BVDV体外感染与细胞SUMO通路关系的初步研究

发布时间：2018-04-10 02:35

本文选题：牛病毒性腹泻病毒　切入点：牛胚肾细胞　出处：《石河子大学》2015年硕士论文

【摘要】：牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)是一种重要的动物疫病病原,牛感染后临床症状主要表现为腹泻、母畜流产和持续性感染等,造成重大的畜牧业经济损失。该病在全球范围内广泛分布,在我国20多个省、市、自治区都有流行。目前,研究人员对于BVDV感染的致病机制尚不清楚。小泛素样修饰(Small Ubiquitin-related Modifier,SUMO)是一种重要的蛋白质翻译后修饰,能对病毒蛋白进行调控,进而影响病毒的复制等。关于SUMO修饰能否调控BVDV感染尚没有报道。因此,本研究从宿主SUMO通路的角度出发,研究BVDV感染对宿主细胞的影响及其机制,这将有利于我们寻找新型的BVDV防控策略。为此,本研究以BVDV和牛胚肾细胞(Madin-Darby Bovine Kidney,MDBK)为研究对象,主要开展以下的研究工作:1.不同生物型BVDV感染MDBK细胞后类泛素基因转录水平的检测。培养致细胞病变型(CP型)BVDV NADL病毒和非致细胞病变型(NCP型)BVDV shz 132病毒;RT-q PCR反应测定两种病毒拷贝数;Reed-Muench法测定NADL病毒TCID50值。以100 TCID50 NADL病毒和同等拷贝数的shz 132病毒分别感染MDBK细胞不同时间(4 h、12 h、24 h、48 h),RT-q PCR反应检测细胞类泛素基因SUMO1、SUMO2、SUMO3、Ubc9的转录水平。结果显示,NADL和shz 132病毒拷贝数分别是1.13×1011copies/m L和1.77×1011copies/m L,NADL TCID50值是104.9 TCID50/0.1 m L。RT-q PCR分析显示,与对照组相比,两种BVDV病毒感染都能引起MDBK细胞SUMO1、SUMO2、SUMO3、Ubc9基因的相对表达量变化。在shz 132感染的细胞中,SUMO1、SUMO2、SUMO3的相对表达量在各个感染时间都高于或接近于BVDV NADL感染的细胞,且在感染后24 h,以上三个基因的相对表达量下调,差异极显著(P0.01);而Ubc9基因的相对表达量则低于或接近于BVDV NADL感染的细胞,且在感染后24 h,其相对表达量上调,差异极显著(P0.01)。2.SUMO1和Ubc9基因对BVDV复制的影响检测。设计并合成靶向SUMO1和Ubc9基因的si RNA,构建慢病毒干扰载体,与VSVG、PMDL、Rev共转染人胚肾细胞(HEK293FT),包装为慢病毒后感染MDBK细胞。RT-q PCR检测si RNA对SUMO1和Ubc9基因的干扰效率及其对BVDV复制的影响。测序结果说明,成功构建了靶向SUMO1基因和Ubc9基因的慢病毒干扰载体。在HEK293FT细胞中成功地包装为重组慢病毒,荧光倒置显微镜下可见大量绿色荧光蛋白表达。RT-q PCR表明,与对照组相比,SUMO1-sh RNA-1-p LL3.7和SUMO1-sh RNA-2-p LL3.7对SUMO1基因的干扰效率分别为64%和62%;Ubc9-sh RNA-1-p LL3.7、Ubc9-sh RNA-2-p LL3.7和Ubc9-sh RNA-3-p LL3.7对Ubc9基因的干扰效率分别为0%、6%和26%。干扰MDBK细胞SUMO1基因后,病毒的拷贝数是1.1×1016copies/m L;干扰Ubc9基因后,病毒的拷贝数是1.3×1014copies/m L。3.BVDV E0蛋白与细胞类泛素蛋白的相互作用分析。PCR方法扩增细胞类泛素基因SUMO1/SUMO2/SUMO3/Ubc9,进行TA克隆,经菌液PCR、双酶切、测序鉴定,构建重组原核表达载体p GEX-4T-1-SUMO1/SUMO2/SUMO3/Ubc9,转化E.coli BL21(DE3)表达菌,以0.1 m M IPTG诱导表达不同时间,SDS-PAGE鉴定表达效果。同时,诱导表达已鉴定正确的p ET-32a(+)-E0重组表达载体的转化菌,GST-pull down方法分析E0蛋白与类泛素蛋白的直接相互作用。测序结果表明,成功构建了p MD18-T-SUMO1/SUMO2/SUMO3/Ubc9克隆载体和p GEX-4T-1-SUMO1/SUMO2/SUMO3/Ubc9原核表达载体,核苷酸无突变;SDS-PAGE电泳表明,成功诱导表达了SUMO1/SUMO2/SUMO3/Ubc9-GST可溶性融合蛋白,分子量依次为37.3 k Da、36.6k Da、37.6 k Da、43.5 k Da;GST-pull down实验证明BVDV E0蛋白不能结合细胞类泛素蛋白。以上所有的研究结果表明,BVDV感染影响了MDBK细胞SUMO通路,且这种作用与病毒的生物型存在密切联系;BVDV能够利用宿主SUMO通路,以有利于病毒在胞内的生存与增殖;BVDV E0蛋白与细胞类泛素蛋白之间不存在直接相互作用。本项研究结果丰富了BVDV致病机制的理论基础,为BVDV的抗病毒感染提供科学依据。
[Abstract]:Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) is an important animal pathogen, infection of cattle after clinical symptoms of diarrhea, female abortion and persistent infection, animal husbandry caused great economic losses. This disease is widely distributed in the global scope, in more than 20 provinces in China. City, autonomous region are popular. At present, the researchers in the pathogenesis of BVDV infection is not clear. Small ubiquitin like modifier (Small Ubiquitin-related, Modifier, SUMO) is an important posttranslational modification of viral proteins for regulation, thereby affecting the replication of the virus. A modified SUMO can control BVDV the infection is not reported. Therefore, this study from the perspective of the host SUMO pathway, study the effect of BVDV infection on host cells and its mechanism, which will help us to find new strategies for prevention and control of BVDV. Therefore, this study With BVDV and bovine embryonic kidney cells (Madin-Darby Bovine Kidney, MDBK) as the research object, mainly carry out the following research work: the detection of ubiquitin gene transcription of 1. biotypes of BVDV infected MDBK cells. Cultured cytopathic cell type (CP type) BVDV NADL and non cytopathic virus type (NCP type) BVDV SHZ 132 RT-q virus; PCR reaction for the determination of two kinds of virus copy number; Determination of NADL virus TCID50 Reed-Muench. At 100 TCID50 NADL and the same virus copy number SHZ 132 viruses were used to infect MDBK cell at different time (4 h, 12 h, 24 h, 48 h, RT-q) PCR reaction to detect cell ubiquitin gene SUMO1, SUMO2, SUMO3, the transcriptional level of Ubc9. The results showed that NADL and SHZ 132 virus copy number were 1.13 * 1011copies/m and 1.77 * 1011copies/m L L NADL TCID50 TCID50/0.1 m L.RT-q value is 104.9 PCR analysis showed that, compared with the control group, two BVDV virus sense Dye can cause MDBK cell SUMO1, SUMO2, SUMO3, the relative expression of Ubc9 gene variation. In 132 SHZ infected cells, SUMO1, SUMO2, SUMO3 expression in each time of infection are higher than or close to BVDV NADL infected cells, and in 24 h after infection, the relative expression of the above three genes were down regulated, extremely significant difference (P0.01); and the relative expression of Ubc9 gene was lower than or close to BVDV NADL infected cells, and in 24 h after infection, the relative expression, significant differences (P0.01.2.SUMO1) and the effect of Ubc9 gene on BVDV replication and synthesis of target detection. To Si RNA SUMO1 and Ubc9 gene, construct lentivirus vector, and VSVG, PMDL, Rev were transfected into human embryonic kidney cells (HEK293FT), Si RNA MDBK cell interference detection efficiency of SUMO1.RT-q PCR and Ubc9 gene and its effect on the replication of BVDV virus infection was slow. After sequencing packing node The results illustrate that the successful construction of lentivirus vector targeting SUMO1 gene and Ubc9 gene in HEK293FT cells successfully. The recombinant lentiviral packaging, the fluorescent microscope showed a large amount of green fluorescent protein expression of.RT-q PCR showed that compared with the control group, RNA-1-p LL3.7 and SUMO1-sh interference efficiency of SUMO1-sh RNA-2-p LL3.7 of SUMO1 gene respectively. 64% and 62%; Ubc9-sh RNA-1-p LL3.7, Ubc9-sh RNA-2-p and Ubc9-sh LL3.7 interference efficiency RNA-3-p LL3.7 of Ubc9 gene were 0%, 6% and 26%. of MDBK cells after SUMO1 gene interference, the virus copy number is 1.1 * 1016copies/m L; Ubc9 gene interference, the virus copy number is 1.3 * 1014copies/m L.3.BVDV E0 interaction protein cell and ubiquitin like protein ubiquitin gene amplification cells SUMO1/SUMO2/SUMO3/Ubc9.PCR analysis method, TA cloning, the bacterium PCR, double enzyme digestion, sequencing, structure Construction of Recombinant Prokaryotic expression vector p GEX-4T-1-SUMO1/SUMO2/SUMO3/Ubc9, E.coli BL21 (DE3) expression in transformed bacteria, induced the expression of different time in 0.1 M M IPTG, SDS-PAGE was identified. At the same time, identified the correct P induced expression of ET-32a (+) -E0 recombinant expression vector transformed bacteria, the direct interaction between E0 protein and ubiquitin like protein GST-pull down analysis method. The sequencing result showed that the successful construction of P MD18-T-SUMO1/SUMO2/SUMO3/Ubc9 clone vector and P GEX-4T-1-SUMO1/SUMO2/SUMO3/Ubc9 prokaryotic expression vector, the nucleotide mutation; SDS-PAGE electrophoresis showed that SUMO1/SUMO2/SUMO3/Ubc9-GST successfully induced the expression of soluble fusion protein, the molecular weight was 37.3 K Da, 36.6k Da K, 37.6 Da, 43.5 K Da; GST-pull down BVDV proved that E0 protein could not bind cellular ubiquitin like protein. All of the above research results show that the effect of BVDV infection of MDBK cells S The UMO pathway is closely linked with the virus and the effect of biological type; BVDV can use the host SUMO pathway, survival and proliferation to help the virus inside the cell; there is no direct interaction between BVDV E0 protein and cellular ubiquitin protein. The results of this study enriches the theory basis of the pathogenic mechanism of BVDV, to provide science the basis for antiviral infection of BVDV.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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