A型流感病毒核蛋白基因的克隆与表达

发布时间：2018-02-28 21:04

本文关键词： A型流感病毒核蛋白重组质粒原核表达　出处：《郑州大学》2005年硕士论文　论文类型：学位论文

【摘要】：背景与目的 A型流感病毒含有8条分离的负义RNA基因组片断,核蛋白(nucleoprotein, NP)由节段5编码。NP与病毒的RNA及多聚酶共同装配成RNP复合体,通过NP与RNA、其它病毒蛋白成分及感染细胞上某些大分子之间的相互作用,影响病毒的转录、复制、装配及转运功能,而病毒的复制与转录直接关系到对宿主的侵害,是病毒感染与致病的重要因素。另外核蛋白是细胞毒T淋巴细胞(Cytotoxic T lymphocytes, CTL)识别的主要抗原,CTL通过识别感染细胞表面的由MHC Ⅰ类分子提呈的病毒抗原肽,有利于清除病毒及控制感染。由于核蛋白基因相当保守,其抗原肽诱导的细胞免疫反应能够针对同型不同亚型的流感病毒株产生交叉反应。当前的流感病毒亚单位疫苗主要利用表面抗原诱导体液免疫,抗体的株特异性及表面抗原的易变性使疫苗具有很大局限性。因此若能将核蛋白或其抗原肽作为疫苗成分来诱导产生CTL反应,将有可能提供更加广泛、更加有效的保护作用。本实验拟利用流感病毒株A/Swine/Henan/703/2001(H3N2)构建原核重组质粒pGEX-4T-1-NP,并诱导该原核重组质粒在大肠杆菌中表达核蛋白,从而为进一步探讨核蛋白与宿主免疫反应的关系、利用核蛋白构建病毒亚单位疫苗及其它与核蛋白相关的病毒功能方面研究提供实验基础。材料与方法本实验从流感病毒株A/Swine/Henan/703/2001(H3N2)中提取总RNA,通过RT-PCR扩增到带有EcoRI酶切位点的NP全长基因序列;将NP基因片段与pGEM-T Easy克隆质粒连接,将重组子转化JM109感受态菌,利用Amp抗性、蓝白筛选
[Abstract]:Background and purpose. Influenza A virus contains eight isolated negative sense RNA genomic fragments. The nucleoprotein nucleoprotein (NPN) is encoded by segment 5. NP is assembled into a RNP complex with the virus's RNA and polymerase. Through the interaction between NP and RNAs, other viral protein components and some macromolecules in infected cells, the transcription, replication, assembly and transport of viruses are affected, and the replication and transcription of viruses are directly related to the invasion of the host. Nuclear protein is the main antigen recognized by cytotoxic T lymphocytes (CTLs) by recognizing viral antigenic peptides presented by MHC class I molecules on the surface of infected cells. Help to clear the virus and control infection. Because the nucleoprotein gene is quite conserved, The cellular immune response induced by its antigenic peptide can cross-react with influenza virus strains of the same type and different subtypes. The current influenza virus subunit vaccine mainly uses surface antigen to induce humoral immunity. The specificity of antibody and the variability of surface antigen make the vaccine have great limitation, so if the nucleoprotein or its antigen peptide can be used as vaccine component to induce the CTL reaction, it will be possible to provide more extensive and effective protective effect. In this experiment, the prokaryotic recombinant plasmid pGEX-4T-1-NPN was constructed by using influenza virus strain A / Swine / Henan / 703 / 2001 H3N2, and the prokaryotic recombinant plasmid was induced to express the nucleoprotein in Escherichia coli, so as to further study the relationship between the nucleoprotein and host immune reaction. Construction of virus subunit vaccine using nucleoprotein and other viral function related to nucleoprotein provide experimental basis. Materials and methods. In this experiment, total RNAs were extracted from influenza virus strain A / Swine / Henan / 703 / 2001 H3N2, and the full-length NP gene sequence with EcoRI restriction site was amplified by RT-PCR. The NP gene fragment was ligated with pGEM-T Easy clone plasmid, the recombinant plasmid was transformed into JM109 receptive bacteria, Amp resistance was used and blue-white screening was carried out.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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