【摘要】：豬輪狀病毒(PoRV)是引起幼齡仔豬發(fā)生急性胃腸炎的主要病原,一般通過接觸病畜的排泄物后經(jīng)糞口途徑進(jìn)行傳播,呈地方性流行。目前在我國流行范圍較廣的是A群PoRV,在豬群中流行率為3.3%~67.3%,其中G9型毒株呈再度流行趨勢。PoRV單純感染或與其它病原(豬流行性腹瀉病毒、豬傳染性胃腸炎病毒、豬圓環(huán)病毒等)混合感染,直接或間接地影響豬群生產(chǎn)力,嚴(yán)重制約了我國養(yǎng)豬業(yè)的健康發(fā)展,造成巨大經(jīng)濟(jì)損失。因此,深入了解PoRV在機(jī)體內(nèi)的復(fù)制過程,研究病毒蛋白與宿主蛋白之間的相互作用機(jī)制,對于有效控制疫病流行、降低對養(yǎng)豬業(yè)的危害極為迫切和重要。本研究將實驗室分離的Rotavirus A pig/China/NMTL/2008/G9P[23]毒株(簡稱NMTL株)作為研究對象,該毒株對豬有較強致病力,豬也是輪狀病毒主要的儲存宿主,存在動物病原傳染給人的潛在危險,因此具有重要的研究價值。PoRV VP6蛋白是病毒粒子最主要的結(jié)構(gòu)蛋白,位于病毒粒子三層衣殼結(jié)構(gòu)的中間層。它與病毒脫殼釋放至胞漿中的雙層病毒粒子(DLPs)的轉(zhuǎn)錄活性有關(guān)。根據(jù)研究報道,VP6蛋白在病毒感染細(xì)胞中與不同細(xì)胞蛋白發(fā)生一系列相互作用,在病毒的復(fù)制過程中起關(guān)鍵作用,但對參與這個過程的細(xì)胞蛋白以及有關(guān)功能仍不十分清楚。在本研究中,采用RT-PCR方法擴(kuò)增出PoRV NMTL株VP6基因c DNA全長,將VP6 PCR產(chǎn)物克隆至原核表達(dá)載體p GEX-6P-1中,并轉(zhuǎn)化至DH5α感受態(tài)細(xì)胞,獲得p GEX-PoRV-VP6重組質(zhì)粒,測序正確后轉(zhuǎn)化至大腸桿菌表達(dá)菌BL21(DE3),IPTG誘導(dǎo)融合蛋白表達(dá),獲得GST-VP6重組蛋白。利用GST pull-down技術(shù)和MALDI-TOF/TOF質(zhì)譜分析鑒定,發(fā)現(xiàn)了3種與PoRV VP6蛋白存在相互作用的細(xì)胞蛋白,分別是:β-肌動蛋白(beta-actin)、原肌球蛋白1型(TPM1)以及40S核糖體蛋白S16(RPS16),并通過免疫共沉淀(Co-IP)試驗加以驗證。此外,還對PoRV VP6蛋白與β-肌動蛋白相互作用進(jìn)行了后續(xù)研究:用si RNA干擾敲低細(xì)胞β-肌動蛋白表達(dá)量后,感染PoRV,用western blot方法檢測細(xì)胞裂解物中VP6蛋白表達(dá)量變化,用高通量內(nèi)涵篩選系統(tǒng)檢測釋放至培養(yǎng)液上清中病毒的感染率。通過熒光定量q RT-PCR方法,檢測接毒后不同時間釋放至培養(yǎng)液上清中的病毒含量,確定PoRV一輪復(fù)制周期所需時間。結(jié)合電鏡觀察和VP6蛋白表達(dá)量檢測結(jié)果,確定PoRV的不同復(fù)制階段。分別在PoRV不同復(fù)制階段(接毒后0h/2h/4h/6h),用肌動蛋白聚合抑制劑Cyto D處理感染細(xì)胞,用熒光定量q RT-PCR方法檢測接毒后8h釋放至培養(yǎng)液上清中的病毒含量差異,確定抑制肌動蛋白聚合作用對PoRV增殖釋放的影響。雙免疫熒光染色后在共聚焦顯微鏡下觀察PoRV VP6蛋白與?-肌動蛋白在感染細(xì)胞中是否共定位,用免疫電鏡觀察PoRV VP6蛋白在細(xì)胞內(nèi)的運送途徑。用氯化銫不連續(xù)密度梯度離心方法制備PoRV DLPs,通過肌動蛋白結(jié)合蛋白(ABPs)spin-down試驗確定PoRV DLPs能否在體外增強肌動蛋白的聚合作用。通過以上研究發(fā)現(xiàn),β-肌動蛋白、原肌球蛋白1型、40S核糖體蛋白S16,是PoRV感染過程中與VP6蛋白相互作用的細(xì)胞蛋白。這3種蛋白廣泛存在于各種組織細(xì)胞中,在各物種進(jìn)化過程中高度保守,病毒在體內(nèi)增殖,利用容易獲得的細(xì)胞蛋白,有助于增強病毒對細(xì)胞的適應(yīng)性,擴(kuò)大病毒感染的組織范圍,也為RV的跨種間傳播提供了可能。用PoRV感染si RNA干擾敲低β-肌動蛋白表達(dá)量的細(xì)胞,病毒VP6蛋白合成和病毒粒子釋放顯著降低。MA104細(xì)胞經(jīng)si ACTB轉(zhuǎn)染72h后,感染PoRV NMTL株48h,細(xì)胞裂解物中PoRV VP6蛋白表達(dá)量與對照組相比顯著降低;將上述處理后收集到的上清樣品感染MA104細(xì)胞18h后,si ACTB處理組PoRV感染陽性細(xì)胞數(shù)量,明顯少于未處理組、Mock組、si FAM組等對照組。以上研究證實,PoRV VP6與?-肌動蛋白相互作用,能夠有效介導(dǎo)病毒感染,在胞漿中運輸DLPs、起始m RNA轉(zhuǎn)錄翻譯合成病毒蛋白、組裝和釋放TLPs過程中起重要作用。雙免疫熒光染色實驗發(fā)現(xiàn),帶有不同熒光標(biāo)記的PoRV VP6蛋白和β-肌動蛋白,在病毒感染細(xì)胞的胞漿中共定位。免疫電鏡觀察發(fā)現(xiàn),在PoRV感染早期,帶有膠體金標(biāo)記的VP6蛋白位于肌動蛋白微絲上,此后VP6蛋白分布于感染細(xì)胞核糖體、線粒體、內(nèi)質(zhì)網(wǎng)、細(xì)胞核中。研究結(jié)果表明,PoRV DLPs在感染細(xì)胞中依賴肌動蛋白網(wǎng)絡(luò)結(jié)構(gòu),運送至細(xì)胞內(nèi)的特定位置進(jìn)行復(fù)制。這些發(fā)現(xiàn)揭示了PoRV VP6蛋白在感染細(xì)胞內(nèi)的運輸途徑,以及參與PoRV復(fù)制的細(xì)胞成分。ABPs spin-down試驗體外證實,PoRV VP6蛋白結(jié)合至肌動蛋白微絲上,但DLPs并不能增強肌動蛋白的聚合活性。通過這2種蛋白之間的相互作用,PoRV DLPs劫持了細(xì)胞的肌動蛋白骨架系統(tǒng),使它在細(xì)胞中像“貨物”一樣被運輸至特定的細(xì)胞器中,以便于病毒復(fù)制。但在體內(nèi)PoRV感染過程中,VP6蛋白是否招募其它宿主細(xì)胞蛋白(如:本研究已證實的VP6互作蛋白TPM1)協(xié)同刺激肌動蛋白聚合,還需要后續(xù)實驗驗證。在PoRV感染細(xì)胞過程中,病毒與宿主細(xì)胞之間存在著多種多樣的蛋白相互作用,形成密不可分的蛋白網(wǎng)絡(luò),對于調(diào)控病毒復(fù)制、啟動宿主天然免疫反應(yīng),都發(fā)揮著重要的功能。本論文的研究發(fā)現(xiàn),將有助于進(jìn)一步揭示PoRV VP6蛋白及其互作蛋白如何參與介導(dǎo)和調(diào)控病毒復(fù)制過程,對于深入闡明PoRV復(fù)雜的致病機(jī)制,預(yù)防和控制腹瀉相關(guān)疾病傳播,提供可靠的科學(xué)依據(jù)和研究參考。
[Abstract]:Porcine rotavirus (PoRV) is the main pathogen causing acute gastroenteritis in piglets. At present, the prevalence rate of G9 strain in swine population is 3. 3% ~ 67. 3%. PoRV's simple infection or mixed infection with other pathogens (porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine circovirus, etc.) directly or indirectly affects pig population productivity, severely restricts the healthy development of pig industry in China, and causes huge economic loss. Therefore, it is very urgent and important to study the replication process of PoRV in organism and study the mechanism of interaction between virus protein and host protein. Rotavirus A pig/ China/ NMTL/ 2008/ G9P[23] strain (NMTL strain) isolated from the laboratory was used as the research object. The strain has strong pathogenicity to the pig, and the pig is the main storage host of rotavirus, and there is a potential danger to the human being. Therefore, it has important research value. PoRV VP6 protein is the most important structural protein of viral particles and is located in the middle layer of three-layer capsid structure of virus particles. It is related to the transcription activity of the double-layer virus particles (DLPs) released into the cytoplasm of the virus. According to the study, VP6 protein has a series of interactions with different cell proteins in virus-infected cells, playing a key role in the replication of viruses, but the cellular proteins involved in this process, as well as related functions, remain unclear. In this study, the full length of VP6 gene of PoRV NMTL strain was amplified by RT-PCR, and VP6 PCR products were cloned into prokaryotic expression vector pGEX-6P-1 and transformed to DH5 Jurkat cells to obtain p-GEX-PoRV-VP6 recombinant plasmid, and then transformed into E. coli expression bacteria BL21 (DE3) after sequencing. The fusion protein was induced by IPTG to obtain GST-VP6 recombinant protein. Three kinds of cellular proteins interacting with PoRV VP6 protein were identified by GST pull-down technique and Capture-TOF/ TOF mass spectrometry. The results showed that there were three kinds of cellular proteins interacting with PoRV VP6 protein, namely: actin-actin (beta-actin), tropomyosin type 1 (TPM1), and 40S ribosomal protein S16 (RPS16), and verified by co-precipitation (Co-IP) test. In addition, the interaction between the PoRV VP6 protein and the actin-actin was studied. The expression of VP6 protein in the cell lysate was detected by western blot. The high-throughput screening system was used to detect the infection rate of virus released to the culture solution. The time required for the PoRV round replication cycle was determined by the fluorescence quantitative q RT-PCR method, the virus content released to the culture broth at different times after the incubation. The different replication phases of PoRV were determined by combining the results of electron microscope observation and VP6 protein expression. The effects of actin cytoskeleton on the proliferation of PoRV were determined by using actin polymerization inhibitor Cyto D to treat infected cells at different replication stages of PoRV (post-toxin 0h/ 2h/ 4h/ 6h). The PoRV VP6 protein was observed under confocal microscope after double immunofluorescence staining. Whether actin was co-located in infected cells, and the transport pathway of PoRV VP6 protein in cells was observed by immunoelectron microscopy. PoRV DLPs were prepared by non-continuous density gradient centrifugation with cesium chloride and the ability of PoRV DLPs to enhance actin in vitro was determined by the actin-binding protein (ABPs) spin-down assay. It has been found by the above studies that actin-actin, tropomyosin type 1, 40S ribosomal protein S16 is a cellular protein interacting with VP6 protein in the process of PoRV infection. The three proteins are widely present in various histiocytes, and are highly conserved in the evolution of each species, virus is proliferated in the body, the cell protein which is easy to obtain can be utilized, the adaptability of the virus to the cells can be enhanced, and the tissue range of the virus infection is expanded, It is also possible to provide inter-seed propagation for the RV. A significant reduction in viral VP6 protein synthesis and viral particle release was observed with poRV-infected si RNA interfering with the expression of low-actin-actin-expressing cells. The expression level of PoRV VP6 in cell lysates was significantly lower than that in the control group after treatment with si ACTB for 72h, and the number of positive cells of PoRV-infected cells in the si ACTB treated group was significantly lower than that in untreated group after the treatment was infected with MA104 cells for 18h. Mock group, si FAM group and other control group. In the above studies, PoRV VP6 and? Actin interaction, which can effectively mediate viral infection, plays an important role in the transport of DLPs in cytoplasm, the transcription of the starting m RNA, the synthesis of viral proteins, the assembly and release of TLPs. Two immunofluorescent staining experiments showed that the PoRV VP6 protein with different fluorescent labels and the actin-actin were co-located in the cytoplasm of the virus infected cells. Immunoelectron microscopy showed that the VP6 protein with colloidal gold was located on actin microfilaments early in PoRV infection, after which VP6 protein was distributed in the ribosomes, mitochondria, ER and nucleus of infected cells. The results show that PoRV DLPs are dependent on actin network structure in infected cells and are transported to specific sites within the cells for replication. These findings reveal the transport pathways of PoRV VP6 proteins in infected cells, as well as cellular components involved in PoRV replication. The ABPs spin-down assay demonstrated in vitro that the PoRV VP6 protein binds to actin microfilaments, but DLPs do not enhance actin polymerization activity. Through the interaction between these two proteins, the PoRV DLPs abducted the actin cytoskeleton system of the cells so that it was in the cells. "Goods" the same is transported to a specific cell organelle to facilitate viral replication. However, in vivo PoRV infection, VP6 protein was used to recruit other host cell proteins (e.g., VP6 interacting protein TPM1 demonstrated in this study) to stimulate actin polymerization, as well as follow-up experimental verification. In the process of poRV infection, there is a wide variety of protein interactions between the virus and the host cell, which forms an inseparable protein network, and plays an important role in regulating the replication of the virus and starting the innate immune response of the host. The study of this paper will help to further reveal how PoRV VP6 protein and its interacting protein can be involved in mediating and regulating the virus replication process. It provides a reliable scientific basis and reference for further elucidating the pathogenesis of PoRV complicated pathogenesis, preventing and controlling the spread of diarrhea-related diseases.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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