论文部分内容阅读
目的:构建pGL3-IL-12p40promoter和pGL3-IL-10promoter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:通过提取小鼠T细胞基因组DNA,分别PCR扩增IL-12p40和IL-10基因的启动子序列,与pGL3-Basic连接成重组体pGL3-IL-12p40promoter和pGL3-IL-10promoter,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:成功构建了pGL3-IL-12p40promoter和pGL3-IL-10promoter两个荧光素酶报告基因载体,分别在IFN-γ或IL-4的诱导下,能启动细胞内荧光素酶的表达。结论:pGL3-IL-12p40promoter和pGL3-IL-10promoter的荧光素酶报告基因载体的成功构建为研究IL-12和IL-10蛋白的表达调控机制和以IL-12/IL-10为检测指标的巨噬细胞活化状态的判断,以其为靶标的抗肿瘤药物的快速高通量筛选提供了重要的研究工具。
OBJECTIVE: To construct two luciferase reporter vectors, pGL3-IL-12p40promoter and pGL3-IL-10promoter, and to identify their biological activity. Methods: The promoter sequences of IL-12p40 and IL-10 gene were amplified by PCR from genomic DNA of mouse T cells and ligated with pGL3-Basic into recombinant pGL3-IL-12p40promoter and pGL3-IL-10promoter respectively. The positive clones were amplified and screened. The constructed luciferase reporter vector was identified by restriction enzyme digestion, sequencing and biological activity. RESULTS: Two luciferase reporter plasmids, pGL3-IL-12p40promoter and pGL3-IL-10promoter, were successfully constructed and were transfected with luciferase respectively under the induction of IFN-γ or IL-4. Conclusion: The successful construction of luciferase reporter gene vectors of pGL3-IL-12p40promoter and pGL3-IL-10promoter To study the regulatory mechanism of IL-12 and IL-10 protein expression and the detection of IL-12 / IL- The judgment of macrophage activation status provides an important research tool for its rapid high-throughput screening of antitumor drugs.