利用T7噬菌体展示系统筛选禽流感（H5N1）非结构蛋白NS1相互作用的细胞蛋白

发布时间：2018-02-08 20:50

本文关键词： 禽流感 NS1蛋白 T7噬菌体展示技术阳性克隆验证　出处：《辽宁大学》2009年硕士论文　论文类型：学位论文

【摘要】：禽流感,是由A型流感病毒(Avian influenza virus,AIV)引起的从呼吸系统病变到全身败血症的一种高度接触性急性传染病,鸡、火鸡、鸭等家禽及野禽均可感染。亚洲、美洲、欧洲的许多国家和地区都曾发生过此病,给养禽业造成了巨大的损失。禽流感不仅可以在家禽中传播,严重影响养殖业的生存和发展,而且还可以由家禽传染给人类,直接威胁人类的健康,对人类的公共卫生也造成了相当大的危害。近期,国内及周边国家动物禽流感和人禽流感疫情频繁发生,仅国内禽流感病毒H5N1核酸阳性死亡病例就达到五例。高致病性禽流感(Highly pathogenic avian influenza,HPAI)已被世界动物卫生组织(OIE)列为A类传染病,并被列入国际生物武器公约动物传染病名单,我国也将其列为一类动物疫病。 NS1蛋白(nonstructural protein 1)是A型流感病毒唯一的非结构蛋白,由第八个基因编码,可编码202-237个氨基酸(Amino Acid,aa),不同毒株有差异,是一个具有多种活性的调控因子。NS1蛋白仅存在于病毒感染的细胞内,在感染的早期,大量存在于细胞核中,而在感染的晚期,NS1蛋白也可出现于胞浆中,且能刺激机体产生抗NS1蛋白的抗体。宿主细胞中存在dsRNA(双链RNA)是病毒感染和复制的明显信号,可诱导机体建立抗病毒应答。A型流感病毒NS1蛋白在病毒感染细胞中高水平表达,它的主要功能是抑制IFN-a/b(干扰素)介导的抗病毒作用,促进病毒基因表达,抑制宿主mRNA加工。近年来,国内外关于NS1蛋白的的进化、结构、调节病毒的致病性及其应用方面进行了深入的研究,通过对其相互作用蛋白的筛选和鉴定,可以进一步揭示A型流感病毒的致病机理,也为禽流感病毒感染人并引起死亡的机理提供重要的参考价值。本实验利用RT-PCR方法扩增AIV NS1基因并克隆到pET-28a载体上,转化大肠杆菌BL21(DE3)感受态细胞。在IPTG诱导下获得预期蛋白的表达,通过Ni-NTA树脂蛋白纯化系统对NS1蛋白进行纯化。应用T7噬菌体展示技术,以通过原核表达的方式得到的核心蛋白NS1作为靶分子,对噬菌体人肺细胞cDNA文库进行筛选,对筛选到的克隆进行DNA序列分析及同源性搜索。经鉴定得到能与NS1蛋白相互作用的蛋白为:Homo sapiens nucleolar and coiled-bodyphosphoprotein 1 (NOLC1) ,利用免疫共沉淀和哺乳动物双杂交的方法,在体内验证筛选到的阳性克隆。筛选到的蛋白,人核仁磷酸化蛋白NOLC1,是一个高度磷酸化的蛋白,呈颗粒状存在于间期细胞核核仁,当细胞进行有丝分裂时,NOLC1颗粒瓦解,均匀分布到细胞质中,与RNA聚合酶Ι相互结合,参与核仁的发生,调控rRNA基因的转录,对细胞生长;炎症发生;肝癌的发生、发展有重要的调控作用。本实验为进一步研究NS1蛋白的生物学功能提供重要依据,为未来研制抗流感药物提供新的线索。
[Abstract]:Avian influenza, a highly contagious acute infectious disease from respiratory diseases to systemic septicemia, is caused by avian influenza virus type A. chickens, turkeys, ducks, and wild birds can be infected. This disease has occurred in many countries and regions in Europe, causing enormous losses to the poultry industry. Avian influenza not only can spread among poultry, seriously affecting the survival and development of the poultry industry, but also can be transmitted from poultry to human beings. As a direct threat to human health, it has also caused considerable harm to human public health. Recently, domestic and neighboring countries have experienced frequent outbreaks of avian and human avian influenza. In China alone, there have been five deaths of avian influenza virus H5N1 nucleic acid positive. Highly pathogenic avian influenza avian influenzavirus HPAI has been classified as a Class A infectious disease by the World Organization for Animal Health (OIE) and included in the list of animal infectious diseases under the International Biological weapons Convention. Our country also listed it as a kind of animal disease. NS1 protein nonstructural protein 1 is the only nonstructural protein of influenza A virus. It is encoded by the eighth gene and encodes 202-237 amino acid amino acids. NS1 protein exists only in the cells infected with virus, and in the nucleus in the early stage of infection, and in the cytoplasm in the late stage of infection. The presence of dsRNAs (double-stranded RNAs) in host cells is an obvious signal of viral infection and replication, which can induce the establishment of high level expression of NS1 protein of influenza virus type A. Its main function is to inhibit IFN-a / b (interferon) -mediated antiviral effect, to promote viral gene expression, and to inhibit host mRNA processing. In recent years, the evolution and structure of NS1 protein at home and abroad, The pathogenicity of influenza A virus and its application have been studied deeply. By screening and identifying the interacting proteins, the pathogenic mechanism of influenza A virus can be further revealed. It also provides important reference value for the mechanism of human infection and death caused by avian influenza virus. In this study, AIV NS1 gene was amplified by RT-PCR and cloned into pET-28a vector, and transformed into Escherichia coli BL21DDE3) competent cells. The expected protein expression was obtained under IPTG induction. The NS1 protein was purified by Ni-NTA resin protein purification system. Using T7 phage display technique, the cDNA library of phage phage human lung cells was screened by using the core protein NS1 obtained by prokaryotic expression as the target molecule. DNA sequence analysis and homology search were carried out on the selected clones. The protein that could interact with NS1 protein was identified as:: Homo sapiens nucleolar and coiled-bodyphosphoprotein 1 (NOLC1). The method of immunoprecipitation and mammalian two-hybrid was used. The selected positive clones were verified in vivo. The screened protein, the human nucleolar phosphorylation protein NOLC1, is a highly phosphorylated protein that exists in the interphase nuclear nucleoli in a granular form. When the cells undergo mitosis, the NOLC1 particles disintegrate and distribute evenly into the cytoplasm. In combination with RNA polymerase I, it participates in nucleolus formation, regulates the transcription of rRNA gene, and promotes cell growth, inflammation and liver cancer. This study provides an important basis for the further study of the biological function of NS1 protein and provides a new clue for the development of antiinfluenza drugs in the future.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：S852.65;R373

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