Abstract：Abstract: A pair of gene-specific primers was designed based on the RNA-seq data, and a full-length cDNA fragment of 1 017 bp, designated MIMDH2, was obtained from Mortierella isabellina strain M6-22 by PCR amplification using cDNA as template. Sequence analysis showed that it comprised an open reading frame encoding 338 amino acids. The deduced amino acid sequence shared the highest identity of 71.26% with that of Aspergillus fumigates, and it also contained the conserved coenzyme and substrate binding sites, as well as catalytic active sites. The cDNA sequence was further subcloned into expression vector pET32a (+) to generate recombinant plasmid pET32a-MIMDH2, which was subsequently transformed into Escherichia coli BL21 for inducing expression. The expressed protein was purified by Ni-NTA affinity chromatography. Enzymatic activity analysis demonstrated that the specific activity of the recombinant protein was 271.33 U/mg. All these results demonstrated that the amplified cDNA fragment MIMDH2 is a novel and potential malate dehydrogenase gene and its expressed protein has the catalytic activity of malate dehydrogenase.
引用本文:
崔锦锦, 季秀玲, 林连兵, 魏云林, 张 琦. 一个深黄被孢霉苹果酸脱氢酶基因的克隆与表达[J]. 生命科学研究, 2017, 21(3): 208-212. CUI Jin-Jin, JI Xiu-Ling, LIN Lian-Bing, WEI Yun-Lin, ZHANG Qi. Cloning and Expression of a Novel Malate Dehydrogenase Gene from Mortierella isabellina. Life Science Research, 2017, 21(3): 208-212.
2017,Vol. 21
