本文選題：豬繁殖與呼吸綜合征病毒　切入點(diǎn)：nsp11　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：豬繁殖與呼吸綜合征病毒(PRRSV)是嚴(yán)重影響全球養(yǎng)豬業(yè)的重要病原之一。近來(lái)年,PRRSV的非結(jié)構(gòu)蛋白(nsp)在其基因組轉(zhuǎn)錄、復(fù)制調(diào)控和拮抗機(jī)體的天然免疫應(yīng)答中的作用受到廣泛關(guān)注。本研究分析了 PRRSVnsp11對(duì)宿主細(xì)胞蛋白p21的降解作用及其生物學(xué)意義,探討了病毒的nsp9和nsp2與宿主細(xì)胞的白細(xì)胞介素增強(qiáng)子結(jié)合因子2 (ILF2)的相互作用及其對(duì)病毒復(fù)制的調(diào)控效應(yīng),以期揭示nsp11、nsp9和nsp2在PRRSV復(fù)制過(guò)程中的作用,為理解PRRSV復(fù)制調(diào)控的分子機(jī)制提供科學(xué)依據(jù)。采用Western Blot分析了 PRRSV感染MARC-145細(xì)胞中p21蛋白的變化。結(jié)果顯示,PRRSV感染呈劑量依賴性下調(diào)MARC-145細(xì)胞中p21蛋白水平。流式細(xì)胞術(shù)分析表明,PRRSV 可通過(guò)下調(diào)p21蛋白而促進(jìn)細(xì)胞進(jìn)入S期。分別用血清饑餓、胸苷雙阻斷和諾苯達(dá)唑處理,將MARC-145細(xì)胞同步化至G0/G1、S和G2/M期后接種PRRSV,測(cè)定病毒滴度。結(jié)果顯示,同步化至S期的細(xì)胞有利于PRRSV復(fù)制。進(jìn)一步采用CRISPR/Cas9系統(tǒng)證實(shí)敲除MARC-145細(xì)胞p21基因有利于病毒復(fù)制。將表達(dá)PRRSV各個(gè)非結(jié)構(gòu)蛋白和結(jié)構(gòu)蛋白的質(zhì)粒與表達(dá)p21的質(zhì)粒共同轉(zhuǎn)染HEK 293FT細(xì)胞,進(jìn)行Western Blot分析。結(jié)果顯示,nsp11可明顯下調(diào)p21蛋白。構(gòu)建失活核酸內(nèi)切酶活性和去泛素化酶活性的nsp11突變體,與表達(dá)p21的質(zhì)粒共同轉(zhuǎn)染HEK293FT細(xì)胞。結(jié)果表明,nsp11的核酸內(nèi)切酶活性對(duì)其下調(diào)p21蛋白至關(guān)重要。Real-time PCR分析表明,PRRSV感染和表達(dá)nsp11的質(zhì)粒轉(zhuǎn)染細(xì)胞后,p21mRNA水平均無(wú)明顯變化。降解試驗(yàn)顯示nsp11通過(guò)蛋白酶體途徑降解p21,泛素化分析證實(shí)nsp11通過(guò)非泛素依賴的蛋白酶體途徑降解p21。以上結(jié)果表明,PRRSV利用其nsp11的核酸內(nèi)切酶活性下調(diào)細(xì)胞p21蛋白,促進(jìn)細(xì)胞進(jìn)入S期,從而利于病毒自身復(fù)制。利用慢病毒表達(dá)系統(tǒng)結(jié)合免疫沉淀技術(shù)及質(zhì)譜分析,篩選出52個(gè)與PRRSV nsp9相互作用的細(xì)胞蛋白,選取ILF2進(jìn)行下一步研究。將表達(dá)ILF2的質(zhì)粒和表達(dá)nsp9或nsp2的質(zhì)粒共同轉(zhuǎn)染HEK 293FT細(xì)胞,通過(guò)免疫共沉淀(Co-IP)證實(shí)外源ILF2與nsp9和nsp2均存在相互作用。進(jìn)一步在過(guò)表達(dá)nsp9或PRRSVJXwn06感染的MARC-145細(xì)胞中證實(shí)內(nèi)源性ILF2與nsp9或nsp2存在相互作用。利用Co-IP證實(shí)nsp9的RdRp結(jié)構(gòu)域與ILF2相互作用,而nsp2的不同截短體均與ILF2相互作用。此外,PRRSV感染可使MARC-145細(xì)胞和PAMs中的部分ILF2從細(xì)胞核移位至細(xì)胞質(zhì),與nsp9和nsp2共定位。在MARC-145細(xì)胞中,利用siRNA敲低和慢病毒過(guò)表達(dá)試驗(yàn)證實(shí)ILF2可負(fù)調(diào)控PRRSV復(fù)制。以上結(jié)果表明,細(xì)胞蛋白ILF2與PRRSVnsp9和nsp2均存在相互作用,且這種相互作用不利于病毒自身復(fù)制。綜上所述,PRRSV利用其nsp11的核酸內(nèi)切酶活性通過(guò)非泛素依賴的蛋白酶體途徑降解細(xì)胞內(nèi)p21蛋白水平,促進(jìn)細(xì)胞進(jìn)入S期,從而有利于病毒復(fù)制。細(xì)胞蛋白ILF2與PRRSV的nsp9和nsp2存在相互作用,且ILF2對(duì)病毒復(fù)制具有負(fù)調(diào)控效應(yīng)。以上結(jié)果有助于進(jìn)一步理解PRRSV與細(xì)胞間的相互作用及nsp 11、nsp9和nsp2在PRRSV復(fù)制過(guò)程中的作用,為闡明PRRSV復(fù)制調(diào)控的分子機(jī)制提供了科學(xué)依據(jù)。
[Abstract]:Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of serious impact on the global pig industry. In recent years, non structural protein PRRSV (NSP) in its genome transcription, replication and regulation of the innate immune response in the role of antagonistic organism has attracted extensive attention. This study analyzed the degradation effect of PRRSVnsp11 on host cells protein p21 and its biological significance of interleukin Nsp9 and Nsp2 virus and host cell factor enhancer binding factor 2 (ILF2) and its interaction effects on viral replication, in order to reveal the role of nsp11, Nsp9 and Nsp2 in the PRRSV replication process, to provide a scientific basis for understanding the molecular mechanisms of PRRSV copy control. Using Western Blot to analyze the changes of p21 protein in MARC-145 cells infected with PRRSV. The results showed that PRRSV infection was dose dependent downregulation of p21 protein level in MARC-145 cells by flow cytometry. FCM analysis showed that PRRSV can downregulate p21 protein and promote cells to enter S phase respectively. Serum starvation, thymidine double block and Nobel albendazole treatment, MARC-145 cells were synchronized to G0/G1, S and G2/M after PRRSV inoculation, the virus titer was determined. The results show that the synchronization to S phase cells for PRRSV replication. Further using CRISPR/Cas9 system confirmed that knockdown of MARC-145 cells with p21 gene for virus replication. The expression of PRRSV and expression plasmid all non structural and structural proteins of p21 co transfected with plasmid HEK 293FT cells, Western Blot analysis. The results show that nsp11 can decrease p21 protein inactivation. Construction of nucleic acid enzyme activity and deubiquitinating enzyme activity of nsp11 mutants, and the expression of p21 plasmid was co transfected into HEK293FT cells. The results showed that the nsp11 endonuclease activity on the expression of p21 protein is.Real-time PCR analysis showed that PRRSV infection and expression of nsp11 plasmid transfected cells after p21mRNA levels did not change significantly. The degradation test showed that nsp11 degradation via the proteasome pathway p21, ubiquitination analysis confirmed that nsp11 through the ubiquitin dependent proteasome degradation pathway of p21. above the PRRSV using its nsp11 endonuclease activity by p21 cells protein, promote cells to enter S phase, which is conducive to viral replication. The expression system combined with immunoprecipitation technology and mass spectrometry using lentivirus, screened 52 PRRSV and Nsp9 interacting protein, ILF2 is selected for the next study. The expression and expression of ILF2 plasmid Nsp9 or Nsp2 co transfected with plasmid HEK 293FT cells by CO immunoprecipitation (Co-IP) confirmed that exogenous ILF2 with Nsp9 and Nsp2 have interaction. Further in the over expression of Nsp9 or PRRSVJXwn06 infected MARC-145 cells The interaction between endogenous ILF2 and Nsp9 or Nsp2 confirmed. By Co-IP confirmed the interaction of Nsp9 domain and ILF2 RdRp structure, and different truncated Nsp2 were interacted with ILF2. In addition, PRRSV infection can make the ILF2 and PAMs in MARC-145 cells from the nucleus translocated to the cytoplasm, CO localization with Nsp9 and Nsp2. In MARC-145 cells, using siRNA knockdown and overexpression of lentivirus test confirmed that ILF2 can negatively regulate PRRSV replication. These results indicate that cell protein ILF2 with PRRSVnsp9 and Nsp2 have interaction, and this interaction is not conducive to the virus replication. In summary, the protein level of p21 proteasome degradation pathway of PRRSV cells using nsp11 the endonuclease activity through the ubiquitin dependent, promote cells to enter S phase, which is conducive to viral replication. The interaction between ILF2 and PRRSV cell protein Nsp9 and Nsp2, and ILF2 Viral replication plays a negative regulatory role. The above results will help further understand the interaction between PRRSV and cells, as well as the role of NSP 11, Nsp9 and Nsp2 in the process of PRRSV replication, and provide a scientific basis for elucidating the molecular mechanism of PRRSV replication regulation.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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相關(guān)期刊論文 前1條

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本文編號(hào)：1577080