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当前位置: 首页 > 文章 > 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 畜牧与兽医 2018 (6) 41-46
Position: Home > Articles > Preparation and identification of sheep fetal fibroblasts expressing shRNA targeting at BVDV Animal Husbandry & Veterinary Medicine 2018 (6) 41-46

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

作  者：
魏军昌;陈创夫;乔军;侯晓旭;马齐蔓;姚洋;李村院;李晓悦;倪伟;胡圣伟
关键词：
shRNA;转基因细胞;BVDV;抗病毒
摘  要：
为了获得具有抗牛病毒性腹泻病毒(BVDV)能力的绵羊胎儿成纤维细胞,采用组织块和酶消化法分离培养绵羊胎儿成纤维细胞,利用脂质体转染法将shRNA转基因表达载体转染绵羊胎儿成纤维细胞,经抗性筛选获得抗性细胞克隆,利用PCR方法对获得的细胞克隆进行鉴定,用BVDV侵染转基因细胞,利用real-time PCR和病毒滴定测定法分析不同细胞克隆抑制BVDV复制的能力。结果显示:试验成功构建了靶向BVDV表达shRNA的转基因表达载体pNeo-2loxp-u6-sh BVDV,经抗性筛选共获得22个抗性细胞克隆;22个细胞克隆中12个细胞克隆含有目的基因,为转基因细胞,其中5个转基因细胞克隆具有明显的抗BVDV复制的能力,最高抗病毒效率达到90.7%。结论:在细胞水平能有效抑制BVDV病毒增殖的转基因绵羊胚胎成纤维细胞的获得,为进一步利用体细胞核移植技术制备转基因绵羊提供了合适的供体细胞。
译  名：
Preparation and identification of sheep fetal fibroblasts expressing shRNA targeting at BVDV
作  者：
WEI Junchang;CHEN Chuangfu;QIAO Jun;HOU Xiaoxu;MA Qiman;YAO Yang;LI Cunyuan;LI Xiaoyue;NI Wei;HU Shengwei;College of Life Sciences,Shihezi University;College of Animal Science and Technology,Shihezi University;
单  位：
WEI Junchang%CHEN Chuangfu%QIAO Jun%HOU Xiaoxu%MA Qiman%YAO Yang%LI Cunyuan%LI Xiaoyue%NI Wei%HU Shengwei%College of Life Sciences,Shihezi University%College of Animal Science and Technology,Shihezi University
关键词：
shRNA;;transgenic cells;;BVDV;;antiviral property
摘  要：
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 p Neo-2 loxp-u6-sh BVDV which expressed shRNA targeting at BVDV,and that twenty two resistant 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.

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