SARS-CoV冠状病毒M蛋白作为一类新的胞内病原相关分子模式上调Ⅰ型干扰素表达

发布时间：2018-05-03 13:09

本文选题：SARS-CoV冠状病毒 + 膜蛋白　；参考：《北京协和医学院》2016年博士论文

【摘要】：天然免疫反应是机体抵抗外源性病原体入侵感染的第一道防线。天然免疫系统主要通过受染细胞中的模式识别受体(PRR)来识别病原体来源的病原相关分子模式(PAMP)。大部分的模式识别受体位于细胞膜或内体膜上以识别病原体相关的NA、DNA或者人工合成的双链RNA类似物如polyI:C,同时还有一些模式识别受体游离存在于细胞质中,识别病毒的DNA核酸。然而,目前对于侵入细胞内病原体所释放出的蛋白质是否可以作为一类胞内的病原相关分子模式(PAMP来发挥激活先天免疫反应作用还不是很清楚。实验室前期的研究发现,在HEK293细胞中,转染严重急性呼吸综合症冠状病毒(SARS-CoV)编码的M基因可以诱导Ⅰ型干扰素(p干扰素)表达上调,然而具体机制并未得到深入的研究。在本论文研究中,我们发现转染SARS-CoV冠状病毒M基因可以上调HEK293T,HEK293ET以及永生化小鼠骨髓巨噬细胞J2-M(?)中IFNβ的基因表达,但在A549和Vero细胞中,M基因未能上调IFNβ启动子的表达水平,说明M基因上调IFNβ的现象可能具有组织细胞特异性。并且M基因同时激活了诱导Ⅰ型干扰素表达的TBK1-IRF3以及NFκB信号通路。通过构建M基因不翻译的突变M-stop,并与正常M蛋白表达质粒进行功能比较,结果发现是M蛋白而不是M的mRNA诱导IFNβ的基因表达。此外,对干扰素表达通路中上游信号分子进行系统检测,发现M蛋白激活IFNβ的基因表达与TLR受体通路中的衔接蛋白MyD88, TIRAP和TICAM2相关,而并没诱导激活RIG-I和MDA5介导的信号通路。我们还通过免疫共沉淀的方法检测了M蛋白与TLR受体通路MyD88和TRAM衔接蛋白的相互作用,结果为阴性,说明M蛋白可能通过间接的方式调控了TLR受体通路的衔接蛋白。由于蛋白表达实验中发现M蛋白的条带有拖尾现象,我们试图检测M蛋白是否可能发生了泛素化修饰,通过突变其预测可能发生泛素化的赖氨酸位点并构建突变进行表达检测,同时对M蛋白与HA-Ub共转后进行相互作用检测,以及免疫沉淀M蛋白后进行质谱检测,然而我们得到的都是阴性结果,说明M蛋白在Western blotting实验中检测到的拖尾条带不是泛素化修饰,可能发生了其他的翻译后修饰。为进一步分析M蛋白发挥作用是否发生在细胞内,我们用蛋白分泌抑制剂brefeldin A(BFA)阻断M蛋白由胞内向胞外的运输,结果显示加入BFA后并没有抑制M蛋白诱导IFNP的基因表达。用TLR2和TLR4受体的抑制剂阻断可能的胞外信号转导途径,也没有削弱M蛋白诱导IFNβ的基因表达,证明了M蛋白诱导IFNp活化的驱动力产生自细胞内部。构建TRAF3基因的siRNA进行瞬时转染和稳转细胞系的构建,其并没有影响M蛋白诱导IFNβ的转录表达过程,因而M蛋白诱导IFNβ的活化过程不依赖于TRAF3信号分子。SARS-CoV冠状病毒的假病毒感染细胞后,诱导IFNβ的表达上调依赖于M蛋白,M蛋白点突变(V68A)产生的假病毒并不具有诱导IFNβ的表达上调的现象。同时,我们根据M蛋白亲水区和疏水跨膜区构建了M蛋白的缺失突变体,发现M蛋白的疏水跨膜区对于上调IFNβ的功能可能具有重要作用。本研究首次证明了来自于病毒编码的蛋白可以作为一个细胞内的病原相关分子模式,发挥诱导Ⅰ型干扰素的表达上调的作用,其激活途径与TLR受体信号通路相关但不依赖于TRAF3。本研究证实病毒蛋白可作为胞内病原相关分子模式的概念。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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