本文關(guān)鍵詞： 禽流感 NS1蛋白 T7噬菌體展示技術(shù) 陽(yáng)性克隆驗(yàn)證　出處：《遼寧大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：禽流感,是由A型流感病毒(Avian influenza virus,AIV)引起的從呼吸系統(tǒng)病變到全身敗血癥的一種高度接觸性急性傳染病,雞、火雞、鴨等家禽及野禽均可感染。亞洲、美洲、歐洲的許多國(guó)家和地區(qū)都曾發(fā)生過(guò)此病,給養(yǎng)禽業(yè)造成了巨大的損失。禽流感不僅可以在家禽中傳播,嚴(yán)重影響?zhàn)B殖業(yè)的生存和發(fā)展,而且還可以由家禽傳染給人類,直接威脅人類的健康,對(duì)人類的公共衛(wèi)生也造成了相當(dāng)大的危害。近期,國(guó)內(nèi)及周邊國(guó)家動(dòng)物禽流感和人禽流感疫情頻繁發(fā)生,僅國(guó)內(nèi)禽流感病毒H5N1核酸陽(yáng)性死亡病例就達(dá)到五例。高致病性禽流感(Highly pathogenic avian influenza,HPAI)已被世界動(dòng)物衛(wèi)生組織(OIE)列為A類傳染病,并被列入國(guó)際生物武器公約動(dòng)物傳染病名單,我國(guó)也將其列為一類動(dòng)物疫病。 NS1蛋白(nonstructural protein 1)是A型流感病毒唯一的非結(jié)構(gòu)蛋白,由第八個(gè)基因編碼,可編碼202-237個(gè)氨基酸(Amino Acid,aa),不同毒株有差異,是一個(gè)具有多種活性的調(diào)控因子。NS1蛋白僅存在于病毒感染的細(xì)胞內(nèi),在感染的早期,大量存在于細(xì)胞核中,而在感染的晚期,NS1蛋白也可出現(xiàn)于胞漿中,且能刺激機(jī)體產(chǎn)生抗NS1蛋白的抗體。宿主細(xì)胞中存在dsRNA(雙鏈RNA)是病毒感染和復(fù)制的明顯信號(hào),可誘導(dǎo)機(jī)體建立抗病毒應(yīng)答。A型流感病毒NS1蛋白在病毒感染細(xì)胞中高水平表達(dá),它的主要功能是抑制IFN-a/b(干擾素)介導(dǎo)的抗病毒作用,促進(jìn)病毒基因表達(dá),抑制宿主mRNA加工。近年來(lái),國(guó)內(nèi)外關(guān)于NS1蛋白的的進(jìn)化、結(jié)構(gòu)、調(diào)節(jié)病毒的致病性及其應(yīng)用方面進(jìn)行了深入的研究,通過(guò)對(duì)其相互作用蛋白的篩選和鑒定,可以進(jìn)一步揭示A型流感病毒的致病機(jī)理,也為禽流感病毒感染人并引起死亡的機(jī)理提供重要的參考價(jià)值。 本實(shí)驗(yàn)利用RT-PCR方法擴(kuò)增AIV NS1基因并克隆到pET-28a載體上,轉(zhuǎn)化大腸桿菌BL21(DE3)感受態(tài)細(xì)胞。在IPTG誘導(dǎo)下獲得預(yù)期蛋白的表達(dá),通過(guò)Ni-NTA樹脂蛋白純化系統(tǒng)對(duì)NS1蛋白進(jìn)行純化。應(yīng)用T7噬菌體展示技術(shù),以通過(guò)原核表達(dá)的方式得到的核心蛋白NS1作為靶分子,對(duì)噬菌體人肺細(xì)胞cDNA文庫(kù)進(jìn)行篩選,對(duì)篩選到的克隆進(jìn)行DNA序列分析及同源性搜索。經(jīng)鑒定得到能與NS1蛋白相互作用的蛋白為:Homo sapiens nucleolar and coiled-bodyphosphoprotein 1 (NOLC1) ,利用免疫共沉淀和哺乳動(dòng)物雙雜交的方法,在體內(nèi)驗(yàn)證篩選到的陽(yáng)性克隆。 篩選到的蛋白,人核仁磷酸化蛋白NOLC1,是一個(gè)高度磷酸化的蛋白,呈顆粒狀存在于間期細(xì)胞核核仁,當(dāng)細(xì)胞進(jìn)行有絲分裂時(shí),NOLC1顆粒瓦解,均勻分布到細(xì)胞質(zhì)中,與RNA聚合酶Ι相互結(jié)合,參與核仁的發(fā)生,調(diào)控rRNA基因的轉(zhuǎn)錄,對(duì)細(xì)胞生長(zhǎng);炎癥發(fā)生;肝癌的發(fā)生、發(fā)展有重要的調(diào)控作用。本實(shí)驗(yàn)為進(jìn)一步研究NS1蛋白的生物學(xué)功能提供重要依據(jù),為未來(lái)研制抗流感藥物提供新的線索。
[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.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：S852.65;R373

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本文編號(hào)：1496243