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转基因山羊抑制口蹄疫病毒3D基因

DOI: 10.1360/052012-335, PP. 404-410

Keywords: 山羊,口蹄疫病毒3D基因,shRNA,&ldquo,睡美人&rdquo,转座子

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Abstract:

利用shRNA与高效的“睡美人”(sleepingbeauty,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Southernblot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.

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