日本乙型脑炎病毒NS4B蛋白与NS3解旋酶的相互作用研究
发布时间：2018-08-05 15:28
【摘要】：日本乙型脑炎病毒(JEV)属于黄病毒科(Flaviviridae)黄病毒属(Flavivirus),是引起日本乙型脑炎的病原。该病毒能引起人出现明显的脑炎症状,怀孕母猪感染后会出现高热、流产、死胎和木乃伊胎,公猪则出现睾丸炎。目前,日本乙型脑炎病毒流行于亚洲25个国家或地区,全世界约有60%的人口生活在日本乙型脑炎病毒的流行区,日本乙型脑炎病毒带来的公共卫生问题仍然十分严峻。现阶段对JEV复制过程中复制复合体的形成机制及其作用机制尚不是十分明确,已知JEV的所有非结构蛋白均参与到复制复合体的构成,NS3解旋酶能够解旋双链RNA,而NS4B蛋白是黄病毒中唯一一个在内质网膜内外均有较长片段的一个非结构蛋白,对黄病毒复制复合体的形成具有关键作用。本研究通过确定JEV NS4B的拓扑结构,用免疫共沉淀实验及GST pull down实验,对NS4B膜外区与NS3解旋酶之间的相互作用进行了验证,并验证了过表达NS4B及NS4B膜外区蛋白对JEV复制的影响。研究的内容及结果如下:1.JEV NS4B蛋白与NS3解旋酶的相互作用通过PCR扩增JEV NS4B基因和NS3解旋酶基因,将该基因连接至pcDNA3.1(+)、pcDNA4.0等真核表达载体上,并通过间接免疫荧光技术验证其表达情况。将验证表达的真核表达质粒共转染至HEK 293T细胞,36h后收集细胞样品,利用免疫共沉淀实验验证NS3解旋酶与NS4B蛋白之间的相互作用。结果表明,NS3解旋酶与NS4B之间确实存在互作。2.JEV NS4B蛋白跨膜拓扑结构的的鉴定为确定NS4B蛋白的拓扑结构,我们采用TMHMM、Tmpred、HMMTOP、SOSUI、PHD、DAS等六种蛋白跨膜区分析软件对JEV NS4B的氨基酸序列进行分析,根据NS4B氨基酸序列的疏水性,确定NS4B蛋白含有5个疏水区。依据软件分析结果,对NS4B蛋白进行分段表达,将5个疏水区分别连接至pEGFP-C1载体上,并将其转染至Hela细胞中,利用间接免疫荧光技术验证其亚细胞定位,最终确定NS4B含有三段跨膜区,其中软件预测的疏水区1和疏水区2位于内质网膜内,并不参与NS4B的跨膜;第118-175位氨基酸是NS4B蛋白的膜外区,最有可能参与NS3的互作。3.JEV NS4B蛋白膜外区与NS3解旋酶的相互作用扩增JEV NS4B蛋白的膜外区基因,并将其连接至真核表达质粒pEGFP-N1上,将该质粒与pcDNA3.1(+)-NS3 helicase共转染至HEK 293T细胞中,通过免疫共沉淀实验验证NS3解旋酶与NS4B膜外区的相互作用,结果显示,NS3解旋酶与NS4B的膜外区存在相互作用。扩增NS4B膜外区基因,并将其连接至原核表达载体pGEX-6p-1上,在大肠杆菌BL21(DE3)中表达的重组蛋白,通过GST亲和层析纯化获得较纯的NS4B膜外区蛋白,采用GST pull down实验验证NS4B与NS3解旋酶之间的直接相互作用,结果显示,NS3解旋酶与NS4B的膜外区之间的相互作用是直接作用。4.NS4B蛋白对JEV复制的影响克隆NS4B的膜外区基因,并构建真核表达质粒pEGFP-N1-NS4B(118-175),将该质粒转染至Hela细胞中,24h后感染JEV P3毒株,收集感染后的样品,采用空斑实验验证NS4B膜外区对JEV病毒增殖的影响。结果显示,过表达NS4B及NS4B的膜外区在一定程度上能够抑制病毒复制,但其抑制率还需进一步确认。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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