畜牧与兽医2018，Vol.50Issue(6)：41-46,6.
表达靶向牛病毒性腹泻病毒shRNA的绵羊胎儿成纤维细胞的制备和鉴定
Preparation and identification of sheep fetal fibroblasts expressing shRNA targeting at BVDV
摘要
Abstract
Sheep fetal fibroblasts possess the ability of resisting against bovine viral diarrhea virus (BVDV).In this study,sh3 and sh8 which efficiently inhibit the replication of BVDV were used to construct a shRNA transgenic expression vector.Tissue explants and the enzyme digestion method were used to isolate and cultivate sheep fetal fibroblasts.The liposome transfection method was used to transfect the shRNA transgenic expression vector into the sheep fetal fibroblasts,and resistant cell clones were obtained by resistance selection.Then,PCR was used to identify the obtained cell clones.The transgenic cells were infected with BVDV,and Real-time PCR and virus titration were employed to analyze the ability of inhibiting the replication of BVDV in different cell clones.The results showed that the construction was successful of the transgenic expression vector pNeo-2loxp-u6-shBVDV which expressed shRNA targeting at BVDV,and that twenty two re sistant cell clones were identified.Twelve of the cell clones containing the target genes were transgenic cells.And five of these transgenic cells effectively inhibited the replication of BVDV with a maximal antiviral efficiency of 90.7％.Conclusion:The sheep fetal fibroblasts were obtained here to inhibit the replication of BVDV efficiently,which suggested that suitable donor cells could be provided for transgenic sheep by means of the somatic cell nuclear transfer technique.关键词
shRNA/转基因细胞/BVDV/抗病毒Key words
shRNA/transgenic cells/BVDV/antiviral property分类
农业科技引用本文复制引用
魏军昌,胡圣伟,陈创夫,乔军,侯晓旭,马齐蔓,姚洋,李村院,李晓悦,倪伟..表达靶向牛病毒性腹泻病毒shRNA的绵羊胎儿成纤维细胞的制备和鉴定[J].畜牧与兽医,2018,50(6):41-46,6.基金项目
国家自然科学基金项目(31460037,316607180,31360615) （31460037,316607180,31360615）
