本研究以组织块法分离大白猪胎儿成纤维细胞(Porcine fetus fibroblasts,PFF),通过脂质体将含有抗猪瘟病毒基因及绿色荧光蛋白基因(GFP)的重组质粒转入猪胎儿成纤维细胞,探讨转染猪胎儿成纤维细胞的影响因素,如DNA质量、脂质体用量及转染时间.实验发现脂质体2.5uL、质粒1.2μg、转染时间6h时转染率最高,为32.5％.过量的脂质体、质粒及过长的转染时间(＞6h)会引起细胞的皱缩及死亡.本研究旨在为构建抗猪瘟病毒转基因克隆猪提供核供体.%Take large white pig as the study materials, porcine fetus fibroblasts was obtained by the method of tissue culture. Plasmid pGPU6/GFP/Neo of eGFP as report gene and Anti - Swine fever virus gene as aim gene was transfected into porcine fetus fibroblasts by lipofectamineTM 2000. By adjusting transfection time, concentrations of plasmid DNA and liposome to improve transfection rate. Perfect cell transfection efficiency (32. 5% ) was obtained on the condition of the complex of 1. 2μg Plasmid and 2. 5uL lipofectamine TM 2000 transfecting PFF in 6 hours. Both high concentration of LipofectamineTM 2000 and longer incubation time ( >6h) will cause cell shrinkage and death. The present study would provide donor cell for the production of transgenic pig which can resist classicial swine fever vires infection.
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