牛myf6基因真核表达载体的构建及在成肌细胞中的表达  

Construction of Eukaryotic Expression Vector of Bovine myf6 Gene and Expression of the Gene in Myoblasts

作　　者：汤展毅[1] 严云勤[1] 高学军[1] 陆黎敏[1] 朱丹丹[1] 冀志庚[1] 

机构地区：[1]东北农业大学生命科学与生物技术研究中心,哈尔滨150030

出　　处：《中国农业科学》2010年第13期2793-2799,共7页Scientia Agricultura Sinica

基　　金：国家转基因重大专项课题(2008ZX08007-002)

摘　　要：【目的】构建牛myf6基因真核表达载体,并观察myf6真核表达载体转染鲁西黄牛成肌细胞后基因的表达和细胞形态的变化。【方法】在质粒pIRES2-EGFP的多克隆位点插入myf6基因构建真核表达载体pIRES2-EGFP-myf6,用脂质体技术转染鲁西黄牛成肌细胞,通过G418筛选出稳定转染的细胞株。利用Western印记、Real-time PCR技术检测成肌细胞转染前后myf6基因、肌肉肌酸激酶基因和肌球蛋白轻链基因的表达量。【结果】与对照组相比,转染质粒的成肌细胞myf6蛋白和mRNA的表达量提高(P<0.01),肌肉肌酸激酶基因和肌球蛋白轻链基因的mRNA表达量提高(P<0.01)。细胞形态观察显示成肌细胞融合为肌管。【结论】构建的真核表达载体pIRES2-EGFP-myf6能在成肌细胞中高效表达,myf6基因促进了成肌细胞向肌肉细胞分化。【Objective】 The objective of the study is to construct the eukaryotic expression vector of bovine myf6 gene and analyse the change of gene expression and cell morphology of transfected myoblasts. 【Method】 Eukaryotic expression vector pIRES2-EGFP-myf6 was constructed by insert myf6 in the MCS. Liposome techinigue was used to transfect Luxi cattle myoblasts ,and by G418 selection,a stable transfected cell line was gained. The changes of gene expression of myoblasts were analyzed by western blot and real-time PCR.【Result】 Compared with the control group,the results showed that,after myoblasts transfected by plasmid,the protein and mRNA expression levels of myf6 were both increased （P0.01）,muscle creatine kinase gene （P0.01） and myosin light chain gene mRNA expression were both increased （P0.01）. Morphology observations indicated that myoblast had fused to myotubes. 【Conclusion】Eukaryotic expression vector pIRES2-EGFP-myf6 can highly express in myoblasts. The results suggest that myf6 gene can promote myoblasts differentiation.

关 键 词：牛myf6 稳定转染 成肌细胞 

分 类 号：S823[农业科学—畜牧学] Q78[农业科学—畜牧兽医]