猪瘟病毒NS3互作蛋白TRAF5及对猪瘟病毒增殖的影响

发布时间：2018-08-26 09:16

【摘要】：猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的对养猪业危害严重的传染病。我国通过兔化弱毒疫苗的使用有效地控制了猪瘟的流行,然而非典型猪瘟的出现使得猪瘟的防控形势依然严峻。非典型猪瘟造成猪群的持续带毒排毒,但外表健康,这种持续性感染是造成我国猪瘟仍然发生的一个重要原因。在这个过程中,CSFV与宿主细胞的长期共存是CSFV能够持续性感染的主要原因之一,一方面CSFV利用宿主的物质来进行自身的组装,另一方面通过诱导调控宿主的相关反应,来创造利于自身增殖的细胞微环境,从而达到免疫逃逸的结果。因此,研究CSFV免疫逃逸的相关机制,有助于了解CSFV持续性感染的原因,为猪瘟的防控提供科学依据,而肿瘤坏死因子受体相关因子(tumor necrosis factor receptor-associated factors,TRAF)作为天然免疫通路中的重要分子,在多个免疫通路中发挥着重要作用。本文研究了CSFV NS3蛋白与TRAF5蛋白之间的相互作用,进而探究了TRAF5对CSFV增殖的影响,获得了以下结果。(1)证实CSFV NS3蛋白与宿主TRAF5蛋白之间存在直接的相互作用。成功构建TRAF5基因和CSFV NS3基因的相关表达载体,通过外源性和内源性Co-IP试验,都检测到细胞内的TRAF5-NS3蛋白复合体,通过GST-Pulldown试验,在体外检测到TRAF5-NS3复合体,同时,通过激光共聚焦试验,观察到TRAF5蛋白与CSFV NS3蛋白在PAM细胞中存在共定位现象。(2)证明TRAF5通过诱导IL-10的表达促进CSFV的增殖。成功构建TRAF5过表达稳转细胞系和TRAF5 shRNA干扰稳转细胞系,通过RT-qPCR和Western blot检测发现,TRAF5过表达细胞系中CSFV的增殖能力显著增强,而在TRAF5干扰细胞系中,CSFV的增殖受到明显的抑制;在随后对细胞因子的检测中发现,TRAF5过表达细胞系中IL-10表达量显著升高,而在干扰细胞系中却被明显抑制;在接种CSFV后,TRAF5过表达细胞系中IL-10被进一步上调,而在干扰细胞系中,IL-10的表达依然受到了明显抑制。综上所述,本研究证实了CSFV NS3蛋白能够与TRAF5蛋白发生相互作用,并且发现TRAF5通过诱导IL-10的表达促进CSFV的增殖。
[Abstract]:Classical swine fever (Classical swine fever,CSF) is a serious infectious disease caused by (Classical swine fever virus,CSFV (swine fever virus). The use of rabbitized attenuated vaccine has effectively controlled the prevalence of swine fever in China. However, the emergence of atypical swine fever has made the situation of prevention and control of swine fever still severe. Atypical swine fever caused persistent detoxification of swine flocks, but the appearance of this persistent infection is an important reason for the occurrence of swine fever in China. In this process, the long-term coexistence of CSFV and host cells is one of the main reasons for persistent infection of CSFV. On the one hand, CSFV makes use of host substances to assemble itself, on the other hand, it regulates the host response by inducing. To create a microenvironment conducive to self-proliferation, thus achieving immune escape results. Therefore, to study the mechanism of immune escape of CSFV is helpful to understand the cause of persistent infection of CSFV and to provide scientific basis for prevention and control of swine fever. Tumor necrosis factor receptor related factor (tumor necrosis factor receptor-associated factors,TRAF (TNF- receptor) is an important molecule in innate immune pathway. Play an important role in multiple immune pathways. In this paper, the interaction between CSFV NS3 protein and TRAF5 protein was studied, and the effect of TRAF5 on CSFV proliferation was investigated. The following results were obtained. (1) the direct interaction between CSFV NS3 protein and host TRAF5 protein was confirmed. The expression vectors of TRAF5 gene and CSFV NS3 gene were successfully constructed. The intracellular TRAF5-NS3 protein complex was detected by exogenous and endogenous Co-IP tests, and the TRAF5-NS3 complex was detected in vitro by GST-Pulldown assay. The co-localization of TRAF5 protein and CSFV NS3 protein in PAM cells was observed by laser confocal test. (2) it was proved that TRAF5 could promote the proliferation of CSFV by inducing the expression of IL-10. TRAF5 overexpression stable cell line and TRAF5 shRNA interference stable transformed cell line were successfully constructed. By RT-qPCR and Western blot detection, the proliferative ability of CSFV in the overexpression cell line was significantly enhanced, but the proliferation of CSFV was significantly inhibited in TRAF5 interference cell line. In the subsequent cytokines detection, the expression of IL-10 was significantly increased in the overexpression cell line of TRAF5, but was significantly inhibited in the interfering cell line, and the expression of IL-10 in the overexpression cell line of TRAF5 was further up-regulated after inoculation of CSFV. However, the expression of IL-10 in interfering cell lines was still significantly inhibited. In conclusion, this study confirmed that CSFV NS3 protein can interact with TRAF5 protein, and it was found that TRAF5 promotes the proliferation of CSFV by inducing the expression of IL-10.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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