牛myf6基因真核表达载体的构建及在成肌细胞中的表达.pdfVIP

中国农业科学 2010, 43(13):2793-2799 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2010.13.020 牛myf6基因真核表达载体的构建及在成肌细胞中的表达 汤展毅，严云勤，高学军，陆黎敏，朱丹丹，冀志庚 （东北农业大学生命科学与生物技术研究中心, 哈尔滨 150030 ） 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。 关键词：牛myf6；稳定转染；成肌细胞 Construction of Eukaryotic Expression Vector of Bovine myf6 Gene and Expression of the Gene in Myoblasts TANG Zhan-yi, YAN Yun-qin, GAO Xue-jun, LU Li-min, ZHU Dan-dan, JI Zhi-geng (Research Center of Life Science and Biotchnology, Northeast Agricultural University, Harbin 150030) Abstract: 【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 (P ＜0.01), muscle creatine kinase gene (P ＜0.01) and my