沼泽型水牛卵泡抑素基因的真核表达研究

Eukaryotic Expression of Swamp Buffalo Follistatin

试验通过候选基因法,选定卵泡抑素(follistatin,FS)作为影响水牛繁殖性能的主要候选基因,通过基因工程的方法,用XhoⅠ、SacⅡ双酶切广西大学动物遗传育种与繁殖实验室克隆的添加有ACC的Kozaka序列的pMD-bFS质粒和pEG-FP-N1,构建pEGFP-bFS真核表达质粒,将其与阳离子脂质体混匀后,分别转染体外培养的水牛胎儿成纤维(BFF)细胞和仓鼠肾(BHK)细胞系,经过48h培养后,在相差荧光显微镜下观察绿色荧光蛋白的表达水平,用RT-PCR和Western blotting方法对转入质粒表达进行定性鉴定。结果显示,本研究成功构建了添加有ACC的Kozaka序列的pEGFP-bFS真核表达质粒,该重组质粒在BFF和HBK两种细胞均表达,但在HBK细胞系的表达量稍高。本研究结果将为下一步制备具有生物活性的bFS工程疫苗,研究bFS对水牛繁殖性能的影响提供参考。

In this study,follistatin gene was chosen as the candidate gene affecting the reproductive ability in buffalo.Recombinant plasmid pEGFP-bFS was conducted and expressed in both buffalo fetal fibroblast（BFF） and BHK cells.pMD-bFS and pEGFP-N1 were digested with XhoⅠ,Sac Ⅱ,then eukaryotic expression vector-pEGFP-bFS was constructed.Buffalo fetal fibroblast and BHK cells were cultured with pEGFP-bFS intermixing LipofectamineTM 2000 in carbon dioxide incubator with 37 ℃,5% CO2,100% humidity.After 24 h,expression levels of recombinant vector were verified through observing directly under phase contrast fluorescence microscope,RT-PCR and Western blotting.The results indicated that bFST expressed in both buffalo fibroblast and BHK cells.This study was a very significance to produce bioactive bFS engineered plasmid and research effects of bFS on reproductive ability.