發(fā)布時(shí)間：2019-05-17 04:27

【摘要】：蛋白質(zhì)組學(xué)是當(dāng)今生命科學(xué)研究的重要工具之一。本論文綜合運(yùn)用穩(wěn)定同位素標(biāo)記和亞細(xì)胞結(jié)構(gòu)分離等生化技術(shù)并結(jié)合基于新一代質(zhì)譜的高通量蛋白質(zhì)分析技術(shù),對(duì)EV71和HBV這兩種重要病毒感染所引起的宿主細(xì)胞蛋白變化進(jìn)行了系統(tǒng)研究。EV71病毒能引起嬰幼兒嚴(yán)重的中樞神經(jīng)系統(tǒng)疾病,是我國(guó)乃至亞太地區(qū)公共健康安全的重大隱患之一。目前關(guān)于EV71病毒與宿主的相互作用組學(xué)研究報(bào)道較少,制約了我們對(duì)于病毒的了解。因此在本論文的第二章,我們系統(tǒng)地研究了病毒感染不同階段的宿主細(xì)胞蛋白質(zhì)組的動(dòng)態(tài)變化。我們將不同的同位素標(biāo)記的SILAC培養(yǎng)的細(xì)胞經(jīng)過(guò)EV71感染,在8小時(shí)與20小時(shí)后提取宿主蛋白質(zhì),利用高分辨液質(zhì)聯(lián)用技術(shù)定量分析到了4114個(gè)宿主蛋白的感染前后的動(dòng)態(tài)變化情況,發(fā)現(xiàn)其中有約17%的蛋白在不同的時(shí)間點(diǎn)發(fā)生了顯著變化。通過(guò)對(duì)這些發(fā)生顯著變化的蛋白進(jìn)行分析,我們找到了受EV71感染后宿主細(xì)胞蛋白的一些變化規(guī)律,包括發(fā)現(xiàn)5個(gè)生物學(xué)進(jìn)程和7個(gè)蛋白家族在感染后發(fā)生了顯著的變化,尤其是histone蛋白家族在感染后持續(xù)下降。線粒體蛋白中有很多發(fā)生了明顯的上下調(diào),但是絕大部分只在某一個(gè)感染時(shí)間點(diǎn)發(fā)生變化。分層聚類(lèi)分析結(jié)果也證實(shí)了這一點(diǎn)。此外,一些與病毒感染相關(guān)的蛋白家族,例如泛素化和SUMO化相關(guān)蛋白、膜泡運(yùn)輸?shù)鞍缀湍[瘤壞死因子等蛋白,也發(fā)生了明顯變化。通過(guò)進(jìn)一步蛋白功能驗(yàn)證和篩選,我們發(fā)現(xiàn)宿主線粒體蛋白CHCH2對(duì)EV71的復(fù)制可能具有重要的影響,而這種影響可能是通過(guò)負(fù)調(diào)控宿主天然免疫信號(hào)通路來(lái)實(shí)現(xiàn)的。我國(guó)有超過(guò)9300萬(wàn)的乙肝患者,對(duì)于HBV病毒的研究有著重要的現(xiàn)實(shí)意義。在本論文的第三章中,我們采用一種源自Huh7細(xì)胞的HBV穩(wěn)定表達(dá)細(xì)胞系Huh7.37,系統(tǒng)地研究了全細(xì)胞和亞細(xì)胞結(jié)構(gòu)中蛋白質(zhì)的表達(dá)變化。我們運(yùn)用核質(zhì)分離技術(shù)純化了細(xì)胞核組分,運(yùn)用蔗糖密度梯度超速離心技術(shù)富集了線粒體組分,并結(jié)合三重雙甲基化標(biāo)記和質(zhì)譜分析技術(shù),發(fā)現(xiàn)在全細(xì)胞組分、細(xì)胞核組分和線粒體組分中發(fā)生明顯改變的信號(hào)通路和蛋白家族均不相同。我們?cè)诰�粒體組分中發(fā)現(xiàn)了19個(gè)過(guò)氧化物酶體蛋白發(fā)生了明顯的變化,這暗示了Huh7.37細(xì)胞中線粒體和過(guò)氧化物酶體之間的聯(lián)系變得更加緊密。在亞細(xì)胞結(jié)構(gòu)蛋白質(zhì)組與全蛋白質(zhì)組的比較過(guò)程當(dāng)中,我們發(fā)現(xiàn)約有9.6%的核胞質(zhì)共存蛋白在病毒復(fù)制過(guò)程中可能進(jìn)入細(xì)胞核,而29.8%的線粒體胞質(zhì)共存蛋白在這個(gè)過(guò)程中可能進(jìn)入線粒體,這表明HBV的復(fù)制可能改變了這些蛋白在細(xì)胞中的定位。STRING分析顯示,有30個(gè)蛋白在細(xì)胞核中的表達(dá)量明顯下調(diào),而它們?cè)谌?xì)胞和線粒體中的變化不明顯,甚至反而有所上調(diào),因此我們推測(cè)這些蛋白可能在亞細(xì)胞結(jié)構(gòu)間發(fā)生了穿梭。此外,我們初步探究了20個(gè)宿主蛋白對(duì)于HBV復(fù)制的影響,發(fā)現(xiàn)其中有10個(gè)蛋白能影響HBeAg的表達(dá),但是僅有DDX1能同時(shí)影響HBsAg的表達(dá)。
[Abstract]:Proteomics is one of the most important tools in life science. In this paper, the changes of host cell protein caused by two important viral infections of EV71 and HBV were systematically studied by using biochemical techniques such as stable isotope labeling and subcellular structure separation, and combining with high-throughput protein analysis based on a new generation of mass spectrometry. The EV71 virus can cause severe central nervous system diseases in infants, and is one of the major hidden dangers of public health and safety in China and the Asia-Pacific region. The current study on the interaction between the EV71 virus and the host has reported less and restricted our understanding of the virus. Therefore, in the second chapter of this thesis, we systematically study the dynamic changes of host cell protein groups in different stages of viral infection. The cells of SIMILAC cultured with different isotopic labels were infected with EV71, and the host protein was extracted after 8 hours and 20 hours, and the dynamic changes of 4114 host proteins were quantitatively analyzed by high-resolution liquid chromatography. It was found that about 17% of the proteins had a significant change at different time points. By analyzing these proteins with significant changes, we found a number of changes in host cell proteins after EV71 infection, including the discovery of five biological processes and seven protein families with significant changes after infection, In particular the histone protein family continues to decline after infection. Many of the mitochondrial proteins have been significantly down-regulated, but most of them change only at a certain point of infection. The results of the hierarchical clustering analysis also confirmed this. In addition, some protein families associated with viral infections, such as ubiquitination and SUMO-related proteins, membrane vesicle transport proteins, and tumor necrosis factor proteins, have also changed significantly. By further protein function validation and screening, we have found that the host mitochondrial protein CHCH2 may have an important effect on the replication of the EV71, which may be achieved by negatively regulating the host's natural immune signal path. There are more than 93 million hepatitis B patients in China, and it is of great practical significance for the study of HBV. In the third chapter of this thesis, we used an HBV-stable expression cell line Huh7.37 derived from Huh7 cells, and systematically studied the expression of the protein in the whole cell and subcellular structure. The nuclear component was purified by the nuclear separation technique, and the mitochondrial fraction was enriched by the sucrose density gradient ultracentrifugation technique, and the whole cell fraction was found in combination with the triple double-methylation labeling and the mass-spectrum analysis technique. The signal pathway and the protein family that are significantly altered in the nuclear component and the mitochondrial component are not the same. We found a significant change in the mitochondrial fraction of the 19 peroxisome proteins, suggesting that the association between the mitochondria and the peroxidase in the Huh7.37 cell becomes more compact. During the comparison of the subcellular structural proteome and the whole proteome, we found that about 9.6% of the nuclear cytoplasmic co-existing proteins might enter the nucleus during the viral replication, while 29.8% of the mitochondrial cytoplasmic co-existing proteins may enter the mitochondria during this process, This suggests that the replication of HBV may alter the localization of these proteins in the cells. The STRING analysis showed that there were 30 proteins down-regulated in the cell nucleus, and their changes in the whole cell and the mitochondria were not significant, and even up-regulated, so we speculate that these proteins may be shuttling between the subcellular structures. In addition, we explored the effect of 20 host proteins on the replication of HBV, and found that 10 of them can affect the expression of HBeAg, but only DDX1 can affect the expression of HBsAg at the same time.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R373

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