DDX5与猪瘟病毒NS5A蛋白的相互作用及对猪瘟病毒复制的影响

发布时间：2018-08-14 14:35

【摘要】：猪瘟(Classical swine fever,CSF),是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种以急性、高热、免疫抑制和全身泛发性出血为主要特征的猪的烈性传染病,又名猪霍乱,俗称“烂肠瘟”。猪瘟是接触性高度传染性疫病,传播快、致死率高,不同年龄段和品种的猪均可感染,世界动物卫生组织(OIE)将猪瘟定为一种必须上报的动物传染病,我国也将其列为一类动物疫病。猪瘟病毒是有囊膜的单股正链RNA病毒,属于黄病毒科瘟病毒属成员。NS5A蛋白是猪瘟病毒的一个非结构蛋白,对于猪瘟病毒的复制起到重要作用,也是猪瘟病毒复制复合体的组成部分。本文主要围绕CSFV NS5A蛋白和宿主细胞胞内蛋白DDX5(RNA解旋酶DDX5,RNA-helicase p68),研究两者之间的相互作用以及DDX5蛋白对猪瘟病毒基因组复制的影响,本实验得到以下结果:1.设计引物通过反转录PCR(Reverse Transcription PCR,RT-PCR)在SUVEC(swine umbilical vein endothelial cell line,永生化猪脐静脉血管内皮细胞)中克隆得到DDX5基因,并连上Myc标签,将带Myc标签的DDX5基因连入真核表达载体pCDNA3.1中,得到重组质粒pCDNA3.1-DDX5-Myc,在此基础上同时构建了慢病毒过表达载体CMV-DDX5和带红色荧光真核表达载体pDsREDN1-DDX5,经质粒PCR、双酶切以及测序鉴定正确后用于后续试验。2.将重组质粒pCDNA3.1-DDX5-Myc转入SUVEC细胞通过Western-blot检测DDX5蛋白的表达情况,结果表明DDX5在SUVEC细胞中可以稳定表达。在此基础上,在SUVEC细胞中共转pCDNA3.1-DDX5-Myc与实验室保存的pCDNA3.1-NS5A-Flag质粒,利用Co-IP方法证明了DDX5蛋白和NS5A蛋白的相互作用。3.在SUVEC细胞中,共转pDsREDN1-DDX5与实验室保存的pDsGFPN1-NS5A,利用激光共聚焦方法证明了NS5A和DDX5在细胞中存在共定位现象。4.通过实时荧光定量PCR检测通过慢病毒介导过表达DDX5对CSFV基因组复制的影响,证明DDX5对猪瘟病毒基因组的复制有着明显的促进作用。本研究得到了重组质粒pCDNA3.1-DDX5-Myc、CMV-DDX5和pCDNA3.1-DDX5-RedN1,并确定了DDX5和NS5A存在相互作用,在细胞中存在共定位,并进一步在基因水平证明了DDX5对猪瘟病毒基因组的复制有着促进作用,这为研究宿主细胞蛋白DDX5在猪瘟病毒增殖中的作用提供了新的科学资料。
[Abstract]:CSFV) is a severe infectious disease of pigs characterized by acute, high fever, immunosuppression and systemic generalized hemorrhage. It is also known as swine cholera, commonly known as "plague". Swine fever is a highly contagious infectious disease of contact, with high mortality and rapid transmission. Pigs of different ages and breeds can be infected. The (OIE) of the World Organization of Animal Health has designated hog fever as an animal epidemic that must be reported. Our country also listed it as a kind of animal disease. Swine Fever virus (CSFV) is a single-stranded positive RNA virus with envelope. The NS5A protein is a non-structural protein of CSFV, which plays an important role in the replication of CSFV. It is also part of the CSFV replication complex. In this paper, the interaction between CSFV NS5A protein and host cell intracellular protein DDX5 (RNA helicase DDX5 CSFV NS5A p68) and the effect of DDX5 protein on the genomic replication of CSFV were studied. The results were as follows: 1. The DDX5 gene was cloned from SUVEC (swine umbilical vein endothelial cell line, immortalized porcine umbilical vein endothelial cells by reverse transcription-polymerase chain reaction (RT-PCR), and the DDX5 gene with Myc tag was inserted into the eukaryotic expression vector pCDNA3.1. The recombinant plasmid pCDNA3.1-DDX5-Myc was obtained, and the lentivirus overexpression vector CMV-DDX5 and the red fluorescent eukaryotic expression vector pDsREDN1-DDX5 were constructed simultaneously. The recombinant plasmid pCDNA3.1-DDX5-Myc was transferred into SUVEC cells to detect the expression of DDX5 protein by Western-blot. The results showed that DDX5 could be expressed stably in SUVEC cells. On the basis of these results, pCDNA3.1-DDX5-Myc was co-transformed into pCDNA3.1-NS5A-Flag plasmids preserved in laboratory in SUVEC cells. The interaction between DDX5 protein and NS5A protein was proved by Co-IP method. In SUVEC cells, the co-transfer of pDsREDN1-DDX5 and pDsGFPN1-NS5A was carried out. The co-localization of NS5A and DDX5 in the cells was proved by laser confocal method. The effect of lentivirus mediated overexpression of DDX5 on the replication of CSFV genome was detected by real-time fluorescence quantitative PCR. It was proved that DDX5 could promote the replication of CSFV genome. In this study, the recombinant plasmids pCDNA3.1-DDX5-Mycton-CMV-DDX5 and pCDNA3.1-DDX5-RedN1 were obtained, and the interaction between DDX5 and NS5A was confirmed, and the co-localization in cells was confirmed. Furthermore, it was proved at the gene level that DDX5 could promote the replication of CSFV genome, and the recombinant plasmid pCDNA3.1-DDX5-CMV-DDX5 and pCDNA3.1-DDX5-RedN1 were obtained. This provides new scientific data for the study of the role of host cell protein DDX5 in the proliferation of classical swine fever virus (CSFV).
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651

【相似文献】