Liang Yu, Liang De-sheng, Xue Zhi-gang, Long Zhi-gao, Wu Ling-qian, Pan Qian, Hu Yi-qiao, Dai He-ping, Xia Kun, Xia Jia-hui
National Laboratory of Medical Genetics, Central South University, Changsha, Hunan, PR China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005 Oct;22(5):493-6.
To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.
The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.
Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.
The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.
构建含微小抗肌萎缩蛋白-增强绿色荧光蛋白（minidystrophin-EGFP）融合基因的人源载体，并研究其在Cos-7细胞中的表达。
采用PCR克隆法构建重组人源载体pHrnDysG。从正常人抗肌萎缩蛋白基因cDNA（GenBank NM04006）中PCR扩增抗肌萎缩蛋白基因的三个片段。将这三个片段连接以产生微小抗肌萎缩蛋白基因。将增强绿色荧光蛋白（EGFP）基因融合到微小抗肌萎缩蛋白基因的C末端，然后通过将融合基因克隆到pHrneo中最终获得pHrnDysG。通过脂质体转染将重组构建体转染到Cos-7细胞后，使用荧光显微镜和RT-PCR检测微小抗肌萎缩蛋白-EGFP融合基因的表达。
限制性酶切分析和测序证实pHrnDysG载体成功构建。将重组pHrnDysG转染到Cos-7细胞后，RT-PCR表明融合基因成功转录，并且在细胞膜处观察到绿色荧光。
由pHrneo载体介导的微小抗肌萎缩蛋白-EGFP融合基因可在Cos-7细胞中表达，其产物在细胞膜中的定位与全长抗肌萎缩蛋白相同。这些结果表明重组人源载体pHrnDysG可能潜在地用于杜氏肌营养不良症的基因治疗研究。
