本文選題：丙型肝炎病毒(HCV) + 包膜E2蛋白�。� 參考：《第二軍醫(yī)大學》2006年碩士論文

【摘要】：丙型肝炎是由丙型肝炎病毒(hepatitis C virus,HCV)感染引起的一種世界性傳染病,目前全球HCV感染者近1億7千萬。HCV急性感染后約40-60%以上患者發(fā)展為慢性感染,其中部分病人可發(fā)展為肝硬化或肝癌。迄今為止,對HCV慢性感染尚無高效的特異性治療方法。 HCV屬于黃病毒家族,是單股正鏈RNA病毒,基因組全長約9.5kb,有一個大的開放閱讀框,可翻譯成一個約3000個氨基酸的多聚蛋白前體,該前體蛋白在宿主信號肽酶和病毒自身蛋白酶的加工下,產(chǎn)生結構蛋白(C,E1,E2,)和非結構蛋白(P7,NS2,NS3,NS4A,NS4B,NS5A,NS5B等)。 HCV的包膜糖蛋白E2分布于病毒顆粒表面,可能與病毒和宿主間免疫應答及介導病毒進人靶細胞等過程密切相關,因此成為預防HCV感染研究中的主要角色。E2蛋白能與肝細胞表面的多種分子結合,其中,人CD81分子(hCD81)已被公認為是HCV的重要受體。雖然CD81介導HCV感染的機制尚不清楚,但是體外試驗表明,CD81分子是HCV感染原代肝細胞以及肝源性腫瘤細胞所必需的。因此,靶向人CD81的模擬分子對于HCV感染的治療有著應用前景,這也是目前HCV治療藥物領域的一個研究熱點。 在本實驗中,我們首先分別在哺乳動物細胞中表達了HCV E2蛋白和在大腸桿菌中表達了人CD81分子大胞外環(huán)。再以hCD81單抗為配基,從噬菌體12肽庫中篩選hCD81模擬小肽,通過實驗證明能競爭抑制HCV E2和hCD81的結合。 第一部分 HCV包膜E2蛋白和人CD81分子大胞外環(huán)的表達及HCV假病毒的構建 用中國倉鼠卵巢細胞(CHO)分泌表達HCV包膜E2蛋白。首先根據(jù)1b基因型HCV包膜E2蛋白編碼基因的核苷酸序列設計并合成引物,以聚合酶反應(PCR)擴增HCV包膜E2蛋白外段基因;將neo基因插入真核表達質(zhì)粒pCI-neo的內(nèi)含子基因的剪切供體與剪切受體位點之間,而將HCV包膜E2蛋白基因置于Deo基因內(nèi)含子的3′端,構建了新型的哺乳動物細胞表達質(zhì)粒載體pCIDA-neo-E2。該質(zhì)粒載體利用真核啟動子高水平調(diào)控HCV包膜E2基因的表達,借助于內(nèi)含子的剪切功能篩選用的標記基因(neo)僅低水平表達。以表達質(zhì)粒pCIDA-neo-E2
[Abstract]:Hepatitis C is a worldwide infectious disease caused by hepatitis C virus infection. At present, more than 40 to 60% of the patients with HCV infection in the world develop chronic infection after acute infection. Some of these patients can develop cirrhosis or liver cancer. So far, there is no effective specific treatment for chronic HCV infection. HCV belongs to the family of yellow viruses and is a single-stranded positive strand RNA virus. The genome is about 9.5 kb in length and has a large open reading frame that can be translated into a precursor of about 3,000 amino acids. The precursor protein was processed by the host signal peptidase and the virus itself protease to produce the structural protein (Con E1) and the nonstructural protein (P7 NS2 + NS3), NS4AN4B, NS5B, NS5B, and so on. The envelope glycoprotein E2 of HCV is distributed on the surface of virus particles, which may be closely related to the immune response between virus and host and the process of mediating virus entry into human target cells. Therefore, the E2 protein can bind to a variety of molecules on the surface of hepatocytes. Human CD81 molecule hCD81) has been recognized as an important receptor of HCV. Although the mechanism of HCV infection mediated by CD81 is not clear, the in vitro experiments show that the CD81 molecule is necessary for HCV to infect primary hepatocytes and hepatogenic tumor cells. Therefore, mimic molecules targeting human CD81 have a promising application in the treatment of HCV infection, which is also a research hotspot in the field of drug therapy for HCV. In this study, we first expressed HCV E2 protein in mammalian cells and large extracellular ring of human CD81 molecule in Escherichia coli. Using hCD81 monoclonal antibody as ligand, hCD81 mimic peptide was screened from phage 12 peptide library. It was proved that the binding of HCV E2 and hCD81 could be inhibited by competition. Part I: expression of HCV envelope E2 protein and large extracellular ring of human CD81 molecule and construction of HCV pseudovirus The HCV envelope E2 protein was secreted by Chinese hamster ovarian cells (Cho). Firstly, primers were designed and synthesized according to the nucleotide sequence of 1b genotype HCV envelope E2 protein coding gene, and the outer segment of HCV envelope E2 protein gene was amplified by polymerase reaction. A novel mammalian cell expression plasmid pCIDA-neo-E2 was constructed by inserting the neo gene between the splicing donor and the splicing receptor site of the intron gene of the eukaryotic expression plasmid pCI-neo and placing the HCV envelope E2 gene at the 3'end of the intron of the Deo gene. The plasmid vector uses eukaryotic promoter to regulate the expression of E2 gene in HCV capsule at high level, and the marker gene (neoplasm) used for screening intron shear function is only low level expression. Expression plasmid pCIDA-neo-E2
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R373;Q78

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相關期刊論文 前1條

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