本文關鍵詞： GDF基因 慢病毒 山羊 原代成纖維細胞　出處：《中國畜牧獸醫(yī)》2017年09期 　論文類型：期刊論文

【摘要】：試驗旨在構建山羊生長分化因子9(growth differentiation factor 9,GDF9)慢病毒載體,并使其在山羊原代成纖維細胞中穩(wěn)定表達。利用全基因合成法克隆了綿羊GDF9基因的CDS全長片段,長約1 362bp,編碼453個氨基酸。利用雙酶切和連接,將GDF9基因片段亞克隆至慢病毒載體中,構建了過表達GDF9的慢病毒載體。將慢病毒載體與慢病毒包裝質粒共轉染293T細胞后,獲得了滴度為1×106 TU/mL的慢病毒。利用制備的GDF9慢病毒感染山羊原代成纖維細胞后,觀察熒光表明,60%以上的細胞能夠觀察到紅色熒光,說明制備的慢病毒對山羊原代成纖維細胞具有較高的感染效率。經過嘌呤霉素篩選后,所有的細胞均能觀察到紅色熒光。實時熒光定量PCR分析表明,制備的細胞系中GDF9表達水平比對照組高12.17倍,說明成功獲得了穩(wěn)定表達GDF9的山羊成纖維細胞系。本試驗結果為進一步研究GDF9基因的生物學功能,以及山羊未來的種質資源創(chuàng)新奠定基礎。
[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.
【作者單位】： 河南省農業(yè)科學院畜牧獸醫(yī)研究所河南省畜禽繁育與營養(yǎng)調控重點實驗室;
【基金】：轉基因生物新品種培育子課題——優(yōu)質轉基因(fat-1)肉山羊新品種培育(2014ZX08008003-007) 家畜“卵泡波”對外源激素的響應機制研究(162102110035) 羊促卵泡素(FSH)敏感性的分子調控機制研究(2016YQ18)
【分類號】：Q78;S827

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