本文選題：牛病毒性腹瀉病毒　切入點(diǎn)：牛胚腎細(xì)胞　出處：《石河子大學(xué)》2015年碩士論文

【摘要】：牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV)是一種重要的動(dòng)物疫病病原,牛感染后臨床癥狀主要表現(xiàn)為腹瀉、母畜流產(chǎn)和持續(xù)性感染等,造成重大的畜牧業(yè)經(jīng)濟(jì)損失。該病在全球范圍內(nèi)廣泛分布,在我國(guó)20多個(gè)省、市、自治區(qū)都有流行。目前,研究人員對(duì)于BVDV感染的致病機(jī)制尚不清楚。小泛素樣修飾(Small Ubiquitin-related Modifier,SUMO)是一種重要的蛋白質(zhì)翻譯后修飾,能對(duì)病毒蛋白進(jìn)行調(diào)控,進(jìn)而影響病毒的復(fù)制等。關(guān)于SUMO修飾能否調(diào)控BVDV感染尚沒有報(bào)道。因此,本研究從宿主SUMO通路的角度出發(fā),研究BVDV感染對(duì)宿主細(xì)胞的影響及其機(jī)制,這將有利于我們尋找新型的BVDV防控策略。為此,本研究以BVDV和牛胚腎細(xì)胞(Madin-Darby Bovine Kidney,MDBK)為研究對(duì)象,主要開展以下的研究工作:1.不同生物型BVDV感染MDBK細(xì)胞后類泛素基因轉(zhuǎn)錄水平的檢測(cè)。培養(yǎng)致細(xì)胞病變型(CP型)BVDV NADL病毒和非致細(xì)胞病變型(NCP型)BVDV shz 132病毒;RT-q PCR反應(yīng)測(cè)定兩種病毒拷貝數(shù);Reed-Muench法測(cè)定NADL病毒TCID50值。以100 TCID50 NADL病毒和同等拷貝數(shù)的shz 132病毒分別感染MDBK細(xì)胞不同時(shí)間(4 h、12 h、24 h、48 h),RT-q PCR反應(yīng)檢測(cè)細(xì)胞類泛素基因SUMO1、SUMO2、SUMO3、Ubc9的轉(zhuǎn)錄水平。結(jié)果顯示,NADL和shz 132病毒拷貝數(shù)分別是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分析顯示,與對(duì)照組相比,兩種BVDV病毒感染都能引起MDBK細(xì)胞SUMO1、SUMO2、SUMO3、Ubc9基因的相對(duì)表達(dá)量變化。在shz 132感染的細(xì)胞中,SUMO1、SUMO2、SUMO3的相對(duì)表達(dá)量在各個(gè)感染時(shí)間都高于或接近于BVDV NADL感染的細(xì)胞,且在感染后24 h,以上三個(gè)基因的相對(duì)表達(dá)量下調(diào),差異極顯著(P0.01);而Ubc9基因的相對(duì)表達(dá)量則低于或接近于BVDV NADL感染的細(xì)胞,且在感染后24 h,其相對(duì)表達(dá)量上調(diào),差異極顯著(P0.01)。2.SUMO1和Ubc9基因?qū)VDV復(fù)制的影響檢測(cè)。設(shè)計(jì)并合成靶向SUMO1和Ubc9基因的si RNA,構(gòu)建慢病毒干擾載體,與VSVG、PMDL、Rev共轉(zhuǎn)染人胚腎細(xì)胞(HEK293FT),包裝為慢病毒后感染MDBK細(xì)胞。RT-q PCR檢測(cè)si RNA對(duì)SUMO1和Ubc9基因的干擾效率及其對(duì)BVDV復(fù)制的影響。測(cè)序結(jié)果說明,成功構(gòu)建了靶向SUMO1基因和Ubc9基因的慢病毒干擾載體。在HEK293FT細(xì)胞中成功地包裝為重組慢病毒,熒光倒置顯微鏡下可見大量綠色熒光蛋白表達(dá)。RT-q PCR表明,與對(duì)照組相比,SUMO1-sh RNA-1-p LL3.7和SUMO1-sh RNA-2-p LL3.7對(duì)SUMO1基因的干擾效率分別為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對(duì)Ubc9基因的干擾效率分別為0%、6%和26%。干擾MDBK細(xì)胞SUMO1基因后,病毒的拷貝數(shù)是1.1×1016copies/m L;干擾Ubc9基因后,病毒的拷貝數(shù)是1.3×1014copies/m L。3.BVDV E0蛋白與細(xì)胞類泛素蛋白的相互作用分析。PCR方法擴(kuò)增細(xì)胞類泛素基因SUMO1/SUMO2/SUMO3/Ubc9,進(jìn)行TA克隆,經(jīng)菌液PCR、雙酶切、測(cè)序鑒定,構(gòu)建重組原核表達(dá)載體p GEX-4T-1-SUMO1/SUMO2/SUMO3/Ubc9,轉(zhuǎn)化E.coli BL21(DE3)表達(dá)菌,以0.1 m M IPTG誘導(dǎo)表達(dá)不同時(shí)間,SDS-PAGE鑒定表達(dá)效果。同時(shí),誘導(dǎo)表達(dá)已鑒定正確的p ET-32a(+)-E0重組表達(dá)載體的轉(zhuǎn)化菌,GST-pull down方法分析E0蛋白與類泛素蛋白的直接相互作用。測(cè)序結(jié)果表明,成功構(gòu)建了p MD18-T-SUMO1/SUMO2/SUMO3/Ubc9克隆載體和p GEX-4T-1-SUMO1/SUMO2/SUMO3/Ubc9原核表達(dá)載體,核苷酸無(wú)突變;SDS-PAGE電泳表明,成功誘導(dǎo)表達(dá)了SUMO1/SUMO2/SUMO3/Ubc9-GST可溶性融合蛋白,分子量依次為37.3 k Da、36.6k Da、37.6 k Da、43.5 k Da;GST-pull down實(shí)驗(yàn)證明BVDV E0蛋白不能結(jié)合細(xì)胞類泛素蛋白。以上所有的研究結(jié)果表明,BVDV感染影響了MDBK細(xì)胞SUMO通路,且這種作用與病毒的生物型存在密切聯(lián)系;BVDV能夠利用宿主SUMO通路,以有利于病毒在胞內(nèi)的生存與增殖;BVDV E0蛋白與細(xì)胞類泛素蛋白之間不存在直接相互作用。本項(xiàng)研究結(jié)果豐富了BVDV致病機(jī)制的理論基礎(chǔ),為BVDV的抗病毒感染提供科學(xué)依據(jù)。
[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.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

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本文編號(hào)：1729303