本文選題：NS1蛋白　切入點(diǎn)：NOLC1蛋白　出處：《遼寧大學(xué)》2017年碩士論文

【摘要】：A型流感病毒屬正粘病毒科,為單股負(fù)鏈RNA病毒。至今,世界性的大規(guī)模流感疫情的爆發(fā)都是由A型流感病毒引起的,并且新型的禽流感病毒已經(jīng)變得更容易在人與人之間傳播,因此禽流感病毒的傳染性和致病性必須引起廣泛關(guān)注。A型流感病毒的NS1蛋白(nonstructural protein 1)僅存在于被感染的細(xì)胞中,在細(xì)胞中發(fā)揮著抑制宿主pre-mRNA剪接、拮抗干擾素、促進(jìn)病毒復(fù)制等重要功能。人核仁螺旋體磷酸化蛋白(NOLC1)是一個(gè)高度磷酸化的哺乳動(dòng)物蛋白,既參與核仁的發(fā)生和rDNA的轉(zhuǎn)錄,又參與炎癥發(fā)生、細(xì)胞生長(zhǎng)、細(xì)胞凋亡、腫瘤發(fā)生等相關(guān)信號(hào)通路的調(diào)控。在本實(shí)驗(yàn)中我們主要研究NS1蛋白與NOLC1蛋白相互作用對(duì)宿主細(xì)胞增殖、凋亡和病毒復(fù)制的影響及其相關(guān)信號(hào)通路。本研究用慢病毒轉(zhuǎn)染方法構(gòu)建NS1 A549穩(wěn)定轉(zhuǎn)染細(xì)胞株,免疫熒光和Western Blot檢測(cè)穩(wěn)轉(zhuǎn)株NS1蛋白的表達(dá),結(jié)果顯示穩(wěn)轉(zhuǎn)細(xì)胞株構(gòu)建成功。為了深入研究NS1與NOLC1相互作用對(duì)細(xì)胞凋亡的影響,本研究采用ELISA的方法確定了A549、A549-FLAG、A549-FLAG-NS1細(xì)胞中的CK2的表達(dá)水平,采用Real-time PCR和Western Blot的方法檢測(cè)了A549、A549-FLAG、A549-FLAG-NS1細(xì)胞中NOLC1和NF-κB的mRNA及蛋白水平,結(jié)果顯示NS1與NOLC1相互作用降低了CK2的表達(dá)水平,上調(diào)了NOLC1的mRNA水平,抑制了NOLC1的蛋白表達(dá),同時(shí)提高了細(xì)胞中NF-κB的mRNA水平,有文獻(xiàn)報(bào)道CK2、NOLC1和NF-κB是調(diào)控細(xì)胞凋亡的關(guān)鍵因子,結(jié)合本實(shí)驗(yàn)室之前的研究結(jié)果,我們推測(cè)NS1與NOLC1相互作用促進(jìn)細(xì)胞凋亡與其調(diào)控CK2、NOLC1和NF-κB的表達(dá)水平相關(guān),具體的機(jī)制還有待于進(jìn)一步研究。為檢測(cè)NS1與NOLC1相互作用相互作用對(duì)病毒復(fù)制能力的影響,本實(shí)驗(yàn)用H3N2禽流感病毒感染A549、A549-FLAG、A549-FLAG-NS1細(xì)胞,用Real-time PCR檢測(cè)宿主細(xì)胞中病毒NP三種RNA水平來(lái)反映病毒復(fù)制情況,結(jié)果顯示NS1與NOLC1相互作用促進(jìn)了病毒的復(fù)制。為檢測(cè)NS1與NOLC1相互作用對(duì)宿主細(xì)胞增殖能力的影響,本實(shí)驗(yàn)用MTT法對(duì)A549、A549-FLAG、A549-FLAG-NS1細(xì)胞株的增殖能力進(jìn)行檢測(cè),結(jié)果顯示NS1與NOLC1相互作用提高了細(xì)胞的增殖能力。通過(guò)上述研究,我們確定了NS1與NOLC1相互作用影響宿主細(xì)胞的增殖和凋亡,為進(jìn)一步研究相關(guān)的信號(hào)通路,本研究采用Real-time PCR技術(shù)對(duì)A549、A549-FLAG、A549-FLAG-NS1中與細(xì)胞增殖和凋亡相關(guān)的多個(gè)基因進(jìn)行了檢測(cè),結(jié)果顯示其中Wnt信號(hào)通路中的β-catenin和cyclinD基因、Bcl2和CDK4基因與兩種蛋白的相互作用相關(guān),NS1與NOLC1相互作用上調(diào)了β-catenin、cyclinD和Bcl2的mRNA水平,對(duì)CDK4的mRNA水平起下調(diào)作用,NS1可能通過(guò)調(diào)控這些基因及其信號(hào)通路來(lái)調(diào)控細(xì)胞的增殖和凋亡。研究NS1與NOLC1相互作用對(duì)宿主細(xì)胞增殖、凋亡和病毒復(fù)制的影響及相關(guān)信號(hào)通路,有助于我們深入研究NS1與NOLC1相互作用的機(jī)制,為禽流感致病機(jī)理的研究提供了新的思路。
[Abstract]:Influenza A virus belongs to Orthomyxoviridae, which is a single negative strand RNA virus.So far, worldwide outbreaks of large-scale influenza have been caused by influenza A viruses, and new avian influenza viruses have become more easily transmitted from person to person.Therefore, the infectivity and pathogenicity of avian influenza virus (Avian Influenza virus) must attract wide attention. The nonstructural protein 1 (NS1 protein) of influenza virus type A only exists in infected cells. It plays an important role in inhibiting pre-mRNA splicing and antagonizing interferon in the cells.Promote virus replication and other important functions.Human nucleolar spirochete phosphorylated protein (NOLC1) is a highly phosphorylated mammalian protein involved in the regulation of signal pathways such as nucleolus formation and rDNA transcription, inflammation, cell growth, apoptosis, tumorigenesis, and so on.In this study, we studied the effects of interaction between NS1 protein and NOLC1 protein on host cell proliferation, apoptosis and virus replication, and related signaling pathways.In this study, NS1 A549 stable transfection cell line was constructed by lentivirus transfection. The expression of NS1 protein was detected by immunofluorescence and Western Blot. The results showed that the stable transformed cell line was successfully constructed.In order to study the effect of NS1 and NOLC1 interaction on apoptosis, the expression of CK2 in A549A549-FLAG-NS1 cells was determined by ELISA. The mRNA and protein levels of NOLC1 and NF- 魏 B in A549-FLAG-NS1 cells were detected by Real-time PCR and Western Blot.The results showed that the interaction of NS1 and NOLC1 decreased the expression of CK2, up-regulated the mRNA level of NOLC1, inhibited the expression of NOLC1 protein, and increased the mRNA level of NF- 魏 B. CK2NOLC1 and NF- 魏 B were reported to be the key factors in regulating apoptosis.Based on our previous results, we speculate that the interaction between NS1 and NOLC1 promotes apoptosis related to the regulation of CK2NOLC1 and NF- 魏 B expression, and the specific mechanism remains to be further studied.In order to detect the effect of interaction between NS1 and NOLC1 on viral replication, we used H3N2 avian influenza virus to infect A549 FLAGN A549-FLAG-NS1 cell line, and Real-time PCR was used to detect the level of NP three kinds of RNA in host cells to reflect the viral replication.The results showed that the interaction of NS1 and NOLC1 promoted the replication of the virus.In order to detect the effect of NS1 and NOLC1 interaction on the proliferation of host cells, the proliferation ability of A549A549-FLAG-NS1 cell line was detected by MTT method. The results showed that the interaction between NS1 and NOLC1 enhanced the proliferation of A549- FLAG-NS1 cell line.Through the above studies, we have determined that the interaction between NS1 and NOLC1 affects the proliferation and apoptosis of host cells.In this study, Real-time PCR technique was used to detect several genes related to cell proliferation and apoptosis in A549A549-FLAG-NS1, A549-FLAG-NS1, A549-FLAG-NS1 and A549-FLAG-NS1.The results showed that 尾 -catenin and cyclinD genes Bcl2 and CDK4 genes in the Wnt signaling pathway were related to the interaction between the two proteins. The interaction between NS1 and NOLC1 up-regulated the mRNA levels of 尾 -catenin D and Bcl2.NS1 may regulate the proliferation and apoptosis of CDK4 cells by regulating these genes and their signaling pathways.The study of the effect of NS1 / NOLC1 interaction on host cell proliferation, apoptosis and virus replication and related signaling pathways is helpful for us to study the mechanism of NS1 / NOLC1 interaction and provide a new idea for the study of the pathogenesis of avian influenza.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373

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