慢病毒介导稳定表达FABP4的小尾寒羊成纤维细胞系的构建

摘要

构建1株能够稳定表达FABP4的小尾寒羊成纤维细胞系,用于小尾寒羊未来的品种保护、开发与利用,首先利用组织块培养法制备了小尾寒羊原代成纤维细胞,然后利用全基因合成法克隆了绵羊 FABP4基因,并构建了过表达FABP4的慢病毒载体.经过包装后,使用获得的慢病毒感染小尾寒羊原代成纤维细胞,并通过嘌呤霉素筛选稳定转染细胞,最后应用Western Blot鉴定FABP4蛋白的过表达水平.结果表明,经过传代2 ~3次后,得到纯度较高的小尾寒羊原代成纤维细胞.滴度测定表明,当在96孔板中添加病毒原液量为10 -4μL时,观测不到荧光.当添加量为10 -3μL,能够观测到荧光,说明获得慢病毒的滴度为1 ×106TU/mL以上.利用制备的慢病毒感染小尾寒羊原代成纤维细胞,并经过嘌呤霉素筛选后,在荧光显微镜下所有细胞均能观察到绿色荧光.应用Western Blot分析发现,在对照组几乎检测不到 FABP4蛋白的表达,而在病毒感染组则能够清晰的观察到FABP4蛋白的表达,说明成功制备了稳定表达FABP4的小尾寒羊成纤维细胞系.%The aim of the study is to construct a fibroblast of Small-tailed Han sheep which could stably ex-press FABP4.Firstly,the primary fibroblasts of Small-tailed Han sheep were prepared by tissue culture,then the sheep FABP4 gene was cloned by chemical synthesis and the DNA fragment was sub-cloned into a lentivi-ral vector.After packaging,the lentivirus was affected with the primary fibroblasts of Small-tailed Han sheep and purified by puromycin.At last,the expression of FABP4 protein was identified by Western Blot.The results showed that after passing 2 -3 times,the primary fibroblasts of Small-tailed Han sheep were obtained with high purity.The titer assay showed that no fluorescence was observed when the amount of 10 -4μL virus stock solu-tion was added in the 96-well plate, while the fluorescence could be observed when the amount of 10 -3μL vi-rus stock solution was added, indicating that the titer of lentivirus was above 1 × 106TU / mL. After infected by the lentivirus and purified by puromycin,green fluorescence could be observed in all cells.The Western Blot showed that FABP4 protein expression was undetectable in the control group, but FABP4 protein expression was clearly observed in the virus-infected group, which indicated that the stably expressing FABP4 fibroblasts from the Small-tailed Han sheep were constructed successfully.