coli-derived E2 proteins carried HCV E2-specific, glycosylation-and-conformation-independent epitopes. ELISA system for the detection of anti-E2 antibodies in patients’ sera was established using aa 385-730 fragment, which detected anti-E2 in 56% of hepatitis C patients and demonstrated a correlation between anti-E2 and HCV viraemia. By using aa 385-365 fragment, an ELISA system for the evaluation of post-E2-vaccination humoral immune responses was also established, and was successfully applied to recombinant vaccinia virus- and DNA-based vaccine research. By immunizing rabbits with aa 450-565 or aa 385-730 fragment, polyclonal sera were obtained that were able to recognize E2 proteins expressed in both E. coli and mammalian cells, and cross-react between certain HCV subtypes. Two E1 fragments, aa 192-340 and aa 192-326, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. By immunizing rabbits with aa 192-326 fragment, polyclonal sera were obtained that were able to recognize E1 proteins expressed in both E. coli and mammalian cells, suggesting that E. coli-derived E2 proteins carried HCV E1-specific, glycosylation-and-conformation-independent epitopes.This work produced large amounts of high-purity envelope protein antigens and envelope-specific antisera, and established detection systems for envelope antigens as well as antibodies. Meanwhile, in the process of expressing envelope proteins, some useful insights into the mechanisms governing HCV envelope protein expression in E. coli were obtained.更多还原