用噬菌体随机肽库筛选hCD81模拟小肽

发布时间：2018-05-02 14:11

本文选题：丙型肝炎病毒(HCV) + 包膜E2蛋白　；参考：《第二军医大学》2006年硕士论文

【摘要】：丙型肝炎是由丙型肝炎病毒(hepatitis C virus,HCV)感染引起的一种世界性传染病,目前全球HCV感染者近1亿7千万。HCV急性感染后约40-60%以上患者发展为慢性感染,其中部分病人可发展为肝硬化或肝癌。迄今为止,对HCV慢性感染尚无高效的特异性治疗方法。 HCV属于黄病毒家族,是单股正链RNA病毒,基因组全长约9.5kb,有一个大的开放阅读框,可翻译成一个约3000个氨基酸的多聚蛋白前体,该前体蛋白在宿主信号肽酶和病毒自身蛋白酶的加工下,产生结构蛋白(C,E1,E2,)和非结构蛋白(P7,NS2,NS3,NS4A,NS4B,NS5A,NS5B等)。 HCV的包膜糖蛋白E2分布于病毒颗粒表面,可能与病毒和宿主间免疫应答及介导病毒进人靶细胞等过程密切相关,因此成为预防HCV感染研究中的主要角色。E2蛋白能与肝细胞表面的多种分子结合,其中,人CD81分子(hCD81)已被公认为是HCV的重要受体。虽然CD81介导HCV感染的机制尚不清楚,但是体外试验表明,CD81分子是HCV感染原代肝细胞以及肝源性肿瘤细胞所必需的。因此,靶向人CD81的模拟分子对于HCV感染的治疗有着应用前景,这也是目前HCV治疗药物领域的一个研究热点。在本实验中,我们首先分别在哺乳动物细胞中表达了HCV E2蛋白和在大肠杆菌中表达了人CD81分子大胞外环。再以hCD81单抗为配基,从噬菌体12肽库中筛选hCD81模拟小肽,通过实验证明能竞争抑制HCV E2和hCD81的结合。第一部分 HCV包膜E2蛋白和人CD81分子大胞外环的表达及HCV假病毒的构建用中国仓鼠卵巢细胞(CHO)分泌表达HCV包膜E2蛋白。首先根据1b基因型HCV包膜E2蛋白编码基因的核苷酸序列设计并合成引物,以聚合酶反应(PCR)扩增HCV包膜E2蛋白外段基因;将neo基因插入真核表达质粒pCI-neo的内含子基因的剪切供体与剪切受体位点之间,而将HCV包膜E2蛋白基因置于Deo基因内含子的3′端,构建了新型的哺乳动物细胞表达质粒载体pCIDA-neo-E2。该质粒载体利用真核启动子高水平调控HCV包膜E2基因的表达,借助于内含子的剪切功能筛选用的标记基因(neo)仅低水平表达。以表达质粒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
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373;Q78

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