转基因山羊抑制口蹄疫病毒3D基因  

RNA Interference Against FMDV-3DReplication in Transgenic Goat

作　　者：邓守龙[1] 于坤[1] 李文婷[1] 姚玉昌[2] 鲁涛 赵雅琳 袁三平 富俊才[1] 连正兴[1] 

机构地区：[1]中国农业大学动物科技学院,北京100193 [2]东北农业大学动物科学技术学院,哈尔滨150030 [3]青岛森淼生物技术研究所,青岛266101

出　　处：《中国科学：生命科学》2013年第5期404-410,共7页Scientia Sinica(Vitae)

基　　金：转基因生物新品种培育重大专项课题(批准号:2008ZX08008-005;2009ZX0800-020B)资助项目

摘　　要：利用shRNA与高效的"睡美人"(sleeping beauty,SB)转座子系统生产抑制口蹄疫病毒3D基因复制的山羊.对口蹄疫病毒基因(2B,3D)shRNA抑制靶点进行筛选,构建了3条含靶基因shRNA的pLL3.7表达载体,与含靶基因的psiCheck2载体(双荧光素标记)共转染293FT细胞,对合成的shRNA进行筛选.获得的抑制效果好的3D1shRNA(干扰3D基因)插入到SB转座子载体上,将含3D1shRNA的SB转座子与转座酶(2:1,5:1,10:1)通过原核显微注射生产转基因山羊.出生羔羊进行Southern blot与整合位点鉴定,对阳性个体耳成纤维细胞转染含3D基因的pisCheck2载体,观察其抑制率.结果共获得了7只阳性山羊.SB转座子与转座酶的比例为10:1时,生产转基因山羊的效率为21.05%.阳性个体耳成纤维细胞对口蹄疫病毒3D基因有显著抑制效果.In this study, shRNA and sleeping beauty （SB） transposon system were used to produce transgenic goats that inhibit foot-and-mouth disease virus 3D gene replication. Foot-and-mouth disease virus gene （2B and 3D） shRNA was designed and inserted into the shRNA expression vector pLL3.7, pLL3.7-shRNA vectors and psiCheck2 vectors （containing the target gene） were co-transfected 293FT cells for functional verification. Effective shRNA （interference 3D gene） was inserted into the SB transposon vector. The SB transposon containing 3DlshRNA and the transposase enzyme （2 ： 1, 5 ： 1, 10： 1） was performed to make transgenic goats by pronuclear injection. Newly born lambs were examined by Southern blotting and integration sites were identified. Ear fibroblast cells of positive individuals were transfected with the psiCheck2-3D to observe the inhibition rate. Seven positive transgnic goats were obtained. The efficiency of transgenic goat production was 21.05% with the SB transposon and transposase in 10：1. The results show that RNAi effectively inhibits foot-and-mouth disease virus 3D gene replication in ear fibroblasts of positive individuals.

关 键 词：山羊 口蹄疫病毒3D基因 SHRNA “睡美人”转座子 

分 类 号：S855.3[农业科学—临床兽医学]