RNA-Seq技术筛选抗猪繁殖与呼吸综合征病毒的宿主限制性因子及其抗病毒作用验证

发布时间：2018-06-07 08:45

本文选题：猪呼吸与繁殖综合征 + RNA-Seq　；参考：《中国农业科学院》2016年硕士论文

【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种急性、高度传染的病毒性传染病,对世界养猪业造成巨大的经济损失。2006年,我国出现高致病性猪繁殖与呼吸综合征病毒(Highly pathogenic PRRSV,HP-PRRSV),具有很强的感染力和致病性,给我国养猪业带来更大的危害。猪肺泡巨噬细胞(Porcine pulmonary alveolar macrophages,PAMs)是HP-PRRSV的主要靶细胞。I型干扰素(Interferon,IFN)具有抑制PRRSV复制的能力,而IFN发挥抗病毒作用主要是依靠干扰素刺激基因(Interferon stimulated genes,ISGs)编码的宿主限制性因子。寻找抑制HP-PRRSV复制的宿主限制性因子已经成为近年来研究的热点,但IFN刺激产生的限制性因子数量众多,筛选工作量巨大。RNA-Seq技术是新一代测序技术,具有高通量、效率高、灵敏度好的优势。本试验中,利用RNA-Seq技术对IFN处理的PAMs中抗HPPRRSV限制性因子进行高通量筛选。将抗PRRSV的限制性因子ISG15作为指示因子,根据感染PRRSV的PAMs中ISG15的表达变化,选择接毒后12小时作为收样时间。本实验设置了4个不同处理组,分别为IFN、IFN+PRRSV、PRRSV和C(空白对照组)组。用猪IFN-α处理PAMs诱导细胞产生抗病毒状态,再感染HP-PRRSV,进而用RNA-Seq技术检测IFN预处理的PAMs对HP-PRRSV的应答变化。IFN-α处理细胞共检测到346个上调的差异性表达基因(DEGs),其中有93个DEGs在HPPRRSV感染细胞中表达水平显著下降。富集分析这93个下降表达的DEGs,主要分布在免疫应答相关的通路,如“RIG-I样受体信号通路”、“病毒应答”、“刺激应答”。在表达差异的16个抗病毒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个的表达可以被HP-PRRSV抑制。为了验证以上16个抗病毒DGEs是否具有抑制HP-PRRSV复制的作用,采用siRNA干扰技术下调其表达,然后感染HP-PRRSV,分析病毒复制水平。结果发现PKR、OAS1、IFIT1(ISG56)、ISG15、IFI44、ISG20、BST2、IRF7、IFIT2、LOC100627004(IFITM1)、IFITM3、IFIT5和GBP1均能显著抑制PRRSV的复制。谷氧还蛋白1(glutaredoxin 1,GLRX1)是一种具有抗氧化、信号转导、抗凋亡等多功能的蛋白。RNA-Seq检测结果显示IFN可以显著上调GLRX1的表达,但HP-PRRSV感染能显著抑制其表达。siRNA干扰下调PAMs中GLRX1的表达,HP-PRRSV复制显著增加,说明GLRX1能够抑制HP-PRRSV的增殖。比较感染PRRSV猪和健康猪的各组织中的GLRX1表达差异,发现感染PRRSV猪腹股沟淋巴结、心脏、肺门淋巴结中的GLRX1表达量显著下降,而小肠、肺脏和肌肉中的GLRX1表达量显著上升。上述结果表明PRRSV能够影响猪不同组织的GLRX1的表达。本研究采用RNA-Seq技术筛选了抑制HP-PRRSV复制的宿主限制性因子,并验证了其抗HP-PRRSV复制的作用,为研究HP-PRRSV免疫应答和免疫逃逸提供了基础数据。
[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- 伪 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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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