發(fā)布時(shí)間：2018-03-11 05:36

  本文選題：H5N1禽流感病毒　切入點(diǎn)：相互作用網(wǎng)絡(luò)　出處：《天津大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：甲型H5N1高致病性禽流感出現(xiàn)的人畜共患現(xiàn)象已引起社會(huì)恐慌。H5N1病毒侵染機(jī)體，與宿主蛋白相互作用以完成病毒復(fù)制和轉(zhuǎn)錄，為了確定宿主蛋白在H5N1病毒致病過(guò)程中發(fā)揮的作用，，本實(shí)驗(yàn)室前期通過(guò)酵母雙雜交技術(shù)，以病毒蛋白為誘餌，對(duì)人脾文庫(kù)進(jìn)行篩選，篩選出與H5N1禽流感病毒的10個(gè)病毒蛋白相互作用的179個(gè)宿主蛋白。本研究通過(guò)生物信息學(xué)方法構(gòu)建了病毒蛋白與人蛋白質(zhì)相互作用網(wǎng)絡(luò)，發(fā)現(xiàn)這些相互作用多集中在病毒聚合酶復(fù)合體，參與調(diào)解包括細(xì)胞凋亡、免疫應(yīng)答、蛋白轉(zhuǎn)運(yùn)、RNA加工等多個(gè)生物學(xué)過(guò)程；與隨機(jī)網(wǎng)絡(luò)蛋白相比發(fā)現(xiàn)，H5N1相互作用的蛋白在網(wǎng)絡(luò)中的連接度和介度均高于其他蛋白，且更傾向于連接在一起；同時(shí)對(duì)宿主蛋白和宿主蛋白鄰近蛋白進(jìn)行基因富集分析，發(fā)現(xiàn)這些蛋白主要集中在NF-κB、WNT、MAPK、p53及凋亡通路；此外還將H5N1與其他禽流感毒株以及其他病原體的人相互作用蛋白質(zhì)進(jìn)行基因功能分析，為更好的解釋不同流感毒株或不同病原體侵染宿主產(chǎn)生不同機(jī)體反應(yīng)提供思路。 基于相互作用網(wǎng)絡(luò)分析，我們選擇病毒聚合酶PB2與人RNF31這對(duì)相互作用進(jìn)行深入研究。PB2是流感病毒聚合酶的一個(gè)重要亞基，它與病毒毒力相關(guān)并決定病毒的宿主范圍，PB2多個(gè)氨基酸殘基的改變都可以引起宿主范圍的改變；RNF31是E3泛素連接酶的一種，其對(duì)NF-κB轉(zhuǎn)錄活性的調(diào)節(jié)具有重要作用。本研究發(fā)現(xiàn)，RNF31下調(diào)時(shí)抑制對(duì)TNF-α介導(dǎo)的NF-κB轉(zhuǎn)錄活性，下調(diào)NF-κB下游與炎癥相關(guān)因子表達(dá)，抑制TNF-α介導(dǎo)的IκBα的磷酸化，同時(shí)細(xì)胞凋亡增多。 另外本研究通過(guò)間接免疫熒光、核質(zhì)分離發(fā)現(xiàn)PB2與RNF31共定位于胞核，通過(guò)Co-IP證實(shí)PB2與RNF31相互作用發(fā)生在胞核內(nèi)，相互作用結(jié)構(gòu)域分別位于PB2N端1-471肽段和RNF31C端910-1062肽段。同時(shí)，PB2抑制NF-κB轉(zhuǎn)錄活性和NF-κB下游與凋亡相關(guān)的靶基因表達(dá)；干涉RNF31，PB2對(duì)NF-κB轉(zhuǎn)錄活性的抑制作用消失；流式細(xì)胞分析發(fā)現(xiàn)PB2促進(jìn)細(xì)胞凋亡，與下調(diào)RNF31促凋亡結(jié)果相一致，提示PB2是通過(guò)RNF31影響NF-κB轉(zhuǎn)錄活性和其介導(dǎo)的生物學(xué)功能。此外，穩(wěn)定性實(shí)驗(yàn)和胞核Co-IP發(fā)現(xiàn)，RNF31不僅能微弱影響PB2的穩(wěn)定性，也能增強(qiáng)PB2與聚合酶另一個(gè)亞基PB1相互作用。推測(cè)RNF31可能在影響流感聚合酶的形成過(guò)程中發(fā)揮重要作用，為分析流感病毒致病機(jī)制和尋找藥物靶點(diǎn)提供新的思路。
[Abstract]:The emergence of highly pathogenic avian influenza A (H5N1) has caused social panic. H5N1 viruses infect the body and interact with host proteins to complete viral replication and transcription, in order to determine the role of host proteins in the pathogenesis of H5N1. In our laboratory, the human spleen library was screened by yeast two-hybrid technique and viral protein was used as bait. 179 host proteins interacting with 10 viral proteins of H5N1 avian influenza virus were screened. In this study, a network of interactions between viral proteins and human proteins was constructed by bioinformatics. It was found that these interactions were mainly concentrated in virus polymerase complexes and involved in many biological processes, including apoptosis, immune response, protein transporter RNA processing and so on. Compared with random network proteins, it was found that proteins interacting with H5N1 had higher connectivity and intermedium in the network than other proteins, and were more likely to be linked together, and the host proteins and host protein adjacent proteins were also analyzed for gene enrichment. It was found that these proteins were mainly concentrated in NF- 魏 B, WNTG MAPKN, p53 and apoptotic pathways. In addition, the gene function analysis of human interaction proteins between H5N1 and other avian influenza strains and other pathogens was carried out. In order to better explain the different influenza strains or different pathogens infected host to produce different body reaction ideas. Based on the interaction network analysis, we select virus polymerase PB2 and human RNF31 to study the interaction. PB2 is an important subunit of influenza virus polymerase. It is associated with virulence of the virus and determines the host range of the virus. Changes in the amino acid residues of PB2 can cause changes in the host range. RNF31 is one of the E3 ubiquitin ligases. It plays an important role in regulating the transcriptional activity of NF- 魏 B. in this study, we found that the down-regulation of RNF31 inhibits the transcriptional activity of NF- 魏 B mediated by TNF- 偽, down-regulates the expression of down-stream NF- 魏 B and inflammatory related factors, inhibits the phosphorylation of I 魏 B 偽 mediated by TNF- 偽, and increases apoptosis. In addition, PB2 and RNF31 were colocated in the nucleus by indirect immunofluorescence, and the interaction between PB2 and RNF31 was confirmed by Co-IP in the nucleus. The interaction domain was located at the PB2N terminal 1-471 peptide and the RNF31C terminal 910-1062, respectively. At the same time, PB2 inhibited NF- 魏 B transcription activity and NF- 魏 B downstream apoptosis-related target gene expression, and interfered with the inhibition of NF- 魏 B transcriptional activity by RNF31tPB2. Flow cytometry showed that PB2 promoted apoptosis, which was consistent with down-regulation of RNF31, suggesting that PB2 affected NF- 魏 B transcriptional activity and its mediated biological function through RNF31. Stability test and nuclear Co-IP showed that RNF31 not only weakly affected the stability of PB2, but also enhanced the interaction of PB2 with another subunit of polymerase, PB1. It is suggested that RNF31 may play an important role in the formation of influenza polymerase. It provides a new idea for analyzing the pathogenic mechanism of influenza virus and searching for drug target.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R511.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 方立群,曹春香,陳國(guó)勝,雷富民,劉亞嵐,李承毅,楊紅,韓曉娜,閆磊,閆磊,李小文,曹務(wù)春;地理信息系統(tǒng)應(yīng)用于中國(guó)大陸高致病性禽流感的空間分布及環(huán)境因素分析[J];中華流行病學(xué)雜志;2005年11期

本文編號(hào)：1596850