GDF9慢病毒载体的构建及其在山羊原代成纤维细胞中的稳定表达

摘要

This study was aimed to constract growth differentiation factor 9 (GDF9) lentiviral vector,which was stably expressed in goat primary fibroblast.The CDS of sheep GDF9 gene was cloned by gene synthesis,the CDS was 1 362 bp,encoding 453 amino acids.After double enzyme digestion and ligation,the GDF9 fragment was sub-cloned into an empty lentiviral vector.The GDF9 lentiviral vector and the packaging plasmids were co-transfected into 293T cells,and the lentivirus with titer of 1 × 106 TU/mL was obtained.The goat primary fibroblasts were transfected with GDF9 lentivirus,the red fluorescence could be observed in more than 60 ％ of the cells,suggesting the prepared lentivirus had high infection efficiency to goat primary fibroblasts.After purom ycin screening,all cells were able to observe red fluorescence.Real-time quantitative PCR analysis showed that the GDF9 expression level was higher than that of control group.The results indicated that the goat primary fibroblasts stably expressing GDF9 were successfully obtained.This results might lay a foundation for the function study of GDF9 and the goat germplasm resources innovation in the future.%试验旨在构建山羊生长分化因子9 (growth differentiation factor 9,GDF9)慢病毒载体,并使其在山羊原代成纤维细胞中稳定表达.利用全基因合成法克隆了绵羊GDF9基因的CDS全长片段,长约1 362 bp,编码453个氨基酸.利用双酶切和连接,将GDF9基因片段亚克隆至慢病毒载体中,构建了过表达GDF9的慢病毒载体.将慢病毒载体与慢病毒包装质粒共转染293T细胞后,获得了滴度为1×106 TU/mL的慢病毒.利用制备的GDF9慢病毒感染山羊原代成纤维细胞后,观察荧光表明,60％以上的细胞能够观察到红色荧光,说明制备的慢病毒对山羊原代成纤维细胞具有较高的感染效率.经过嘌呤霉素筛选后,所有的细胞均能观察到红色荧光.实时荧光定量PCR分析表明,制备的细胞系中GDF9表达水平比对照组高12.17倍,说明成功获得了稳定表达GDF9的山羊成纤维细胞系.本试验结果为进一步研究GDF9基因的生物学功能,以及山羊未来的种质资源创新奠定基础.