人乙醇脱氢酶ADH1B2在毕赤酵母中的表达及活性分析

Expression of human alcohol dehydrogenase ADH1B2 in Pichia pastoris and its activity analysis

根据GeneBank中人乙醇脱氢酶ADH1B2基因序列设计引物,以人肝脏总RNA为模板,反转录获得人乙醇脱氢酶基因.将克隆基因与表达载体pPICZB连接,测序正确的pPICZB-ADH1B2电转化毕赤酵母GS115;重组毕赤酵母工程菌经0.5%(v/v)甲醇诱导72h后目的蛋白以可溶形式表达,经DEAE-Sepharose FF纯化后蛋白酶活性可达8 000U.mg-1.同时构建重组质粒pCDNA3.1-ADH1B2转染HepG2人肝癌细胞,发现细胞内ROS/GSH水平降低,Akt磷酸化水平降低,细胞活性下降.为临床酒精中毒预防和治疗药物的研发以及ADH在肝脏肿瘤方面的研究提供了理论基础.

Human alcohol dehydrongenase gene ADH1B2 was obtained by RT-PCR using total RNA from liver as template.Recombinant pPICZB-ADH1B2 were constructed and electrotransformed into P.pastoris GS115.DEAE-Sepharose FF was employed to purify the intracellular soluble ADH1B2 protein.Enzyme activity was about 8 000 U/mg,influenced by factors like temperature,pH and ion.Plasmid pcDNA3.1-ADH1B2 was constructed and then transfected into human hepatocarcinoma cell HepG2.The transfected HepG2 cells showed decreased ROS and consequent decreased Akt phosphorylation,resulting in depressed cell viability.This study may help to get more insights into the relationship between cancer and ADH,and lay theoretical basis for clinic prevention of alcoholism and drug development.