鼠源吲哚胺2,3-双加氧酶活性检测方法的建立

 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。

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

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.