泛素化调节因子2真核表达质粒的构建及其在原代肾小管上皮细胞的表达

Abstract

Abstract: The assay was aimed to construct the eukaryotic expression vector pEGFP-N3-Smurf2 carrying Smad ubiquitination regulatory factor 2 (Smurf2) and detect its expression after transfecting to renal tubular epithelial cells (RTECs).The CDS of Smurf2 gene was amplified and cloned from total RNA of RTECs by RT-PCR.Both prokaryotic expression vector pEASY-Zero-Smurf2 and eukaryotic expression vector pEGFP-N3-Smurf2 were constructed and then detected by double digestion and sequencing.pEGFP-N3-Smurf2 was transfected into RTECs with Lipofectamine^TM2000 and the expression of Smurf2 was detected by Real-time PCR and Western blotting.The amplified Smurf2 gene was about 2247 bp in length.Restriction analysis and sequencing proved that Smurf2 was successfully inserted into recombinant plasmid pEGFP-N3.The mRNA and protein of Smurf2 were detected by Real-time PCR and Western blotting.Smurf2 expression vector using green fluorescent protein as a reporter gene was constructed and transfected into RTECs successfully,which provided a powerful tool for the further study of function of Smurf2 gene.

Key words: Smad ubiquitination regulatory factor 2; pEGFP-N3; renal tubular epithelial cell; plasmid; expression

CLC Number:

Q786

GAO Lei, ZHAO Hai-jiao, WU Jin-dong, TIAN Ya-hui, ZHANG Zhong-wen, WU Guo-juan. Construction of Eukaryotic Expression Vector for Smurf2 Gene and its Expression in Renal Tubular Epithelial Cells[J]. , 2013, 40(6): 37-40.

References

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Construction of Eukaryotic Expression Vector for Smurf2 Gene and its Expression in Renal Tubular Epithelial Cells

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