cAMP反应元件萤光素酶报告基因载体的构建

摘要

Objective: To construct luciferase reporter vectors containing cAMP responsive elements(CRE). Meth-ods: Using a luciferase reporter plasmid including gal4 sites as template, we used PCR technique to delete the gal4 sites and to add 4 CRE into the original plasmid. Then the vector pCRE-luc was achieved. Next, to mea-sure its luciferase activity, the vector was cotransfected with a G-protein couple receptor(GPCR) into human em-bryonic kidney 293(HEK293) cells. To verify whether the activity of CRE was associated with Gs pathway, inhibi-tor of protein kinase A(PKA) was applied. Results: The activity of pCRE-luc is directly associated with the acti-vating degree of GPCR, and depends on the Gs signal pathway. Conclusion: The CRE luciferase reporter vector, which can be used to measure the activity of Gs protein coupled receptors, was successfully constructed. Our re-search will provide basis for high-throughput screening drugs related to GPCR targets.%目的：构建含有cAMP反应元件（CRE）的萤光素酶报告基因载体。方法：以含有gal4位点的萤光素酶报告基因质粒为模板，利用PCR方法，在去除gal4位点的同时，连入4个CRE元件得到pCRE-luc；将该载体与G蛋白偶联受体（GPCR）共转染人胚肾细胞HEK293，测定萤光素酶活性；应用蛋白激酶A（PKA）抑制剂确认CRE的活性是否与Gs通路相关。结果：pCRE-luc的活性直接相关于GPCR的激活程度，并依赖Gs信号通路。结论：CRE萤光素酶报告基因载体构建成功，可用于检测Gs偶联GPCR的活性，为基于GPCR的高通量药物筛选奠定了基础。