cAMP反应元件萤光素酶报告基因载体的构建

Construction of cAMP Responsive Element-Containing Luciferase Reporter Vectors

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。

Objective： To construct lueiferase reporter vectors containing cAMP responsive elements（CRE）. Meth ods： Using a lueiferase 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 eotransfected 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.