發(fā)布時間：2018-06-07 08:45

  本文選題：豬呼吸與繁殖綜合征 + RNA-Seq��； 參考：《中國農(nóng)業(yè)科學(xué)院》2016年碩士論文

【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)由豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的一種急性、高度傳染的病毒性傳染病,對世界養(yǎng)豬業(yè)造成巨大的經(jīng)濟損失。2006年,我國出現(xiàn)高致病性豬繁殖與呼吸綜合征病毒(Highly pathogenic PRRSV,HP-PRRSV),具有很強的感染力和致病性,給我國養(yǎng)豬業(yè)帶來更大的危害。豬肺泡巨噬細(xì)胞(Porcine pulmonary alveolar macrophages,PAMs)是HP-PRRSV的主要靶細(xì)胞。I型干擾素(Interferon,IFN)具有抑制PRRSV復(fù)制的能力,而IFN發(fā)揮抗病毒作用主要是依靠干擾素刺激基因(Interferon stimulated genes,ISGs)編碼的宿主限制性因子。尋找抑制HP-PRRSV復(fù)制的宿主限制性因子已經(jīng)成為近年來研究的熱點,但IFN刺激產(chǎn)生的限制性因子數(shù)量眾多,篩選工作量巨大。RNA-Seq技術(shù)是新一代測序技術(shù),具有高通量、效率高、靈敏度好的優(yōu)勢。本試驗中,利用RNA-Seq技術(shù)對IFN處理的PAMs中抗HPPRRSV限制性因子進行高通量篩選。將抗PRRSV的限制性因子ISG15作為指示因子,根據(jù)感染PRRSV的PAMs中ISG15的表達變化,選擇接毒后12小時作為收樣時間。本實驗設(shè)置了4個不同處理組,分別為IFN、IFN+PRRSV、PRRSV和C(空白對照組)組。用豬IFN-α處理PAMs誘導(dǎo)細(xì)胞產(chǎn)生抗病毒狀態(tài),再感染HP-PRRSV,進而用RNA-Seq技術(shù)檢測IFN預(yù)處理的PAMs對HP-PRRSV的應(yīng)答變化。IFN-α處理細(xì)胞共檢測到346個上調(diào)的差異性表達基因(DEGs),其中有93個DEGs在HPPRRSV感染細(xì)胞中表達水平顯著下降。富集分析這93個下降表達的DEGs,主要分布在免疫應(yīng)答相關(guān)的通路,如“RIG-I樣受體信號通路”、“病毒應(yīng)答”、“刺激應(yīng)答”。在表達差異的16個抗病毒DGEs中,包括IRG6(RSAD2)、IFI44、ISG20、PKR、LOC100738990(TRIM5)、BST2、OAS1、IFIT1(ISG56)、IRF7、IFIT2、ISG15、LOC100627004(IFITM1)、ISG12(A)(IFI27)、IFITM3、IFIT5,其中15個的表達可以被HP-PRRSV抑制。為了驗證以上16個抗病毒DGEs是否具有抑制HP-PRRSV復(fù)制的作用,采用siRNA干擾技術(shù)下調(diào)其表達,然后感染HP-PRRSV,分析病毒復(fù)制水平。結(jié)果發(fā)現(xiàn)PKR、OAS1、IFIT1(ISG56)、ISG15、IFI44、ISG20、BST2、IRF7、IFIT2、LOC100627004(IFITM1)、IFITM3、IFIT5和GBP1均能顯著抑制PRRSV的復(fù)制。谷氧還蛋白1(glutaredoxin 1,GLRX1)是一種具有抗氧化、信號轉(zhuǎn)導(dǎo)、抗凋亡等多功能的蛋白。RNA-Seq檢測結(jié)果顯示IFN可以顯著上調(diào)GLRX1的表達,但HP-PRRSV感染能顯著抑制其表達。siRNA干擾下調(diào)PAMs中GLRX1的表達,HP-PRRSV復(fù)制顯著增加,說明GLRX1能夠抑制HP-PRRSV的增殖。比較感染PRRSV豬和健康豬的各組織中的GLRX1表達差異,發(fā)現(xiàn)感染PRRSV豬腹股溝淋巴結(jié)、心臟、肺門淋巴結(jié)中的GLRX1表達量顯著下降,而小腸、肺臟和肌肉中的GLRX1表達量顯著上升。上述結(jié)果表明PRRSV能夠影響豬不同組織的GLRX1的表達。本研究采用RNA-Seq技術(shù)篩選了抑制HP-PRRSV復(fù)制的宿主限制性因子,并驗證了其抗HP-PRRSV復(fù)制的作用,為研究HP-PRRSV免疫應(yīng)答和免疫逃逸提供了基礎(chǔ)數(shù)據(jù)。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) an acute, highly contagious viral disease caused by porcine reproductive and respiratory syndrome virus (PRRS), which has caused enormous economic losses to the world pig industry. Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSVV) appears in China, which has strong infectious and pathogenicity, and brings more harm to the pig industry in China. Porcine pulmonary alveolar macrophagesus (PAMs) is the main target cell of HP-PRRSV, type I interferon (IFN) has the ability to inhibit the replication of PRRSV, while the antiviral effect of IFN is mainly dependent on the host restrictive factor encoded by Interferon stimulated genestimulus (ISGs). The search for host restrictive factors to inhibit HP-PRRSV replication has become a hot topic in recent years, but the number of restrictive factors produced by IFN stimulation is numerous. The sieving workload is huge. RNA-Seq technology is a new generation of sequencing technology with high throughput and high efficiency. The advantage of good sensitivity. In this experiment, RNA-Seq technique was used to screen the high throughput HPPRRSV restrictive factors in PAMs treated with IFN. According to the change of ISG15 expression in PAMs infected with PRRSV, the restriction factor (ISG15) against PRRSV was used as indicator factor, and 12 hours after inoculation was selected as the collecting time. Four different treatment groups were set up in this experiment, namely IFNN IFN PRRSVV PRRSV and C (blank control group). PAMs was treated with porcine IFN- 偽 to induce the antiviral state of the cells. Reinfection of HP-PRRSV.Then the response of PAMs pretreated with IFN to HP-PRRSV was detected by RNA-Seq technique. A total of 346 up-regulated differentially expressed genes were detected in the cells treated with IFN. 93 DEGs expression levels in HPPRRSV infected cells decreased significantly. These 93 decreased expression levels were mainly distributed in immune response-related pathways, such as "RIG-I like receptor signaling pathway", "viral response" and "stimulus response". Among the 16 antiviral DGEs, IRG6, RSAD2, IFI44, ISG20, PKRC100738990, BST2OAS1, ISG56, IRF7LOC100627004, IRF7LOC100627004, the expression of 15 of them can be inhibited by HP-PRRSV. In order to verify whether the 16 antiviral DGEs can inhibit the replication of HP-PRRSV, siRNA interference technique was used to down-regulate its expression, and then infected with HP-PRRSVS to analyze the replication level of the virus. The results showed that both the PRRSV replication was inhibited significantly by PKRASOAS1, IFIT1, ISG56, ISG15, ISG20, BST2, IRF7, LOC100627004, IFITM1, IFITM3, IFIT5 and GBP1. GLRX1) is a multifunctional protein with anti-oxidation, signal transduction and anti-apoptosis. The results of RNA-Seq detection show that IFN can significantly up-regulate the expression of GLRX1. But HP-PRRSV infection could significantly inhibit its expression. SiRNA interference down-regulated the expression of GLRX1 in PAMs and the replication of HP-PRRSv increased significantly, indicating that GLRX1 could inhibit the proliferation of HP-PRRSV. The expression of GLRX1 in inguinal lymph nodes, heart and hilar lymph nodes of infected PRRSV pigs was significantly decreased, while the expression of GLRX1 in small intestine, lung and muscle was significantly increased. These results suggest that PRRSV can affect the expression of GLRX1 in different tissues of pigs. In this study, RNA-Seq technique was used to screen host restrictive factors for inhibiting HP-PRRSV replication, and its role in inhibiting HP-PRRSV replication was verified, which provided basic data for the study of HP-PRRSV immune response and immune escape.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.65

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