丙型肝炎病毒结构基因的表达及其病毒样颗粒研究
本文关键词:丙型肝炎病毒结构基因的表达及其病毒样颗粒研究 出处:《第二军医大学》2005年博士论文 论文类型:学位论文
更多相关文章: 丙型肝炎病毒 病毒样颗粒 核心基因 E2基因杆状病毒 昆虫细胞 融合表达 免疫 抗原性 免疫原性 疫苗
【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
[Abstract]:Hepatitis C virus (HCV) is one of the main causes of acute and chronic hepatitis in humans. There are about 170 million people infected with HCV in the world. About 50% of the infected patients developed chronic, of which about 10% developed cirrhosis. About 1 / 5% of HCV vaccine can be developed into hepatoma. Because of the lack of suitable HCV proliferation system in vitro and a suitable small animal model, the development of HCV vaccine. HCV is a single positive strand RNA virus. The genomic RNA is about 9.5 kb long and has only one open reading frame, ORF, which encodes a polymeric protein precursor of about 3011 amino acids. Under the action of host signal peptidase and viral protease, the precursor protein is divided into structural protein and non-structural protein of virus. The three structural proteins include core protein. Envelope proteins E1 and E2 play a key role in inducing humoral and cellular immune responses and become the target antigens for the study of HCV vaccines. In this study, Bac-to-Bac baculovirus expression system was selected to express truncated HCV core protein and EGFP fusion protein. To investigate the transfection efficiency and expression effect of recombinant baculovirus expression vector, and then express the full-length HCV core protein and truncated E2 fusion protein, and study the antigenicity of the recombinant baculovirus expression vector. At the same time, we constructed three recombinant baculovirus expression vectors containing all the structural genes of HCV, and obtained recombinant baculovirus. To investigate the expression of HCV full-length gene C-E _ 1-E _ 2 in insect cells and the characteristics of its assembly to form VLP, and to study the binding characteristics of VLP with various cells in vitro. Antigenicity and immunogenicity in animals. Fusion expression of hepatitis C virus core antigen and green fluorescent protein gene The truncated core protein gene fragment was amplified by PCR from the plasmid pGEM-HCJ4 containing the full-length cDNA of HCV. It was inserted into transposon carrier pFastBacl. The recombinant transposable plasmid pFastCt.The EGFP gene was amplified by PCR from the expression plasmid pEGFP-N1 containing enhanced green fluorescent protein gene. The fusion vector pFastCt-EGFP. was constructed after subcloned into the HCV core gene of pFastCt. The recombinant plasmid was identified and transformed into Escherichia coli DH10Bac. The shuttle plasmid BacmidCt-EGFP. was transfected into insect Sf9 cells by transposition in bacteria. EGFP was observed by fluorescence microscope to count the number of transfected positive cells. It was found that the transfection efficiency of high molecular weight plasmid BacmidCt-EGFP was much lower than that of small molecular weight plasmid pEGFP-N1. A series of experiments were carried out to compare the effect of transfection with different concentrations of high molecular weight plasmids. The base number of Bacmid was determined for the subsequent transfection experiment. Recombinant baculovirus rBacCt-EGFP was obtained from the supernatant of cell culture transfected with BacmidCt-EGFP. It was identified by SDS-PAGE and western blot. It was found that the fusion protein with molecular weight of about 40000 was successfully expressed. The fusion protein could react with 15 out of 28 HCV positive sera by ELISA method, and the positive rate was 54%. It shows that it has certain antigenicity. Expression and antigenicity of HCV core and truncated envelope protein E2 gene in insect cells Using pGEM-HCJ4 plasmid as template, complete C gene and truncated E2 gene fragments were amplified by PCR, and then ligated with pMD18-T vector respectively. The recombinant plasmids pMD-C and pMD-E2twere digested with BamHI and XbaI, and the pMD-E2t was digested with XbaI and EcoRI. The C gene and the digested C gene were recovered respectively.
【学位授予单位】:第二军医大学
【学位级别】:博士
【学位授予年份】:2005
【分类号】:R373
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