本文選題：SARS-CoV冠狀病毒 + 膜蛋白　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：天然免疫反應(yīng)是機(jī)體抵抗外源性病原體入侵感染的第一道防線。天然免疫系統(tǒng)主要通過(guò)受染細(xì)胞中的模式識(shí)別受體(PRR)來(lái)識(shí)別病原體來(lái)源的病原相關(guān)分子模式(PAMP)。大部分的模式識(shí)別受體位于細(xì)胞膜或內(nèi)體膜上以識(shí)別病原體相關(guān)的NA、DNA或者人工合成的雙鏈RNA類(lèi)似物如polyI:C,同時(shí)還有一些模式識(shí)別受體游離存在于細(xì)胞質(zhì)中,識(shí)別病毒的DNA核酸。然而,目前對(duì)于侵入細(xì)胞內(nèi)病原體所釋放出的蛋白質(zhì)是否可以作為一類(lèi)胞內(nèi)的病原相關(guān)分子模式(PAMP來(lái)發(fā)揮激活先天免疫反應(yīng)作用還不是很清楚。實(shí)驗(yàn)室前期的研究發(fā)現(xiàn),在HEK293細(xì)胞中,轉(zhuǎn)染嚴(yán)重急性呼吸綜合癥冠狀病毒(SARS-CoV)編碼的M基因可以誘導(dǎo)Ⅰ型干擾素(p干擾素)表達(dá)上調(diào),然而具體機(jī)制并未得到深入的研究。在本論文研究中,我們發(fā)現(xiàn)轉(zhuǎn)染SARS-CoV冠狀病毒M基因可以上調(diào)HEK293T,HEK293ET以及永生化小鼠骨髓巨噬細(xì)胞J2-M(?)中IFNβ的基因表達(dá),但在A549和Vero細(xì)胞中,M基因未能上調(diào)IFNβ啟動(dòng)子的表達(dá)水平,說(shuō)明M基因上調(diào)IFNβ的現(xiàn)象可能具有組織細(xì)胞特異性。并且M基因同時(shí)激活了誘導(dǎo)Ⅰ型干擾素表達(dá)的TBK1-IRF3以及NFκB信號(hào)通路。通過(guò)構(gòu)建M基因不翻譯的突變M-stop,并與正常M蛋白表達(dá)質(zhì)粒進(jìn)行功能比較,結(jié)果發(fā)現(xiàn)是M蛋白而不是M的mRNA誘導(dǎo)IFNβ的基因表達(dá)。此外,對(duì)干擾素表達(dá)通路中上游信號(hào)分子進(jìn)行系統(tǒng)檢測(cè),發(fā)現(xiàn)M蛋白激活I(lǐng)FNβ的基因表達(dá)與TLR受體通路中的銜接蛋白MyD88, TIRAP和TICAM2相關(guān),而并沒(méi)誘導(dǎo)激活RIG-I和MDA5介導(dǎo)的信號(hào)通路。我們還通過(guò)免疫共沉淀的方法檢測(cè)了M蛋白與TLR受體通路MyD88和TRAM銜接蛋白的相互作用,結(jié)果為陰性,說(shuō)明M蛋白可能通過(guò)間接的方式調(diào)控了TLR受體通路的銜接蛋白。由于蛋白表達(dá)實(shí)驗(yàn)中發(fā)現(xiàn)M蛋白的條帶有拖尾現(xiàn)象,我們?cè)噲D檢測(cè)M蛋白是否可能發(fā)生了泛素化修飾,通過(guò)突變其預(yù)測(cè)可能發(fā)生泛素化的賴氨酸位點(diǎn)并構(gòu)建突變進(jìn)行表達(dá)檢測(cè),同時(shí)對(duì)M蛋白與HA-Ub共轉(zhuǎn)后進(jìn)行相互作用檢測(cè),以及免疫沉淀M蛋白后進(jìn)行質(zhì)譜檢測(cè),然而我們得到的都是陰性結(jié)果,說(shuō)明M蛋白在Western blotting實(shí)驗(yàn)中檢測(cè)到的拖尾條帶不是泛素化修飾,可能發(fā)生了其他的翻譯后修飾。為進(jìn)一步分析M蛋白發(fā)揮作用是否發(fā)生在細(xì)胞內(nèi),我們用蛋白分泌抑制劑brefeldin A(BFA)阻斷M蛋白由胞內(nèi)向胞外的運(yùn)輸,結(jié)果顯示加入BFA后并沒(méi)有抑制M蛋白誘導(dǎo)IFNP的基因表達(dá)。用TLR2和TLR4受體的抑制劑阻斷可能的胞外信號(hào)轉(zhuǎn)導(dǎo)途徑,也沒(méi)有削弱M蛋白誘導(dǎo)IFNβ的基因表達(dá),證明了M蛋白誘導(dǎo)IFNp活化的驅(qū)動(dòng)力產(chǎn)生自細(xì)胞內(nèi)部。構(gòu)建TRAF3基因的siRNA進(jìn)行瞬時(shí)轉(zhuǎn)染和穩(wěn)轉(zhuǎn)細(xì)胞系的構(gòu)建,其并沒(méi)有影響M蛋白誘導(dǎo)IFNβ的轉(zhuǎn)錄表達(dá)過(guò)程,因而M蛋白誘導(dǎo)IFNβ的活化過(guò)程不依賴于TRAF3信號(hào)分子。SARS-CoV冠狀病毒的假病毒感染細(xì)胞后,誘導(dǎo)IFNβ的表達(dá)上調(diào)依賴于M蛋白,M蛋白點(diǎn)突變(V68A)產(chǎn)生的假病毒并不具有誘導(dǎo)IFNβ的表達(dá)上調(diào)的現(xiàn)象。同時(shí),我們根據(jù)M蛋白親水區(qū)和疏水跨膜區(qū)構(gòu)建了M蛋白的缺失突變體,發(fā)現(xiàn)M蛋白的疏水跨膜區(qū)對(duì)于上調(diào)IFNβ的功能可能具有重要作用。本研究首次證明了來(lái)自于病毒編碼的蛋白可以作為一個(gè)細(xì)胞內(nèi)的病原相關(guān)分子模式,發(fā)揮誘導(dǎo)Ⅰ型干擾素的表達(dá)上調(diào)的作用,其激活途徑與TLR受體信號(hào)通路相關(guān)但不依賴于TRAF3。本研究證實(shí)病毒蛋白可作為胞內(nèi)病原相關(guān)分子模式的概念。
[Abstract]:The natural immune response is the first line of defense against the invasion of exogenous pathogens. The natural immune system mainly identifies the pathogen associated molecular pattern (PAMP) by the pattern recognition receptor (PRR) in the infected cells. Most of the pattern recognition receptors are located on the cell membrane or the inner body membrane to identify the pathogen related N A, DNA, or synthetic double stranded RNA analogues, such as polyI:C, also have some pattern recognition receptors free in the cytoplasm to identify virus DNA nucleic acids. However, whether the proteins released by the intruded intracellular pathogens can be used as a class of intracytoplasmic related molecular patterns (PAMP to activate innate immunity. The effect of pestilence was not very clear. Earlier laboratory studies found that the transfection of M gene encoded by severe acute respiratory syndrome coronavirus (SARS-CoV) in HEK293 cells could induce the up-regulated expression of interferon type I (interferon P), but the specific mechanism was not deeply studied. In this study, we found transfection of SARS-C OV coronavirus M gene can increase the expression of IFN beta in HEK293T, HEK293ET and immortalized mouse bone marrow macrophages J2-M (?), but in A549 and Vero cells, the M gene does not increase the expression level of the IFN beta promoter, indicating that the M gene up-regulated IFN beta may have the tissue cell specificity. TBK1-IRF3 and NF kappa B signaling pathway expressed by type IFN interferon. By constructing a mutant M-stop that is not translated by M gene and comparing with normal M protein expression plasmid, the result was found to be the gene expression of mRNA induced IFN beta of M protein instead of M. In addition, the upstream signal molecules in the interferon expression pathway were systematically detected and the M protein was found. The gene expression of IFN beta was associated with the cohesive protein MyD88, TIRAP and TICAM2 in the TLR receptor pathway, but did not induce the activation of RIG-I and MDA5 mediated signaling pathways. We also detected the interaction between M protein and TLR receptor pathway MyD88 and TRAM cohesive protein by immunoprecipitation. The results were negative, indicating that the M protein may pass through. The indirect method regulates the TLR receptor pathway. Due to the discovery of the trailing phenomenon of M protein in the protein expression experiment, we try to detect the possibility of ubiquitination of M protein and predict the possible ubiquitinating lysine site and construct mutation for expression detection by mutation. Meanwhile, M protein and HA-U are also detected. After B co rotation, interaction detection and immunoprecipitation M protein were detected by mass spectrometry, but we were all negative results, indicating that the trailing strips detected by M protein in Western blotting experiment were not ubiquitination, and other post-translational trimming may occur. In the cell, we use protein secretory inhibitor brefeldin A (BFA) to block the transport of M protein from intracellular to extracellular. The result showed that BFA did not inhibit the gene expression of M protein induced IFNP. The possible extracellular signal transduction pathway was blocked by the inhibitor of TLR2 and TLR4 receptor, and the gene expression of IFN beta induced by M protein was not weakened, and it was proved that the gene expression of IFN beta was induced by M protein. The driving force of IFNp activation induced by M protein was produced from the inner cell. The siRNA of TRAF3 gene was constructed for transient transfection and construction of stable cell line, which did not affect the transcription of IFN beta induced by M protein, so the activation process of IFN beta induced by M protein was not dependent on the pseudo virus infection of the TRAF3 signal molecule.SARS-CoV cov. After the cell, the expression of IFN beta was up to up dependent on M protein, and the pseudo virus produced by M protein point mutation (V68A) did not induce the up-regulated expression of IFN beta. At the same time, we constructed a deletion mutant of the M protein based on the hydrophilic region of M protein and the hydrophobic transmembrane region, and found that the hydrophobic transmembrane region of M protein may have a heavy function to regulate the function of IFN beta. This study has demonstrated for the first time that the protein derived from the virus can be used as an intracellular pathogen associated molecular model to induce the up regulation of the expression of type I interferon, which is related to the TLR receptor signaling pathway but does not depend on the TRAF3. protein as a intracellular pathogen associated molecule. The concept of a pattern.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R392

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