猪L-Myc基因的分子克隆及在细胞重编程中的应用

摘要

[ Objective ] Cloning porcine L-Myc gene and exploring its function in cell reprogramming at the protein level could be helpful to provide a foundation for substituting L-Myc gene for c-Myc gene in the induction of induced pluripotent stem (iPS) cells in porcine. [Method] Through NCBI sequence alignment, porcine L-Myc gene cDNA was obtained by RT-PCR. The homology of L-Myc gene among these three species of porcine, human and mouse was analyzed by bioinformatics. The fusion expression vector pEGFP/L-Myc-Cl was constructed, and the vector-transfected and western blotting were used to detect the expression of the cloned porcine L-Myc gene cDNA at the protein level. Then the L-Myc gene was inserted into the retroviral vector, and different transcription factors, respectively, induced porcine embryo fibroblast (PEF) cells. Through observing the variation of the induced cells and alkaline phosphatase (AP) staining, the important role porcine L-Myc gene plays in cell reprogramming was verified. [Result] A 1 113 bp porcine L-Myc gene cDNA sequence was obtained by RT-PCR, encoding 364 amino acids and the theoretical molecular weight is 40 kD. Bioinformatics analysis showed that a high degree of homology in L-Myc existed among porcine, human and mouse. The western blotting analysis proved that the cloned porcine L-Myc gene cDNA could be expressed at the protein level. After induction, the morphology of induced cells are varied, and the quantity of positive clones induced with the transcription factors Oct4, Sox2, Klf4, and L-Myc (OSKL) was far more than that with Oct4, Sox2, and Klf4 (OSK). [Conclusion] The porcine L-Myc, a transcription factor, was cloned, and the results of analysis showed that porcine L-Myc gene plays anrnimportant role in cell reprogramming process.%[目的]克隆猪L-Myc基因,并在蛋白水平表达的情况下探索其在细胞重编程中的作用,为深入研究猪L-Myc基因替代c-Myc诱导多能干细胞(induced pluripotent stem cell,iPSC)奠定基础.[方法]先通过NCBI序列比对,采用RT-PCR克隆猪L-Myc基因cDNA,生物信息学分析猪L-Myc基因与人和小鼠的同源性,构建融合表达载体pEGFP/L-Myc-Cl,通过载体转染和免疫印记检测猪L-Myc基因cDNA的蛋白水平表达；再将L-Myc基因装入逆转录病毒载体中,分别使用不同的转录因子诱导猪胎儿成纤维细胞(porcine embryo fibroblast,PEF),通过形态变化和碱性磷酸酶(AP)染色验证猪L-Myc基因在细胞重编程中的作用.[结果]①获得了1113 bp的猪L-Myc基因cDNA,编码364个氨基酸,理论分子质量为40 kD;②生物信息学分析显示猪L-Myc基因与人和小鼠高度同源；③免疫印记检测结果说明猪L-Myc基因cDNA能够在蛋白水平表达；④细胞诱导试验和AP染色结果显示转录因子Oct4、Sox2、Klf4和L-Myc (OSKL)组合诱导的细胞阳性克隆率明显高于Oct4、Sox2和Klf4 (OSK)组合的阳性克隆率.[结论]获得了猪L-Myc基因,并且该基因在蛋白水平表达且在细胞重编程过程中起到了重要的作用.