中介素真核表达质粒的构建及其在大鼠近端肾小管上皮细胞中的表达

Construction of intermedin eukaryotic expression vector and expression in rat renal epithelial cells

目的构建带有绿色荧光蛋白的中介素(IMD)真核表达质粒pIRES2-EGFP/IMD,并转染大鼠近端肾小管上皮细胞系(NRK-52E)。方法合成带有IMD基因片段的pMD19-TSimple/IMD质粒,将pMD19-TSimple/IMD质粒和pIRES2-EGFP载体双酶切之后进行高效连接,将IMD亚克隆至pIRES2-EGFP上,对重组表达载体进行酶切、测序鉴定。采用FuGENEHD转染试剂转染NRK-52E细胞,荧光显微镜下观察绿色荧光蛋白。提取细胞mRNA和蛋白,反转录-聚合酶链反应(RT-PCR)方法检测基因表达,Western检测IMD蛋白表达。结果经限制性酶切鉴定及测序分析证实pIRES2-EGFP/IMD载体序列正确;荧光显微镜下可见转染的NRK-52E细胞有绿色荧光蛋白的表达,经RT-PCR和Western测定分析,转染细胞IMD基因及蛋白的表达增加。结论pIRES2-EGFP/IMD载体构建成功,并在NRK-52E细胞内成功表达。

Objective To construct pIRES2-EGFP/IMD, an eukaryotic expression vector with green fluorescent protein of rat Intermedin （IMD） gene, and transfect it into NRK-52E cell line. Methods The full-length eDNA of rat IMD was synthesized, combined to pMD19-T Simple plasmid and identified by sequencing. The pMD19-T Simple/IMD and plasmid pIRES2-EGFP were double-digested with Xho I and Pst I, respectively, and the target gene was cloned into pIRES2-EGFP directionally. The recombinant plasmid plRES2-EGFP/IMD was identified by double digestion and sequencing, and then transfected into NRK-52E by FuGENE HD. Green fluorescent protein was observed by fluorescence microscope. Expression of IMD in pIRES2-EGFP/IMD transferred cells was detected by RT-PCR and Western blot. Results The destination gene, 441bp, was inserted successfully into the plasmid pIRES2-EGFP. After transfection, the expressions of green fluorescent protein were present. Expression of IMD in NRK-52E transfected with pIRES2-EGFP/IMD was much higher than in controls. Conclusion The eukaryotic expression plasmid pIRES2-EGFP/IMD was successfully constructed and expressed in NRK-52E.