IDO启动序列GAS和ISRE荧光素酶报告基因载体构建及其活性检测

Construction and Activity Assay of Luciferase Reporter Vector Containing Promoter Sequence GAS and ISRE of IDO

目的构建pGL3-Enhancer-ISRE4和pGL3-Enhancer-GAS两个荧光素酶报告基因载体,并对其进行生物活性鉴定。方法通过人工合成调控吲哚胺2,3-双加氧酶(IDO)表达的启动序列ISRE4(4个串联的ISRE序列)和GAS7(7个串联的GAS序列),与pGL3-Enhancer连接成重组体pGL3-Enhancer-ISRE4和pGL3-EnhancerGAS7,通过转化扩增,筛选出阳性克隆,并通过酶切、测序及生物学活性检测鉴定构建好的荧光素酶报告基因载体。结果成功地构建了pGL3-Enhancer-ISRE4和pGL3-Enhancer-GAS7两个荧光素酶报告基因载体,在IFN-γ的诱导下,能启动细胞内荧光素酶的表达。结论 pGL3-Enhancer-ISRE4和pGL3-Enhancer-GAS7的荧光素酶报告基因载体的成功构建为研究IDO蛋白的表达调控机制和以IDO为靶标的抗肿瘤免疫耐受药物快速高通量的筛选提供了重要的研究工具。

Objective To construct two luciferase reporter gene vector pGL3-Enhancer-ISRE4 and pGL3-Enhancer-GAS7 , and to identify their biological activity of them. Methods The promoter sequences ISRE4 （4 series ISRE sequence） and GAS7 （7 series GAS sequence） were synthesized which regulate the expression of IDO,then they were cloned into empty vector pGL3-Enhancer respectively to construct two luciferase reporter vectors pGL3-Enhancer-ISRE4 and pGL3-Enhancer-GAS7 ,which were amplified by transformation and identified by restriction enzyme digestion,sequencing,and the biological activity detecting. Re-sults Two luciferase reporter vectors pGL3-Enhancer-ISRE4 and pGL3-Enhancer-GAS7 were successfully constructed,the ex-pression of luciferase could be induced by IFN-γ. Conclusion The two luciferase reporter gene vectors of pGL3-Enhancer-ISRE4 and pGL3-Enhancer-GAS provide a very important tool for studying the regulation mechanisms of IDO protein expression and supplying a fast,high-throughput screening for anti-tumor immune tolerance drug which targeting the IDO.