【作者】 陈静；

【导师】 张改平；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2013， 硕士

【摘要】 RNA干扰（RNA interference，RNAi）现象是一种由双链RNA（double-strand RNA，dsRNA）启动的序列特异性基因沉默机制。RNAi从线虫到人类细胞都广泛存在，是一种先天性抗病毒的自我防御方式，能够抵抗外源基因或病毒的入侵。然而，病毒在受到RNAi抵抗过程中会通过病毒蛋白或非编码的病毒RNA来抑制RNA沉默，即RNA沉默抑制子（RNAsilencing suppressor，RSS），从而使病毒在宿主细胞内复制、增殖，促进病毒感染。目前，已从植物、动物和人类病毒中鉴定了20多种RSSs，并且这些RSSs中大部分都是干扰素的拮抗蛋白。猪繁殖与呼吸综合征（Porcine Reproductive and Respiratory Syndrome，PRRS）是一种以母猪繁殖障碍和仔猪呼吸困难为主要特征的病毒性疾病，给养猪业造成重大的经济损失。PRRS是由PRRSV引起的，PRRSV是有囊膜的单股正链RNA病毒，属于动脉炎病毒属（Arterivirus），动脉炎病毒科，套式病毒目(Nidoviruses)。基因组全长约15kb，含有9个开放性阅读框（ORFs），其中，ORF-1编码14中非结构蛋白（nsps），nsps除了具有复制、蛋白酶、聚合酶的功能外，同时还能抑制干扰素的产生；ORF2-7编码病毒的结构蛋白。RNAi作为宿主细胞抑制病毒复制、清除病毒的一种先天性免疫机制，是宿主重要的免疫防御系统，许多持续性感染的病毒都存在一定的机制拮抗RNA诱导的基因沉默。PRRSV存在免疫抑制和持续性感染的免疫学特性，并且PRRSV的感染能抑制干扰素的产生，表明PRRSV可能会存在抑制RNA诱导的基因沉默的作用，从而逃避宿主先天性的抗病毒免疫。本研究利用RNAi技术，将人工合成的dsRNA和shRNA、mi-RNA干扰表达质粒分别转染MARC-145细胞，dsRNA、shRNA和mi-RNA靶向的目标基因是萤火虫荧光素酶报告基因（Luciferase，Luc），经双荧光素酶报告检测系统检测，发现dsRNA、shRNA和mi-RNA均抑制了转染的细胞Luc的表达量，表明在MARC-145细胞上成功构建了dsRNA、shRNA和mi-RNA诱导的基因沉默的模型；PRRSV与RNAi模型共作用于MARC-145细胞，发现PRRSV能够恢复被抑制的Luc的表达量，证实PRRSV能够抑制RNA诱导的基因沉默；PRRSV的nsp1和nsp11分别与RNAi模型共转染MARC-145细胞，nsp1和nsp11同样也能恢复被抑制的Luc的表达量，其中，nsp1可以自水解为nsp1α和nsp1β，nsp1能够恢复被抑制基因表达量主要是通过nsp1α实现的，nsp1α和nsp11可能是RNA沉默抑制子；分别构建nsp1α和nsp11的突变体使nsp1α的木瓜蛋白酶活性和nsp11的核酸内切酶活性都失活，nsp1α和nsp11的突变体分别与RNAi模型共转染MARC-145细胞，然而，这些突变体不能抑制RNAi诱导的基因沉默，这揭示nsp1α的木瓜蛋白酶活性和nsp11的核酸内切酶活性对nsp1α和nsp11发挥RSS活性起关键性作用。在MARC-145细胞上，PRRSV抑制了shRNA、 dsRNA和miRNA诱导的RNA沉默，并且PRRSV的非结构蛋白nsp1和nsp11具有抑制RNA诱导的基因沉默的作用，nsp1α和nsp11是RNA沉默的抑制子，nsp1α的木瓜样蛋白酶的活性和nsp11的核酸内切酶活性分别是nsp1α和nsp11发挥抑制RNA诱导的基因沉默活性所必需的。更多还原

【Abstract】 RNAi is sequence-specific gene silencing mechanism activated by thedouble-stranded RNA（dsRNA）,As a natural defense mechanism to the foreign geneor virus attack, the phenomenon of RNAi existed widly in plant,namatodes worm,drosophila and human cells. However, virus can counteract this anti-viral defense ofthe host by viral proteins and non-coding viral RNAs, RNA silencing suppressor(RSS). Currently, about20RSSs have been identified from plant, animal and humanviruses20, and most of these RSSs are interferon antagonist.Porcine reproductive and respiratory syndrome (PRRS) is an infectious diseasecharacterized by respiratory disorders or abortion in pregnant sows and Pigletsbreathing difficulties, resulting in important economic losses to the swine industry.PRRSV, a small enveloped virus including a single positive-stranded RNA genome, isa member of family Arteriviridae which, along with the Coronaviridae, are classifiedin the order Nidovirales. The genome is about15kb, containing nine open readingframes (ORFs).ORF-1encodes14non-structural proteins (nsps), In addition to thecopy, nsps have protease, polymerase function, and inhibit interferon production.ORF2-7encoed viral structural proteins. The PRRSV exist immunologicalcharacteristics of immunosuppression and persistent infection,which can caused virusimmune escape. RNAi can inhibit of viral replication and clear the virus as a innateimmune defense system. A suboptimal innate immune response and the persistentinfection were the important features of PRRSV infection, so the aims of our presentwork are to address whether PRRSV could suppress the RNA silencing, whether thePRRSV encoded RSSs which help the virus overcome the cellular antiviral RNAsilencing response.In this study, by RNAi technology, the synthetic dsRNA and shRNA or mi-RNAinterference expression plasmid were transfected into MARC-145cells. The fireflyluciferase reporter gene(Luc) is the target gene targeted by dsRNA, shRNA andmi-RNA.Luc expression was suppressed on transfected MARC-145cells by by dualfluorescence detection system. It indicated that the dsRNA, shRNA, andmi-RNA-induced gene silencing model is successfully constructed on MARC-145cells;when PRRSV and RNAi model were acted on MARC-145cells, PRRSV restoreexpression of silenced luciferase reporter gene on MARC-145cells.It showned thatPRRSV can inhibit the shRNA, dsRNA, miRNA-induced gene silencing. Whennsp1or nsp11and RNAi model were con-transfected on MARC-145cells, nsp1andnsp11, also can restore expression of suppressed luciferase. nsp1hydrolysis nsp1αand nsp1β, and nsp1inhibit the shRNA, dsRNA, miRNA-induced gene silencing isachieved by nsp1α. nsp1α and nsp11were RNA silencing suppressor; ThePCPαactivity of nsp1α and the endoribonuclease activity of nsp11are inactivated inthe mutant of Nsp1α and nsp11.The mutant of nsp1α or nsp11and RNAi model were con-transfected on MARC-145cells, however, these mutants can not inhibit theRNAi-induced gene silencing. It demonstrated that the activity of PCPα and theactivity of endoribonuclease were respectively required for nsp1α and nsp11as theRSSs.PRRSV could suppress the shRNA, dsRNA and miRNA-induced RNAsilencing on MARC-145cells. nsp1and nsp11have effect on inhibitting theRNA-induced gene silencing,and nsp1α and nsp11are RNA silencing suppressor. Theactivity of PCPα and the activity of endoribonuclease were respectively required fornsp1α and nsp11as the RSSs.更多还原