CAJ | 학술논문

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-Enhancer-GAS7,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。
목적구건pGL3-Enhancer-ISRE4화pGL3-Enhancer-GAS량개형광소매보고기인재체,병대기진행생물활성감정。방법통과인공합성조공신타알2,3-쌍가양매(IDO)표체적계동서렬ISRE4(4개천련적ISRE서렬)화GAS7(7개천련적GAS서렬),여pGL3-Enhancer련접성중조체pGL3-Enhancer-ISRE4화pGL3-Enhancer-GAS7,통과전화확증,사선출양성극륭,병통과매절、측서급생물학활성검측감정구건호적형광소매보고기인재체。결과성공지구건료pGL3-Enhancer-ISRE4화pGL3-Enhancer-GAS7량개형광소매보고기인재체,재IFN-γ적유도하,능계동세포내형광소매적표체。결론 pGL3-Enhancer-ISRE4화pGL3-Enhancer-GAS7적형광소매보고기인재체적성공구건위연구IDO단백적표체조공궤제화이IDO위파표적항종류면역내수약물쾌속고통량적사선제공료중요적연구공구。
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.