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当前位置: 首页 > 文章 > 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 中国畜牧兽医 2017 (9) 2566-2572
Position: Home > Articles > Construction of GDF9 Lentiviral Vector and Its Stable Expression in Goat Primary Fibroblasts China Animal Husbandry & Veterinary Medicine 2017 (9) 2566-2572

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

作  者：
滑留帅;王璟;陈付英;辛晓玲;楚秋霞;冯亚杰;徐照学;王二耀
单  位：
河南省农业科学院畜牧兽医研究所河南省畜禽繁育与营养调控重点实验室
关键词：
GDF9基因;慢病毒;山羊;原代成纤维细胞
摘  要：
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 362bp,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。
译  名：
Construction of GDF9 Lentiviral Vector and Its Stable Expression in Goat Primary Fibroblasts
作  者：
HUA Liu-shuai;WANG Jing;CHEN Fu-ying;XIN Xiao-ling;CHU Qiu-xia;FENG Ya-jie;XU Zhao-xue;WANG Er-yao;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Institute of Animal Husbandry and Veterinary Science,Henan Academy of Agricultural Sciences;
关键词：
GDF9 gene;;lentivirus;;goat;;primary fibroblasts
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 293 Tcells,and the lentivirus with titer of 1×10~6 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 puromycin 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.

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