MDR1基因单靶点双荧光素酶报告基因系统的建立

目的通过构建以MDR1启动子为启动序列的双荧光素酶报告基因系统并进行活性分析，为MDR1基因表达的单靶点调控研究和逆转剂的筛选提供一种有效的方法。方法从HCT-8细胞中提取DNA并克隆含有MDR1基因启动子中Y—box序列。将该序列重组到萤光素酶报告基因载体pGL-3．Basic的启动区域中，从而构建报告基因载体pGL-MDR1。将pGL-MDR1和pRL-TK载体共转染到HCT-8和HCT-8／VCR细胞中。通过调节不同载体的比例来优化转染效率。利用MDRI基因激活剂（热诱导）和抑制剂（EGCG）等处理来分析其启动转录活性受外界因素的影响。结果通过直接测序法验证了pGL-MDR1含有MDR1基因启动子Y—box序列且没有出现碱基突变。在pGL-MDR1和pRL-TK的转染比例为5：5时，转染效率最高并具有最高的萤光素酶活性。通过MDR1基因激活处理后表现为时间依赖性地激活MDR1基因的表达，而MDR1基因抑制剂的作用则相反。结论MDR1启动子为启动序列的双荧光素酶报告基因系统建立成功。该系统不但可以用于研究活体生物发光成像和MDRI基因表达的机理，而且可用于单靶点的多药耐药抑制剂的筛选。

Establishment of Dual Luciferase Report Gene System Driven by Single Target MDR 1 Promoter

Objective To establish dual luciferase report gene system basic the Y-box sequence of MDR1 gene and analysis it＇ s activity. Method Total DNA was extracted from HCT-8 cells and it was used for amplifying the fragment contained MDR1 gene promoter by PCR. The amplified product was inserted into pGL-3-Basic vector. The pGL-MDR1 and pRL-TK vector were co-transfected into HCT-8 and HCT-8/VCR cells. The activity of MDR1 gene promoter could be assay by measured luciferase. Result The method was optimized by changing ratio of two vectors. Moreover, the inductor （hyperthermia） and inhibitor （EGCG） of MDR1 gene were used for authenticating the method. A 241bp fragment of MDR1 gene promoter was cloned from DNA. Conclusion the dual luciferase report gene system was successful established. The system is useful in bioluminescence imaging technology in vivo, expression of MDR1 gene and screening auticancer drugs.