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目的构建人微小抗肌萎缩蛋白基因(microdystrophin)的真核表达载体,转染大鼠骨髓间充质干细胞(rMSC),研究其体外表达情况。方法限制性酶切PBSK-MICRO质粒的微小抗肌萎缩蛋白基因片段,插入到真核表达质粒pcD-NA3.1(+)的NotI位点内,克隆出真核表达载体pcDNA3.1(+)/micro dystrophin。通过酶切和测序对质粒进行鉴定。用脂质体将重组质粒转染入rMSCs中,经过G418筛选后,用RT-PCR及间接免疫荧光技术检测表达产物。结果pcDNA3.1(+)/micro dystrophin经过NotI、Hind III酶切鉴定及测序证实插入片断正确。提取转染重组质粒rMSCs的总RNA,进行RT-PCR可见mRNA的转录;对G418筛选后rMSCs进行免疫荧光检测可见细胞内亮丽的红色荧光,说明转染后的rMSCs有微小抗肌萎缩蛋白的表达。结论成功构建了人microdystrophin基因真核表达载体,将其转染入rMSCs内有微小抗肌萎缩蛋白的表达,为进一步研究体外修饰自体干细胞移植治疗DMD奠定了基础。
Objective To construct an eukaryotic expression vector of human microdystrophin and transfect rat bone marrow mesenchymal stem cells (rMSCs) to investigate its in vitro expression. Methods The micro-dystrophin gene fragment of PBSK-MICRO plasmid was inserted into the NotI site of the eukaryotic expression plasmid pcD-NA3.1 (+), and the eukaryotic expression vector pcDNA3.1 (+) was cloned. / micro dystrophin Plasmids were identified by digestion and sequencing. The recombinant plasmids were transfected into rMSCs by liposomes, and after screening by G418, the expression products were detected by RT-PCR and indirect immunofluorescence. Results The pcDNA3.1 (+) / micro dystrophin was confirmed by restriction digestion with NotI and Hind III and sequenced. The total RNA was extracted from rMSCs transfected with recombinant plasmid, and the mRNA was transcribed by RT-PCR. The rMSCs were screened by G418 and immunofluorescent staining showed that there was a bright red fluorescence in rMSCs after transfection. . Conclusion The eukaryotic expression vector of human microdystrophin gene was successfully constructed and transfected into rMSCs. The expression of micro-dystrophin was successfully established, which laid the foundation for the further study of autologous stem cell transplantation in vitro.