大鼠肌细胞生成素基因真核表达载体的构建

目的 构建骨骼肌肌细胞生成素(myogenin cDNA)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 cDNA的克隆载体pGEMT—myogenin经限制性内切酶Hind Ⅱ和Xho I双酶切，得到含这两个酶切位点之myogenin cDNA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pcDNA3，构建出重组真核表达载体pcDNA3—myogenin，经凝胶电泳鉴定、酶切鉴定、PCR鉴定及DNA测序证实cDNA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：pcDNA3—myogenin含大小正确的myogenin cDNA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家基因真核表达载体pcDNA3—myogenin。

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR RAT MYOGENIN GENE

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 T4 DNA ligase, and recombinant eukaryotic expression vector was formed. Its length was certificated by agarose gel electrophoresis analysis, digestion with Hind III and Xho I, PCR; and the rightness of the myogenin cDNA sequence was confirmed by sequencing. 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.