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*
投稿时间:2013-11-10  修订日期:2013-12-08
DOI:
中文关键词:  吲哚胺2,3-双加氧酶  启动子序列  荧光素酶报告基因  γ-干扰素  肿瘤免疫耐受
英文关键词:Indoleamine 2, 3-dioxygenase  Promoter sequence  luciferase reporter gene  IFN-γ  Tumor immune tolerance
基金项目:湖南省自然科学基金资助项目(13JJ4078)
作者单位E-mail
江冠民 南华大学附属第一医院 jguanmin@163.com 
匡艳华 南华大学附属第一医院检验科  
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中文摘要:
      目的 构建pGL3-Enhancer-ISRE4和pGL3-Enhancer-GAS7两个荧光素酶报告基因载体并对其进行生物活性的鉴定。方法 通过人工合成调控吲哚胺2,3-双加氧酶(Indoleamine 2, 3-dioxygenase, 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,并在细胞内鉴定了在IFN-γ的诱导下,能启动荧光素酶的表达。结论 具有生物活性的pGL3-Enhancer-ISRE4和pGL3-Enhancer-GAS7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。
英文摘要:
      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. Results We constructed two luciferase reporter vectors pGL3-Enhancer-ISRE4 and pGL3-Enhancer-GAS7 successfully, the expression of luciferase could be induced by IFN-γ.Conclusion The two luciferase reporter gene vectors pGL3-Enhancer-ISRE4 and pGL3-Enhancer-GAS7 were constructed successfully and their biological activity was identified, which provides 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.
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