鼠源吲哚胺2,3-双加氧酶活性检测方法的建立  

Establishment of the mouse indoleamine 2,3-dioxygenase activity assay system

作　　者：郭占领[1] 李娟娟[1] 杨青[1,2] 

机构地区：[1]复旦大学生命科学学院生物化学系,上海200438 [2]云南天然产物与生物制药协同创新中心,昆明650091

出　　处：《复旦学报（医学版）》2016年第3期350-356,共7页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(81373396);高等学校博士学科点专项科研基金(20130071110037);上海市科委生物医药重点课题(12431900204)~~

摘　　要：目的建立鼠源吲哚胺2,3-双加氧酶(mouse indoleamine 2,3-dioxygenase,mIDO)活性检测方法。方法利用基因工程方法表达纯化重组mIDO(recombinant mIDO,rmIDO),建立酶水平mIDO活性检测体系;构建过表达mIDO的小鼠Lewis肺癌(Lewis lung cancer,LLC)细胞株,建立细胞水平mIDO活性检测体系。比较L-1-甲基色氨酸(L-1-methyl tryptophan,L-1-MT)在酶及细胞水平上对mIDO及人源IDO(human IDO,hIDO)的抑制作用,测定抑制类型、抑制常数Ki值及半数抑制浓度(IC50)值。结果 L-1-MT对mIDO和hIDO均有抑制作用,抑制类型及抑制常数Ki值相似,但IC50值在酶水平及细胞水平上均有差异。结论 mIDO酶活性检测体系是筛选IDO抑制剂的有效工具,与hIDO酶活性检测体系联用,能反映种属差异,可更好地利用小鼠模型来替代人类进行IDO抑制剂治疗疾病的研究。Objective To establish a mouse indoleamine 2,3-dioxygenase（mIDO）activity assay system.Methods Recombinant mIDO（rmIDO）expressed and purified by genetic engineering methods was used to set up an enzymatic mIDO activity assay method,and mIDO overexpressing Lewis lung cancer（LLC）cell line was constructed to establish a cellular mIDO activity assay method.The inhibitory activity of Lewis lung cancer（L-1-MT）on mIDO and human IDO（hIDO）in enzymatic and cellular level were investigated respectively,including inhibition type,kinetic parameters Ki and IC50 values. Results L-1-MT showed inhibitory effects on mIDO and hIDO,and inhibition type and Ki values were similar,while IC50 values were distinct in both enzymatic and cellular levels. Conclusions The established mIDO activity assay system is an effective tool for screening IDO inhibitors.It can reflect the species difference when combined with hIDO activity assay system,and is useful for the research on therapeutic effect of IDO inhibitors with mouse model instead of human beings.

关 键 词：吲哚胺2 3-双加氧酶 鼠源 人源 活性检测体系 L-1-甲基色氨酸 

分 类 号：Q331[生物学—遗传学] Q786