【作者】 朱诗应；

【导师】 戚中田； 潘卫； 潘欣；

【作者基本信息】 第二军医大学 ， 微生物学， 2005， 博士

【摘要】 丙型肝炎病毒(Hepatitis Cvirus,HCV)是引起人类急、慢性肝炎的主要致病因子之一,全球HCV感染者约1.7亿人,约50～80%感染者发展成慢性,其中约10～20%发展为肝硬化,约1%～5%可发展为肝癌。由于迄今仍缺乏合适的HCV体外增殖系统及适宜的小动物模型,严重制约了HCV疫苗的研制。HCV为单正链RNA病毒,基因组RNA长约9.5kb,仅有一个开放读码框架(ORF),编码一约3011个氨基酸的多聚蛋白前体,该前体蛋白在宿主信号肽酶及病毒蛋白酶作用下,裂解为病毒的结构蛋白与非结构蛋白。三个结构蛋白包括核心蛋白、包膜蛋白E1和E2在诱导机体体液和细胞免疫应答中起着关键性作用,成为研究HCV疫苗的靶抗原。因此,本研究选择Bac-to-Bac杆状病毒表达系统,首先表达截短的HCV核心蛋白和EGFP融合蛋白,探讨重组杆状病毒表达载体的转染效率和表达效果。再表达全长的HCV核心蛋白和截短的E2融合蛋白,分别研究其抗原性。在此基础上,我们同时构建含HCV全部结构基因的三种重组杆状病毒表达载体,获得重组各种重组杆状病毒,探讨HCV全长结构基因(C-E1-E2)在昆虫细胞中的表达及其装配形成VLP的特点,研究VLP在体外和各种细胞的结合特征、抗原性以及在动物体内的免疫原性。 一、丙型肝炎病毒核心抗原和绿色荧光蛋白基因的融合表达 用PCR方法从含HCV全长cDNA的质粒pGEM-HCJ4中扩增出截短的核心蛋白基因片段(Ct),将其插入转座子载体pFastBacl,得到重组转座质粒pFastCt。用PCR方法从含有增强绿色荧光蛋白基因的表达质粒pEGFP-N1上扩增出EGFP基因,再将其亚克隆到pFastCt中HCV核心基因之后,构建成融合表达载体pFastCt-EGFP。鉴定后将其转化大肠杆菌DH10Bac,经细菌内的转座作用,得到穿梭质粒BacmidCt-EGFP。以之转染昆虫Sf9细胞,通过荧光显微镜观察EGFP来计数转染阳性细胞的数量,发现高分子量质粒BacmidCt-EGFP的转染效率远远不及小分子量质粒pEGFP-N1,通过系列实验比对使用不同浓度高分子量质粒进行转染所取得的效果,为后续的转染实验确定了Bacmid用量的基数。从BacmidCt-EGFP转染的细胞培养上清中获得了重组杆状病毒rBacCt-EGFP,经过SDS-PAGE和western blot鉴定,发现其成功表达了分子量约40000融合蛋白。以ELISA方法检测发现该融合蛋白能与28份HCV阳性血清中的15份发生反应,阳性率为54%,表明其具有一定的抗原性。 二、HCV核心和截短的包膜蛋白E2基因在昆虫细胞中的表达及其抗原性 以pGEM-HCJ4质粒为模板,分别用PCR扩增出完整C基因和截短的E2基因片段,再分别与pMD18-T载体连接,得到重组质粒pMD-C和pMD-E2t。用BamHI和XbaI酶切pMD-C,用XbaI和EcoRI酶切pMD-E2t,分别回收酶切的C基因和更多还原

【Abstract】 Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis. It has been estimated that about 170 million people worldwide are infected with HCV, of whom 50~ 80% will develop chronic liver disease, leading to cirrhosis in 10 to 20% and hepatocellular carcinoma in 1 to 5% of chronically infected individuals. Because lack of tissue culture systems and small animal models, the development of a prophylactic vaccine is very difficult. The linear, single-stranded, positive-sense HCV RNA genome of 9.5 kb contains a single open reading frame (ORF) encoding a polyprotein which is cleaved into the structural proteins and nonstructural proteins by host- and virus-specific proteinases. Three structural proteins including the core protein and two envelope proteins El and E2 play a pivotal role in inducing cellular and humoral immune responses. In this study, first, we expressed a fused protein including HCV truncated core protein with EGFP by using Bac-to-Bac baculovirus expression system, to explore the transfecting efficiency of recombinant baculovirus expression vector, then, we expressed a fused protein including HCV full-length core protein and truncated E2 protein, to study its antigenicity. finally, we studied the HCV-like particles(HCV-VLP) in a recombinant baculovirus that contains the HCV structural gene(core/E1/E2), investigated the binding of HCV-VLP to different cell lines, and evaluated its antigenicity and immunogenicity.1. Expression of hepatitis C virus core protein and EGFP gene in insect cellsHCV truncated core gene amplified by PCR using pGEM-HCJ4 plasmid as the template which contains full-length cDNA clone of HCV, and was inserted into baculovirus expression vector pFastBacl, constructed a recombinant vector, pFastCt. EGFP gene amplified by PCR method, and subcloned into pFastCt, constructed a fusion expression vector, pFastCt-EGFP. After transposition in DHlOBac E .coli., recombinant baculovirus BacmidCt-EGFP were gained. After the transfection of BacmidCt-EGFP to Sf9 cells , using fluorescence microscope observation, we found that the fluorescenced cells transfected with high molecular Bacmid is less than the efficiency of pEGFP-N1. SDS-PAGE and Western blot showed that expressed fusion protein Ct-EGFP had an expected molecular mass of 40 kDa. ELISA results showed that fusion protein Ct-EGFP was able to react with 15 /20 anti-HCV sera samplesfrom HCV patients. The reactivity frequency was 54%.2. Expression of hepatitis C virus core gene and truncated envelope 2 gene in insect cells and its antigenicityTo study the antigenicity of Sf9 insect cells expressed products of HCV different structural gene and obtain HCV core protein and E2 protein, HCV truncated core gene and truncated envelope E2 gene were amplified with PCR method by using plasmid pGEM-HCJ4 as the template, and cloned into pFastBacHTa vector, constructed a fusion expression vector, pFBC-E2t. After transformed into DHlOBac E xoli., we gained recombinant baculovirus transposon-shuttle vector Bacmid C-E2t, and then transfected into Sf9 cells. The expression product was analyed by SDS-PAGE and Western-blot, showed two bands with different molecular weight: 21 kDa band is HCV core protein ,the 33 kDa band is truncated E2 protein, demonstrated that recombinant proteins were expressed and cleaved into the core protein and E2 protein in Sf9 cells. Purified protein could react strongly with monoclonal antibody (mAb) against HCV core protein. ELISA results showed that, fusion protein C-E2t was able to react with 20 /28 anti-HCV sera samples from HCV patients. The reactivity frequency was 71%. using affinity chromatography purified C protein was able to react with 18 /20 anti-HCV sera samples from HCV patients. The reactivity frequency was 64%, The successful expression of recombinant HCV CE2 protein will be helpful in the study of HCV vaccine.3. Expression of hepatitis C virus structural gene and assembly, purification of HCV-like particlesThree fragments(5’NCR- C, 5’NCR- C-E1,5’NCR- C-E1-E2) produced by long template PCR, were separately subcloned into baculovirus donor plasmid pFastBacl, and three expression vectors( pFastA, pFastB, pFastQwere constructed, respectively. The vectors were verified and used to transform DHlOBac. We can see the existence of abundant baculoviruses in the nuclei of transfected cells , and gained recombinant baculovirus transposon-shuttle vector BacmidA, BacmidB, BacmidC to generate recombinant baculoviruses, yielding rBacA , rBacB ,rBacC, respectively. SDS-PAGE and Western blot analysis of lysates from infected Sf-9 cells with either recombinant baculoviruses demonstrated that rBacA showed one novel band of the size of expected HCV C protein which is 21 kD, rBacB demonstrated 2 novel bands更多还原