Cloning and Construction of Eukaryotic Expression Vector for NeuroD2 in Rat,and Its Expression in Mouse MSCs

doi:10.13560/j.cnki.biotech.bull.1985.2016-1087

Abstract

Abstract: This work aims to clone NeuroD2 gene,construct the eukaryotic expression vector pIRES2-AcGFP1-ND2,and analyze its expression in mouse MSCs. The rat NeuroD2 gene was cloned by RT-PCR,and the protein structure,functional domain,cell localization and homology with other species were analyzed. The constructed pIRES2-AcGFP1-ND2 expression vector was transfected to mouse bone marrow mesenchymal stem cells（MSCs）. The expression of the recombinant plasmid in the mouse MSCs was detected by real time-PCR,Western blot and immunofluorescence. The results showed that,the length of CDS in rat NeuroD2 gene was 1149 bp,and it encoded 382 amino acids,belonging to bHLH family. The secondary structure mainly composed of the α-helices and random coil. Tertiary structure of NeuroD2 was a “helix-loop-helix” structure and mainly localized in nucleus. The NeuroD2 protein of the experimental rat（Rattus norvegicus）shared 99%,98% and 97.7% homology with Mus musculus,Macaca mulatta and Homo sapiens,respectively. The results of real-time PCR,Western blot and immunofluorescence indicated that the expression levels of NueorD2 mRNA and protein in transfected group were significantly higher than that in control group（P < 0.05）. In conclusion,the eukaryotic expression vector pIRES2-AcGFP1-ND2 was constructed successfully.

Key words: NeuroD2, gene cloning, bioinformatics, pIRES2-AcGFP1-ND2

JI Pu-zhong, ZHAO Xing-xu, QIN Wen, SONG Xue-wen, ZHANG Yong, ZHAO Hong-bin. Cloning and Construction of Eukaryotic Expression Vector for NeuroD2 in Rat,and Its Expression in Mouse MSCs[J]. Biotechnology Bulletin, 2017, 33(6): 134-141.

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