以胰岛新生相关蛋白启动子为靶点的基因表达上调剂筛选模型的建立与初步应用

Establishment and preliminary application of a screening model for identifying up-regulators of INGAP expression

目的建立以胰岛新生相关蛋白启动子为靶点的高通量筛选模型,用于胰岛新生相关蛋白基因表达上调剂的筛选。方法以叙利亚金黄地鼠基因组DNA为模板扩增胰岛新生相关蛋白核心启动子序列,克隆至pGL4.17质粒载体构建重组质粒。采用脂质体介导的方法将重组质粒和内参质粒pRL-TK共转染金黄地鼠胰岛β细胞HIT-T15,双荧光素酶报告基因系统检测荧光素酶的表达活性。对瞬时转染条件进行优化,以豆蔻酸-佛波醇-乙酸酯作为阳性对照来评价模型的有效性,并利用该细胞模型对本研究所的化合物库进行筛选。结果构建了重组荧光素酶报告基因质粒pGL4.17-INGAP,并成功转染HIT-T15细胞株。对模型进行优化后,应用阳性对照对其进行评价,Z'因子为0.57,适用于进行高通量筛选。用该模型筛选了本所化合物库,其中白藜芦醇显示出较好的上调作用,EC50为1.6μg/ml。结论成功建立了以胰岛新生相关蛋白启动子为靶点的基因表达上调剂筛选模型。

Objective To establish a screening model for identifying up-regulators of INGAP expression and screen active compounds. Methods Syrian golden hamster genome was used as template to amplify the core promoter sequence of INGAP. Then the obtained promoter fragment was cloned into pGL4.17 vector to construct the recombinant plasmid which subsequently was co-transfected into HIT-T15 cell line together with the internal reference plasmid pRL-TK using Lipofectamine 2000. The luciferase activity was detected using dual-luciferase reporter assay system in 96-well format. After optimizing the conditions of transient transfection, PMA was used as positive control to assess the validation of the model. The compounds in the library of our institute were then screened. Results The recombinant plasmid named pGL4.17-INGAP was constructed and successfully transfected into HIT-T15 cell strain. The model was evaluated by positive control after optimization, and Z＇ factor is 0.57, showing that the model was able to use for screening up-regulators. Among the screened compounds, resveratrol showed up-regulated activity with EC50 value of 1.6μg/ml. Conclusion The cellular screening model for identifying up-regulators of INGAP expression was established.