Jiang Hao, Xu Jian-guang, Gu Yu-dong, Hu Shao-nan, Li Ji-feng
Department of Hand Surgery, Huashan Hospital, Fudan University, Shanghai, P.R. China 200040.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2003 May;17(3):227-9.
To construct eukaryotic expression vector of rat myogenin gene for further study on its functions in skeletal muscle denervated atrophy and repair.
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.
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.
pcDNA3-myogenin a eukaryotic expression vector, is successfully constructed.
构建大鼠生肌调节因子基因真核表达载体，为进一步研究其在骨骼肌失神经萎缩及修复中的作用奠定基础。
首先用限制性内切酶HindⅢ和XhoⅠ分别酶切含有生肌调节因子基因全长cDNA且具有HindⅢ和XhoⅠ两个酶切位点的克隆载体及真核表达载体；然后用T4 DNA连接酶将生肌调节因子基因cDNA与酶切后的载体连接，构建重组真核表达载体。通过琼脂糖凝胶电泳分析、酶切及PCR鉴定其长度，测序鉴定生肌调节因子基因cDNA序列的正确性。
琼脂糖凝胶电泳分析、酶切及PCR结果证实插入片段DNA长度正确，与生肌调节因子基因cDNA长度一致，pcDNA3-生肌调节因子基因测序结果与报道序列一致。
成功构建了pcDNA3-生肌调节因子基因真核表达载体。
