深黄被孢霉苹果酸脱氢酶基因的克隆和表达

Cloning and expression of a malate dehydrogenase gene from Mortierella isabellina

目的通过对深黄被孢霉(Mortierella isabellina)M6-22中MDH基因的分离鉴定,为深入了解苹果酸脱氢酶(MDH)的生理特性、结构和功能奠定基础,并进一步探讨生物体中MDH的代谢作用。方法通过基因克隆的方法以深黄被孢霉的cDNA为模板,PCR扩增获得苹果酸脱氢酶基因MIMDH1。结果测序结果显示该序列长990bp,分别编码329个氨基酸。序列分析表明该序列与瓜笄霉菌(Choanephora cucurbitarum)MDH的相同性高达77%。将MIMDH1片段连接到表达载体pET32a(+)中构建重组表达质粒pET32aMIMDH1并转化至大肠埃希菌BL21中诱导表达,SDS-PAGE电泳检测在50kD左右有一条蛋白表达条带,经镍柱亲和层析纯化和酶活分析结果显示所纯化的重组蛋白酶活高达379.28U/mg。结论克隆的cDNA序列MIMDH1是一个新的苹果酸脱氢酶基因,所编码的蛋白具有MDH的活性。

Objective To clone and express the malate dehydrogenase（MDH）gene and lay foundation for further research on its characteristics.Methods With the cDNA of Mortierella isabellina as the template,PCR amplification was performed to get the MDH gene MIMDH1.Results Sequencing results showed that the length of the fragment was 990 bp,encoding 329 amino acids.The deduced amino acid sequence shared an up to 77% of identity with that of Choanephora cucurbitarum.The cDNA sequence was further subcloned into the expression vector pET32a（＋）to generate recombinant plasmid pET32aMIMDH1 which was subsequently transformed into Escherichia coli BL21 for inducing expression.The expressed protein was purified with Ni-NTA affinity chromatography,and enzymatic activity analysis demonstrated that the specific activity of the recombinant protein was 379.28 U/mg.Conclusion The amplified cDNA fragment MIMDH1 is a novel malate dehydrogenase gene with the catalytic activity of malate dehydrogenase.