RNA干扰论文：RNA干扰 ORFV pll3.7载体 Check2载体.docVIP

【关键词】RNA干扰 ORFV pll3.7载体 Check2载体 【英文关键词】RNA interference(RNAi) ORFV pll3.7vector Check2vector RNA干扰论文：利用RNAi技术建立抗羊传染性脓疱皮炎病毒的细胞株 【中文摘要】羊传染性脓疱皮炎(contagious ecthyma)俗称“羊口疮”，是由羊传染性脓疱皮炎病毒(Orf virus,ORFV)引起的山羊、绵羊等中小反刍动物以及人的一种嗜上皮性、接触性传染病。本病传染性强、发病率高、呈世界性流行，虽为温和性疾病，但是，当病畜口、唇等部位病变严重时，将严重影响其吃奶、采食、吞咽，导致衰弱，甚至死亡，有报道称患病羔羊的病死率高达93%。因此本病的发生给养羊业带来巨大的经济损失，并对人类健康构成威胁。本研究选择ORFV复制所必需的DNA聚合酶及在ORFV的增值过程中起到重要作用的VETF-S和VETF-L基因作为RNA干扰的靶基因。首先，设计该靶基因的扩增引物，经PCR扩增后与Check2载体分别经PmeI和NotI双酶切，酶切后连接产生Check2-靶基因重组质粒。同时，利用公用网站按照RNAi序列设计原则，设计RNAi11个靶点序列，与经XhoI和HpaI双酶切后的pll3.7载体连接产生pll3.7-shRNA质粒。然后，利用磷酸钙法进行pll3.7-shRNA质粒和Check2-靶基因重组质粒两种质粒共转染293T细胞，之后利用双荧光检测系统试剂盒检测其RNA干扰效果。干扰效果最好可达到91.0%。最后，筛选获得可分别表达P-D-3，P-L-2，P-S-2干扰小片段的3株山羊成纤维细胞细胞株。并通过测定病毒滴度、测定靶基因蛋白表达量，对siRNA抵抗病毒感染的效力进行了评估。本研究对今后该病的抗病研究、药物研究以及克隆转基因抗病羊等都有很好的参考和应用价值。 【英文摘要】Orf, also knowed as contagious ecthyma, which is caused by orf virus, is a common epitheliotrophic viraldisease of sheep、goats and wild ruminants. Humans are also susceptible to orf. The disease which has highinfectivity and morbidity is found throughout the world. The eating and swallowing of the sick animal will beseriously affected by its mouth and lip′s lesion. It was reported that the prevalence of lamb mortality can behigh up to93%. The disease can cause huge economic loss for ovine husbandry and threaten the health ofhuman.DNA polymerase、VETF-S and VETF-L genes which the virus needed to copy were selected as thetargeted gene. The primers of the targeted gene were designed, then the targeted gene were obtained by PCR,which were connected with Check2vector digested by PmeI and NotI. Check2-targeted gene vectors wereconstructed．In accordance with RNAi sequence design principles in the public Web site, eleven RNAi targetsequences were designed, then the target sequences of Oligo DNA were synthesized and annealed todouble-stranded DNA, which were connected with pll3.7vector digested by XhoI and HpaI. Pll3.7-shRNAvectors were constructed.293T cells were co-transf