酿酒酵母乙醇脱氢酶2 (ADH2) 基因的克隆表达及活性验证

Cloning, expression and evaluation of Saccharomyces cerevisiae ADH2

摘要:

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2 (Alcohol dehydrogenase 2，ADH2) 基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板，通过反转录获得酿酒酵母乙醇脱氢酶2基因，连接到表达载体pTAT上，得到重组表达质粒pTAT-ADH2，将此重组质粒转化到大肠杆菌BL21中，重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后，在体外进行活性检测和小鼠体内进行毒理试验，检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90％的同源性，经SDS-PAGE电泳分析，目的蛋白得到了有效表达，蛋白条带扫描分析表明，表达量占总蛋白的50％左右，纯化得到的蛋白在小鼠体内进行毒理试验，显示出一定的活性。酿酒酵母adh2基因的克隆正确，不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。

Abstract:

In order to clone and express alcohol dehydrogenase II (ADH2) gene from Saccharomyces cerevisiae in E. coli BL21 (DE3) efficiently, we extracted the total RNA as template and obtained ADH2 gene by RT-PCR and connected ADH2 gene to pTAT plasmids to gain recombinant expression plasmid pTAT-ADH2, then transformed this recombinant expression plasmid pTAT-ADH2 into E. coli BL21 (DE3). The recombinant was induced by IPTG to express ADH2. After purification, ADH2 activity was tested in vitro and toxicologic test was done in mouse. Sequence test showed that the acquired fragments exhibited 90% homology to ADH2 gene sequence from GenBank report. The target gene expressed efficiently and took up to approximant 50% of total protein by SDS-PAGE and band scanning analysis. The purified protein exhibited the identified activity through biochemical test and mouse toxicological test. As a result, the acquired ADH2 gene was highly homology to the published sequence and expressed at a high level in E. coli BL21 (DE3), more importantly, ADH2 proved to have ethanol dehydrogenase activity.