大鼠肌细胞生成素基因真核表达载体的构建

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR RAT MYOGENIN GENE

目的 构建骨骼肌肌细胞生成素 (myogenin)基因真核表达载体 ,深入探讨在骨骼肌损伤及骨骼肌失神经萎缩的发生、发展过程中的变化规律 ,以及病理过程。 方法 含 myogenin c DNA的克隆载体 p GEMT- myogenin经限制性内切酶 Hind 和 Xho 双酶切 ,得到含这两个酶切位点之 myogenin c DNA片段 ,T4 连接酶连接到含有这两个酶切位点的真核表达载体 pc DNA3,构建出重组真核表达载体 pc DNA3- myogenin,经凝胶电泳鉴定、酶切鉴定、PCR鉴定及 DNA测序证实 c DNA片段大小和序列的正确性。 结果 经凝胶电泳鉴定、酶切鉴定、PCR鉴定证实 :pc DNA3-myogenin含大小正确的 myogenin c DNA片段 ,DNA测序证实其 DNA序列正确。 结论 成功地构建了肌细胞生成素基因真核表达载体 pc DNA3-

Objective To construct eukaryotic expression vector of rat myogenin gene for further study on its functions in skeletal muscle denervated atrophy and repair. Methods The cloning vectors( containing full length of myogenin cDNA and two restriction sites: Hind Ⅲ and Xho Ⅰ) were first cut by two restriction endonuclease: Hind Ⅲ and Xho Ⅰ, and the same as the eukaryotic expression vector; then, the myogenin cDNA and the digested vector were ligated by T 4 DNA ligase, and recombinant eukaryotic expression vector was formed. Its length was certificated by agarose gel electorphoresis analysis, digestion with Hind Ⅲ and Xho Ⅰ, PCR; and the rightness of the myogenin cDNA sequence was confirmed by sequecing. Results The results of agarose gel electrophoresis analysis, digestion, and PCR confirmed the right length of inserted DNA, which was the same as the myogenin cDNA, and the sequencing result of pcDNA3-myogenin was identical with the reported. Conclusion pcDNA3-myogenin a eukaryotic expression vector, is successfully constructed.