牛c-myc基因的克隆及其在皮肤成纤维细胞中的表达.PDF

生 物 工 程 学 报 Chin J Biotech 2011, June 25; 27(6): 963−968 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn ©2011 CJB, All rights reserved. 生物技术与方法 牛c-myc 基因的克隆及其在皮肤成纤维细胞中的表达 肖佳佳，张凤凤，熊显荣，张涌 西北农林科技大学动物医学院，杨凌 712100 摘 要: 为了构建包含牛c-myc 基因编码序列的重组载体，以胎牛原始生殖嵴为材料，用RT-PCR 方法克隆出牛c-myc 基因的编码序列，将其亚克隆至 pMD19-T 载体，再从酶切鉴定和测序正确的质粒上切下目的片段，定向克隆到 pIRES2-AcGFP1-Nuc 表达载体上，挑选序列正确的重组真核表达质粒转染牛皮肤成纤维细胞，用 RT-PCR 和 Western blotting 分别检测c-myc mRNA 和蛋白的表达。结果表明，从胎牛原始生殖嵴中正确克隆了c-myc 基因的全长编码序列， 所构建的重组质粒能够在皮肤成纤维细胞中有效表达，为进一步研究 c-myc 基因功能以及下一步特定因子诱导牛体细 胞转变为牛iPS 细胞的研究奠定了基础。 关键词: 牛，c-myc 基因，iPS 细胞，真核表达 Cloning of bovine c-myc gene and its expression in skin fibroblast cells Jiajia Xiao, Fengfeng Zhang, Xianrong Xiong, and Yong Zhang College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling 712100, China Abstract: In order to construct a eukaryotic expression vector of bovine c-myc gene, the coding sequence (CDS) of c-myc gene was amplified from bovine primordial genital ridges by RT-PCR. The CDS was subcloned into pMD19-T vector, and then inserted into vector pIRES2-AcGFP1-Nuc. After confirmed by restriction enzyme digestion and sequencing, the recombined plasmid was transfected into skin fibroblast cells. RT-PCR and Western Blotting were used to detect the expression of c-myc mRNA and protein, respectively. The results show that the complete CDS of c-myc gene was cloned from fetal bovine primordial genital ridges. The eukaryotic expression vector of bovine c-myc gene was constructed and efficiently express