日本乙型腦炎病毒NS4B蛋白與NS3解旋酶的相互作用研究
發(fā)布時間：2018-08-05 15:28
【摘要】：日本乙型腦炎病毒(JEV)屬于黃病毒科(Flaviviridae)黃病毒屬(Flavivirus),是引起日本乙型腦炎的病原。該病毒能引起人出現(xiàn)明顯的腦炎癥狀,懷孕母豬感染后會出現(xiàn)高熱、流產(chǎn)、死胎和木乃伊胎,公豬則出現(xiàn)睪丸炎。目前,日本乙型腦炎病毒流行于亞洲25個國家或地區(qū),全世界約有60%的人口生活在日本乙型腦炎病毒的流行區(qū),日本乙型腦炎病毒帶來的公共衛(wèi)生問題仍然十分嚴(yán)峻。現(xiàn)階段對JEV復(fù)制過程中復(fù)制復(fù)合體的形成機(jī)制及其作用機(jī)制尚不是十分明確,已知JEV的所有非結(jié)構(gòu)蛋白均參與到復(fù)制復(fù)合體的構(gòu)成,NS3解旋酶能夠解旋雙鏈RNA,而NS4B蛋白是黃病毒中唯一一個在內(nèi)質(zhì)網(wǎng)膜內(nèi)外均有較長片段的一個非結(jié)構(gòu)蛋白,對黃病毒復(fù)制復(fù)合體的形成具有關(guān)鍵作用。本研究通過確定JEV NS4B的拓?fù)浣Y(jié)構(gòu),用免疫共沉淀實驗及GST pull down實驗,對NS4B膜外區(qū)與NS3解旋酶之間的相互作用進(jìn)行了驗證,并驗證了過表達(dá)NS4B及NS4B膜外區(qū)蛋白對JEV復(fù)制的影響。研究的內(nèi)容及結(jié)果如下:1.JEV NS4B蛋白與NS3解旋酶的相互作用通過PCR擴(kuò)增JEV NS4B基因和NS3解旋酶基因,將該基因連接至pcDNA3.1(+)、pcDNA4.0等真核表達(dá)載體上,并通過間接免疫熒光技術(shù)驗證其表達(dá)情況。將驗證表達(dá)的真核表達(dá)質(zhì)粒共轉(zhuǎn)染至HEK 293T細(xì)胞,36h后收集細(xì)胞樣品,利用免疫共沉淀實驗驗證NS3解旋酶與NS4B蛋白之間的相互作用。結(jié)果表明,NS3解旋酶與NS4B之間確實存在互作。2.JEV NS4B蛋白跨膜拓?fù)浣Y(jié)構(gòu)的的鑒定為確定NS4B蛋白的拓?fù)浣Y(jié)構(gòu),我們采用TMHMM、Tmpred、HMMTOP、SOSUI、PHD、DAS等六種蛋白跨膜區(qū)分析軟件對JEV NS4B的氨基酸序列進(jìn)行分析,根據(jù)NS4B氨基酸序列的疏水性,確定NS4B蛋白含有5個疏水區(qū)。依據(jù)軟件分析結(jié)果,對NS4B蛋白進(jìn)行分段表達(dá),將5個疏水區(qū)分別連接至pEGFP-C1載體上,并將其轉(zhuǎn)染至Hela細(xì)胞中,利用間接免疫熒光技術(shù)驗證其亞細(xì)胞定位,最終確定NS4B含有三段跨膜區(qū),其中軟件預(yù)測的疏水區(qū)1和疏水區(qū)2位于內(nèi)質(zhì)網(wǎng)膜內(nèi),并不參與NS4B的跨膜;第118-175位氨基酸是NS4B蛋白的膜外區(qū),最有可能參與NS3的互作。3.JEV NS4B蛋白膜外區(qū)與NS3解旋酶的相互作用擴(kuò)增JEV NS4B蛋白的膜外區(qū)基因,并將其連接至真核表達(dá)質(zhì)粒pEGFP-N1上,將該質(zhì)粒與pcDNA3.1(+)-NS3 helicase共轉(zhuǎn)染至HEK 293T細(xì)胞中,通過免疫共沉淀實驗驗證NS3解旋酶與NS4B膜外區(qū)的相互作用,結(jié)果顯示,NS3解旋酶與NS4B的膜外區(qū)存在相互作用。擴(kuò)增NS4B膜外區(qū)基因,并將其連接至原核表達(dá)載體pGEX-6p-1上,在大腸桿菌BL21(DE3)中表達(dá)的重組蛋白,通過GST親和層析純化獲得較純的NS4B膜外區(qū)蛋白,采用GST pull down實驗驗證NS4B與NS3解旋酶之間的直接相互作用,結(jié)果顯示,NS3解旋酶與NS4B的膜外區(qū)之間的相互作用是直接作用。4.NS4B蛋白對JEV復(fù)制的影響克隆NS4B的膜外區(qū)基因,并構(gòu)建真核表達(dá)質(zhì)粒pEGFP-N1-NS4B(118-175),將該質(zhì)粒轉(zhuǎn)染至Hela細(xì)胞中,24h后感染JEV P3毒株,收集感染后的樣品,采用空斑實驗驗證NS4B膜外區(qū)對JEV病毒增殖的影響。結(jié)果顯示,過表達(dá)NS4B及NS4B的膜外區(qū)在一定程度上能夠抑制病毒復(fù)制,但其抑制率還需進(jìn)一步確認(rèn)。
[Abstract]:Japanese encephalitis virus (JEV) belongs to the yellow virus (Flaviviridae) family of the family yellow virus (Flavivirus). It is the cause of Japanese encephalitis. The virus can cause obvious symptoms of encephalitis. High fever, abortion, stillbirth and mummification, and orchitis in the boar are caused by the infection of the pregnant sow, and the Japanese encephalitis virus flow is present. In 25 countries or regions of Asia, about 60% of the world's population live in the epidemic area of Japanese encephalitis virus, and the public health problems caused by Japanese encephalitis virus are still very severe. At this stage, the mechanism and mechanism of the replication complex in the process of JEV replication are not yet very clear, and all the non - JEV The structure protein is involved in the composition of the replication complex, and the NS3 helicase can solve the spin double chain RNA, and the NS4B protein is the only non structural protein in the yellow virus, which has a long fragment inside and outside the endoplasmic reticulum. It has a key role in the formation of the replication complex of the yellow virus. The interaction between NS4B membrane and NS3 helicase was verified by precipitation experiment and GST pull down experiment, and the effect of overexpressing NS4B and NS4B membrane protein on JEV replication was verified. The contents and results of the study were as follows: the interaction between 1.JEV NS4B protein and NS3 helicase was amplified by PCR amplification gene and helicase gene The gene was connected to the eukaryotic expression vector such as pcDNA3.1 (+), pcDNA4.0 and other eukaryotic expression vectors, and the expression was verified by indirect immunofluorescence. The expressed eukaryotic expression plasmid was transfected into HEK 293T cells, and the cell samples were collected after 36h. The interaction between NS3 and NS4B protein was verified by the immunoprecipitation test. It is clear that NS3 helicase and NS4B do exist between.2.JEV NS4B protein cross membrane topology structure identification to determine the topological structure of NS4B protein. We use TMHMM, Tmpred, HMMTOP, SOSUI, PHD, DAS and other protein transmembrane analysis software to analyze the amino acid sequence of JEV, and determine the hydrophobicity of the amino acid sequence. The B protein contains 5 hydrophobic regions. According to the software analysis, the NS4B protein is expressed in segments. The 5 hydrophobic regions are connected to the pEGFP-C1 carrier and transfected into the Hela cells. The subcellular location is verified by indirect immunofluorescence technology. Finally, the NS4B contains three segments of the transmembrane region, in which the hydrophobic region is predicted by the software and the hydrophobic area is 1 and hydrophobicity. Area 2 is located in the endoplasmic reticulum and does not participate in the transmembrane of NS4B; the 118-175 amino acid is the outer region of the NS4B protein. It is most likely to participate in the interaction between the NS3's.3.JEV NS4B protein membrane and the NS3 helicase to amplify the outer region gene of the JEV NS4B protein and connect it to the eukaryotic expression plasmid pEGFP-N1, and the plasmid and pcDNA3.1 (+) -NS3 helicase was co transfected into HEK 293T cells. The interaction between NS3 and the outer region of NS4B membrane was verified by the co immunoprecipitation experiment. The results showed that the NS3 helicase was interacting with the outer membrane of NS4B. The amplification of the NS4B membrane outer region gene and its connection to the prokaryotic expression vector pGEX-6p-1, the weight expressed in the BL21 (DE3) of Escherichia coli. Histone was purified by GST affinity chromatography to obtain a pure NS4B membrane protein. The direct interaction between NS4B and NS3 helicase was verified by GST pull down. The results showed that the interaction between NS3 and NS4B was the direct effect of.4.NS4B protein on JEV replication and cloning of the outer membrane gene of NS4B. The eukaryotic expression plasmid pEGFP-N1-NS4B (118-175) was transfected into Hela cells. The JEV P3 strain was infected after 24h, and the infected samples were collected. The effect of NS4B membrane outer region on the proliferation of JEV virus was verified by the plaque test. The results showed that the overexpression of NS4B and NS4B could inhibit the replication of the virus to some extent, but the inhibition rate was in a certain extent. Further confirmation is needed.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65
本文編號：2166213
[Abstract]:Japanese encephalitis virus (JEV) belongs to the yellow virus (Flaviviridae) family of the family yellow virus (Flavivirus). It is the cause of Japanese encephalitis. The virus can cause obvious symptoms of encephalitis. High fever, abortion, stillbirth and mummification, and orchitis in the boar are caused by the infection of the pregnant sow, and the Japanese encephalitis virus flow is present. In 25 countries or regions of Asia, about 60% of the world's population live in the epidemic area of Japanese encephalitis virus, and the public health problems caused by Japanese encephalitis virus are still very severe. At this stage, the mechanism and mechanism of the replication complex in the process of JEV replication are not yet very clear, and all the non - JEV The structure protein is involved in the composition of the replication complex, and the NS3 helicase can solve the spin double chain RNA, and the NS4B protein is the only non structural protein in the yellow virus, which has a long fragment inside and outside the endoplasmic reticulum. It has a key role in the formation of the replication complex of the yellow virus. The interaction between NS4B membrane and NS3 helicase was verified by precipitation experiment and GST pull down experiment, and the effect of overexpressing NS4B and NS4B membrane protein on JEV replication was verified. The contents and results of the study were as follows: the interaction between 1.JEV NS4B protein and NS3 helicase was amplified by PCR amplification gene and helicase gene The gene was connected to the eukaryotic expression vector such as pcDNA3.1 (+), pcDNA4.0 and other eukaryotic expression vectors, and the expression was verified by indirect immunofluorescence. The expressed eukaryotic expression plasmid was transfected into HEK 293T cells, and the cell samples were collected after 36h. The interaction between NS3 and NS4B protein was verified by the immunoprecipitation test. It is clear that NS3 helicase and NS4B do exist between.2.JEV NS4B protein cross membrane topology structure identification to determine the topological structure of NS4B protein. We use TMHMM, Tmpred, HMMTOP, SOSUI, PHD, DAS and other protein transmembrane analysis software to analyze the amino acid sequence of JEV, and determine the hydrophobicity of the amino acid sequence. The B protein contains 5 hydrophobic regions. According to the software analysis, the NS4B protein is expressed in segments. The 5 hydrophobic regions are connected to the pEGFP-C1 carrier and transfected into the Hela cells. The subcellular location is verified by indirect immunofluorescence technology. Finally, the NS4B contains three segments of the transmembrane region, in which the hydrophobic region is predicted by the software and the hydrophobic area is 1 and hydrophobicity. Area 2 is located in the endoplasmic reticulum and does not participate in the transmembrane of NS4B; the 118-175 amino acid is the outer region of the NS4B protein. It is most likely to participate in the interaction between the NS3's.3.JEV NS4B protein membrane and the NS3 helicase to amplify the outer region gene of the JEV NS4B protein and connect it to the eukaryotic expression plasmid pEGFP-N1, and the plasmid and pcDNA3.1 (+) -NS3 helicase was co transfected into HEK 293T cells. The interaction between NS3 and the outer region of NS4B membrane was verified by the co immunoprecipitation experiment. The results showed that the NS3 helicase was interacting with the outer membrane of NS4B. The amplification of the NS4B membrane outer region gene and its connection to the prokaryotic expression vector pGEX-6p-1, the weight expressed in the BL21 (DE3) of Escherichia coli. Histone was purified by GST affinity chromatography to obtain a pure NS4B membrane protein. The direct interaction between NS4B and NS3 helicase was verified by GST pull down. The results showed that the interaction between NS3 and NS4B was the direct effect of.4.NS4B protein on JEV replication and cloning of the outer membrane gene of NS4B. The eukaryotic expression plasmid pEGFP-N1-NS4B (118-175) was transfected into Hela cells. The JEV P3 strain was infected after 24h, and the infected samples were collected. The effect of NS4B membrane outer region on the proliferation of JEV virus was verified by the plaque test. The results showed that the overexpression of NS4B and NS4B could inhibit the replication of the virus to some extent, but the inhibition rate was in a certain extent. Further confirmation is needed.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65
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