本文關鍵詞：丙型肝炎病毒結(jié)構(gòu)基因的表達及其病毒樣顆粒研究　出處：《第二軍醫(yī)大學》2005年博士論文　論文類型：學位論文

  更多相關文章： 丙型肝炎病毒 病毒樣顆粒 核心基因 E2基因桿狀病毒 昆蟲細胞 融合表達 免疫 抗原性 免疫原性 疫苗

【摘要】：丙型肝炎病毒(Hepatitis Cvirus,HCV)是引起人類急、慢性肝炎的主要致病因子之一,全球HCV感染者約1.7億人,約50～80%感染者發(fā)展成慢性,其中約10～20%發(fā)展為肝硬化,約1%～5%可發(fā)展為肝癌。由于迄今仍缺乏合適的HCV體外增殖系統(tǒng)及適宜的小動物模型,嚴重制約了HCV疫苗的研制。HCV為單正鏈RNA病毒,基因組RNA長約9.5kb,僅有一個開放讀碼框架(ORF),編碼一約3011個氨基酸的多聚蛋白前體,該前體蛋白在宿主信號肽酶及病毒蛋白酶作用下,裂解為病毒的結(jié)構(gòu)蛋白與非結(jié)構(gòu)蛋白。三個結(jié)構(gòu)蛋白包括核心蛋白、包膜蛋白E1和E2在誘導機體體液和細胞免疫應答中起著關鍵性作用,成為研究HCV疫苗的靶抗原。因此,本研究選擇Bac-to-Bac桿狀病毒表達系統(tǒng),首先表達截短的HCV核心蛋白和EGFP融合蛋白,探討重組桿狀病毒表達載體的轉(zhuǎn)染效率和表達效果。再表達全長的HCV核心蛋白和截短的E2融合蛋白,分別研究其抗原性。在此基礎上,我們同時構(gòu)建含HCV全部結(jié)構(gòu)基因的三種重組桿狀病毒表達載體,獲得重組各種重組桿狀病毒,探討HCV全長結(jié)構(gòu)基因(C-E1-E2)在昆蟲細胞中的表達及其裝配形成VLP的特點,研究VLP在體外和各種細胞的結(jié)合特征、抗原性以及在動物體內(nèi)的免疫原性。 一、丙型肝炎病毒核心抗原和綠色熒光蛋白基因的融合表達 用PCR方法從含HCV全長cDNA的質(zhì)粒pGEM-HCJ4中擴增出截短的核心蛋白基因片段(Ct),將其插入轉(zhuǎn)座子載體pFastBacl,得到重組轉(zhuǎn)座質(zhì)粒pFastCt。用PCR方法從含有增強綠色熒光蛋白基因的表達質(zhì)粒pEGFP-N1上擴增出EGFP基因,再將其亞克隆到pFastCt中HCV核心基因之后,構(gòu)建成融合表達載體pFastCt-EGFP。鑒定后將其轉(zhuǎn)化大腸桿菌DH10Bac,經(jīng)細菌內(nèi)的轉(zhuǎn)座作用,得到穿梭質(zhì)粒BacmidCt-EGFP。以之轉(zhuǎn)染昆蟲Sf9細胞,通過熒光顯微鏡觀察EGFP來計數(shù)轉(zhuǎn)染陽性細胞的數(shù)量,發(fā)現(xiàn)高分子量質(zhì)粒BacmidCt-EGFP的轉(zhuǎn)染效率遠遠不及小分子量質(zhì)粒pEGFP-N1,通過系列實驗比對使用不同濃度高分子量質(zhì)粒進行轉(zhuǎn)染所取得的效果,為后續(xù)的轉(zhuǎn)染實驗確定了Bacmid用量的基數(shù)。從BacmidCt-EGFP轉(zhuǎn)染的細胞培養(yǎng)上清中獲得了重組桿狀病毒rBacCt-EGFP,經(jīng)過SDS-PAGE和western blot鑒定,發(fā)現(xiàn)其成功表達了分子量約40000融合蛋白。以ELISA方法檢測發(fā)現(xiàn)該融合蛋白能與28份HCV陽性血清中的15份發(fā)生反應,陽性率為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基因在昆蟲細胞中的表達及其抗原性 以pGEM-HCJ4質(zhì)粒為模板,分別用PCR擴增出完整C基因和截短的E2基因片段,再分別與pMD18-T載體連接,得到重組質(zhì)粒pMD-C和pMD-E2t。用BamHI和XbaI酶切pMD-C,用XbaI和EcoRI酶切pMD-E2t,分別回收酶切的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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R373

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