FNDC5真核表达载体的构建及其N-连接糖基化修饰位点的预测  

Construction of Eukaryotic Expression Vector and Prediction of N-Glycosylation Sites of FNDC5

作　　者：聂永伟[1] 戴柏 张思源[1] 诺明途[1] 张静[1] 梁浩[1] 刘东军[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010070

出　　处：《基因组学与应用生物学》2016年第4期827-831,共5页Genomics and Applied Biology

基　　金：转基因生物新品种培育重大专项(No.2014ZX08008-002)资助

摘　　要：为构建小鼠FNDC5的真核表达载体p EYFP-N1-FNDC5,预测FNDC5的N-连接糖基化修饰位点。在质粒p EYFP-N1的多克隆位点插入小鼠FNDC5构建真核表达载体p EYFP-N1-FNDC5,用脂质体转染He La细胞。转染48 h后,用免疫印迹法检测融合蛋白的表达,使用激光共聚焦显微镜观察FNDC5融合蛋白的亚细胞定位。利用Net NGlyc 1.0 Server程序,预测FNDC5蛋白N-连接糖基化修饰位点。结果显示:与对照组相比,真核表达载体p EYFP-N1-FNDC5能够表达FNDC5融合蛋白,且FNDC5融合蛋白定位于细胞膜。FNDC5的Ⅲ型纤连蛋白结构域区存在两个N-连接糖基化修饰位点。上述结果表明FNDC5真核表达载体构建及表达成功,FNDC5存在N-连接糖基化修饰位点。The objective of this study is to construct eukaryotic expression vector p EYFP-N1-FNDC5 and predict N-glycosylation sites of FNDC5. Eukaryotic expression vector p EYFP-N1-FNDC5 was constructed by inserting mice FNDC5 in the MCS of p EYFP-N1. Liposome technique was used to transfect p EYFP-N1-FNDC5 into He La cell. After transfection for 48 h, the expression of FNDC5 fusion protein was analyzed by Western blotting, the subcellular localization of FNDC5 fusion protein was observed by confocal microscopy, the N-glycosylation sites of FNDC5 was predicted by Net NGlyc 1.0 server program. The results show that compared with the control,eukaryotic expression vector p EYFP-N1-FNDC5 express FNDC5 fusion protein, and FNDC5 fusion protein located in cell membrane. There are two N-glycosylation sites on fibronectin type Ⅲ domain-containing segment of FNDC5. These results demonstrated that the eukaryotic expression vector p EYFP-N1-FNDC5 had been constructed successfully. FNDC5 have N-Glycosylation sites.

关 键 词：FNDC5基因 真核表达载体 FNDC5亚细胞定位 N-连接糖基化位点 

分 类 号：Q78[生物学—分子生物学]