人MUC5AC启动子荧光报告基因的构建与分析

摘要

目的：构建人MUC5AC启动子荧光报告基因并进行分析。方法 PCR技术克隆人MUC5AC启动子[(-1300)~(+48) bp]长度片段的基因，将纯化的PCR产物与T载体连接，然后将连接产物转化大肠杆菌DH5α感受态细胞，并利用酶切和测序进行重组质粒鉴定；将该重组质粒克隆至pGL3-ehancer荧光报告基因载体上，用脂质体转染法将荧光报告基因转染至人支气管上皮细胞(16HBE细胞)，分别用1 ng/mL的重组细胞因子IL-4和IL-13刺激细胞24 h，并设不做任何处理的对照组；用Promega双荧光素酶报告基因检测试剂盒检测相对荧光素酶活性(Rlus1/Rlus2)。结果人MUC5AC启动子荧光报告基因构建成功，IL-4和IL-13刺激组的相对荧光素酶活性均高于对照组[IL-4(21.6667±2.08167)，IL-13(26.5678±1.52753)，对照组(8.33±1.52753)；F=89.815，P=0.000]。结论 MUC5AC启动子核心区域为(-1300)~(+48) bp范围，IL-4和IL-13可作用于该区域并促进MUC5AC高表达。%Objective To construct and analyze the luciferase reporter gene of human MUC5AC promoter. Methods Fragment of human MUC5AC promoter [(-1 300)~(+48) bp] was cloned by PCR technology. Purified prod-uct of PCR was connected with T vector. The connection product was transformed into Escherichia coli DH5αcompetent cells and identified by enzyme digestion and sequencing. The recombinant plasmid was cloned into luciferase reporter vector of pGL3-ehancer and transfected into 16HBE cells. Then recombinant cytokines of IL-4 and IL-13 were re-spectively used to stimulate cells at concentration of 1 ng/mL for 24 hours. Meanwhile, cells without any stimulation were used as control. After 24 hours, cells were tested for relative luciferase activity (Rlus1/Rlus2) by dual luciferase reporter gene detection kit of Promega. Results Human MUC5AC promoter was constructed successfully. Relative lu-ciferase activity (Rlus1/Rlus2) of cells with stimulation of IL-4 and IL-13 was higher than that of control cells [IL-4 (21.666 7±2.081 67), IL-13 (26.567 8±1.527 53), (8.33±1.527 53);F=89.815,P=0.000]. Conclusion The core region of MUC5AC promoter is in (-1 300)~(+48) bp section. IL-4 and IL-13 have function in such region and promote expres-sion of MUC5AC.