【摘要】：豬瘟(Classical swine fever,CSF)是由豬瘟病毒(Classical swine fever virus,CSFV)引起的對養(yǎng)豬業(yè)危害嚴重的傳染病。我國通過兔化弱毒疫苗的使用有效地控制了豬瘟的流行,然而非典型豬瘟的出現(xiàn)使得豬瘟的防控形勢依然嚴峻。非典型豬瘟造成豬群的持續(xù)帶毒排毒,但外表健康,這種持續(xù)性感染是造成我國豬瘟仍然發(fā)生的一個重要原因。在這個過程中,CSFV與宿主細胞的長期共存是CSFV能夠持續(xù)性感染的主要原因之一,一方面CSFV利用宿主的物質(zhì)來進行自身的組裝,另一方面通過誘導調(diào)控宿主的相關反應,來創(chuàng)造利于自身增殖的細胞微環(huán)境,從而達到免疫逃逸的結果。因此,研究CSFV免疫逃逸的相關機制,有助于了解CSFV持續(xù)性感染的原因,為豬瘟的防控提供科學依據(jù),而腫瘤壞死因子受體相關因子(tumor necrosis factor receptor-associated factors,TRAF)作為天然免疫通路中的重要分子,在多個免疫通路中發(fā)揮著重要作用。本文研究了CSFV NS3蛋白與TRAF5蛋白之間的相互作用,進而探究了TRAF5對CSFV增殖的影響,獲得了以下結果。(1)證實CSFV NS3蛋白與宿主TRAF5蛋白之間存在直接的相互作用。成功構建TRAF5基因和CSFV NS3基因的相關表達載體,通過外源性和內(nèi)源性Co-IP試驗,都檢測到細胞內(nèi)的TRAF5-NS3蛋白復合體,通過GST-Pulldown試驗,在體外檢測到TRAF5-NS3復合體,同時,通過激光共聚焦試驗,觀察到TRAF5蛋白與CSFV NS3蛋白在PAM細胞中存在共定位現(xiàn)象。(2)證明TRAF5通過誘導IL-10的表達促進CSFV的增殖。成功構建TRAF5過表達穩(wěn)轉細胞系和TRAF5 shRNA干擾穩(wěn)轉細胞系,通過RT-qPCR和Western blot檢測發(fā)現(xiàn),TRAF5過表達細胞系中CSFV的增殖能力顯著增強,而在TRAF5干擾細胞系中,CSFV的增殖受到明顯的抑制;在隨后對細胞因子的檢測中發(fā)現(xiàn),TRAF5過表達細胞系中IL-10表達量顯著升高,而在干擾細胞系中卻被明顯抑制;在接種CSFV后,TRAF5過表達細胞系中IL-10被進一步上調(diào),而在干擾細胞系中,IL-10的表達依然受到了明顯抑制。綜上所述,本研究證實了CSFV NS3蛋白能夠與TRAF5蛋白發(fā)生相互作用,并且發(fā)現(xiàn)TRAF5通過誘導IL-10的表達促進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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651

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