hAng-1真核表达载体的构建及其在MSCs中的表达

Construction of Eukaryotic Expression Plasmid of pcDNA3.1/human Angiopoietin-1 and its Expression in Mesenchymal Stem Cells

目的:通过基因重组技术,构建人血管生成素-1(human angiopoietin 1,hAng-1)真核表达载体体系,并将其转染至大鼠的骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)内进行培养,进而验证hAng-1的表达。方法:将hAng-1编码序列(互补脱氧核糖核酸)通过酶切,插入至pcDNA3.1(+)质粒的多克隆位点,构建质粒pcDNA 3.1(+)/hAng-1真核表达质粒;重组质粒经脂质体介导转染鼠MSCs。应用逆转录聚合酶联反应(RT-PCR)、蛋白免疫印迹法(Western blot)等方法检测hAng-1的表达情况。结果:pcDNA 3.1(+)/hAng-1真核表达质粒转染鼠MSCs后,应用流式细胞仪检测,转染效率约为15%。同时应用RT-PCR能够检测出目的基因mRNA,Western blot能够检出hAng-1的蛋白表达。结论:本实验通过基因重组技术,构建的pcDNA3.1(+)/hAng-1真核表达载体能够在转染的鼠MSCs中表达,且表达较为持续,为hAng-1基因应用于基因治疗的研究奠定了基础。

Objective：To establish angiopoietin 1（human angiopoietin-1,hAng-1） eukaryotic expression vector by genetic recombination and to cultivate the cells hAng-1into rat bone marrow mesenchymal stem cells（marrow mesenchymal stem cells,MSCs） in order to detect hAng-1 expression.Methods：To insert the gene hAng-1 into pcDNA3.1（＋） plasmid multicloning sites to construct pcDNA3.1（＋）/hAng-1 Eukaryotic expression plasmid,and then we transfected recombinant plasmid into rat MSCs by liposome.We used RT-PCR and Western blot to detect hAng-1 expression.Results：After transfecting rat MSCs,we got the transfection efficiency,which was about 15% by flow cytometer.The objective mRNA was detected by RT-PCR,and hAng-1protein expression was detected by Western blot.Conclusions：In this research,we constructed the eukaryotic expression vector pcDNA3.1（＋）/hAng-1 by reconstructing genes,and it could be expressed for a long time so that it could lay the foundation for application on the clinical.