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

发布时间：2018-02-26 20:13

本文关键词： GDF基因慢病毒山羊原代成纤维细胞　出处：《中国畜牧兽医》2017年09期 　论文类型：期刊论文

【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。
[Abstract]:The aim of this study was to construct goat growth differentiation factor 9 differentiation factor 9 (GDF9) lentivirus vector and to stably express it in goat primary fibroblasts. The full-length CDS fragment of sheep GDF9 gene was cloned by whole gene synthesis method. The GDF9 gene fragment was subcloned into the lentivirus vector by double enzyme digestion and ligation. The lentivirus vector expressing GDF9 was constructed. The lentivirus vector and the lentivirus packaging plasmid were co-transfected into 293T cells. A lentivirus titer of 1 脳 10 ~ 6 TU/mL was obtained. After infected goat primary fibroblasts with the prepared GDF9 lentivirus, the observed fluorescence showed that red fluorescence could be observed in more than 60% of the cells. The results showed that the prepared lentivirus had high infection efficiency on goat primary fibroblasts, and red fluorescence could be observed in all the cells after purine mycin screening. Real-time fluorescence quantitative PCR analysis showed that, The expression level of GDF9 in the prepared cell line was 12.17 times higher than that in the control group, which indicated that the goat fibroblast cell line with stable expression of GDF9 was successfully obtained. The results showed that the biological function of GDF9 gene was further studied. And the future goat germplasm resources innovation laid the foundation.
【作者单位】：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;
【基金】：转基因生物新品种培育子课题——优质转基因(fat-1)肉山羊新品种培育(2014ZX08008003-007) 家畜“卵泡波”对外源激素的响应机制研究(162102110035) 羊促卵泡素(FSH)敏感性的分子调控机制研究(2016YQ18)
【分类号】：Q78;S827

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