大鼠EPO真核表达载体的构建及其在骨髓间充质干细胞中的表达

Construction of eukaryotic expression vector of rat EPO and its expression in bone marrow mesenchymal stem cells

目的构建表达大鼠促红细胞生成素(erythropoietin,EPO)的真核表达质粒pEGFP-N1/EPO,并检测其在体外的表达。方法从大鼠肾脏获得EPO基因片段并用RT-PCR方法扩增,将其连接于真核表达质粒pEGFP-N1,测序鉴定后,用脂质体包裹转染大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs),采用免疫组化和RT-PCR检测EPO的表达。结果经酶切和测序鉴定证实本实验构建的重组表达质粒正确,该质粒在体外转染大鼠MSCs细胞后可表达EPO蛋白。结论成功构建的pEGFP-N1/EPO重组质粒能在体外表达EPO蛋白。

Objective To construct eukaryotic expression plasmid pEGFP-N1/EPO and to detect its expression in vitro in order to provide experimental evidence for further study on the treatment of hypoxic-ischemic brain damage.Methods The total RNA was extracted from rat kidney.EPO cDNA fragment was obtained by RT-PCR amplication,inserted into pEGFP-N1 vector and identified by restriction endonuclease digestion and nucleotide sequencing.MSCs cells were transfected with the plasmid using Lipofectamine 2000.The expression of EPO protein was detected by immunohistochemistry technique and RT-PCR. Results Confirmed by restric- tion endonuclease digestion and sequencing, the recombinant plasmid contained the sequence of rat EPO gene was successfully constructed. After transfection with the plasmid, EPO protein could be expressed in MSCs cells. Conclusion The constructed euka~,ogc expression vector pEGFP-N1/EPO can express rat EPO in vitro.