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目的构建大鼠酸敏感离子通道1(ASIC1)基因启动子荧光素酶报告质粒pGL3-ASIC1-promoter,并进行功能的鉴定。方法设计、合成ASIC1启动子引物,采用聚合酶链反应(PCR)技术从大鼠全基因组DNA中扩增出ASIC1启动子片段;NheⅠ和XhoⅠ双酶切后将目的片段连接到pGL3-Basic报告载体上;构建的pGL3-ASIC1-promoter重组质粒和pRLTK内参质粒瞬时共转染293T细胞检测ASIC1启动子活性。结果 PCR扩增得到大鼠ASIC1基因启动子片段;成功构建pGL3-ASIC1-promoter报告基因载体,菌落PCR和测序结果表明启动子DNA序列正确。与空质粒pGL3-Basic转染组相比,pGL3-ASIC1-promoter质粒转染组的荧光素酶活性明显增加(P<0.01)。结论成功构建了大鼠ASIC1基因启动子报告基因载体,为探究ASIC1转录表达的调控机制奠定基础。
Objective To construct the luciferase reporter plasmid pGL3-ASIC1-promoter of rat acid sensitive ion channel 1 (ASIC1) gene and to identify its function. Methods The ASIC1 promoter was designed and synthesized. The ASIC1 promoter fragment was amplified from whole rat genomic DNA by polymerase chain reaction (PCR). After digestion with Nhe I and Xho I, the target fragment was ligated into pGL3-Basic reporter vector The constructed pGL3-ASIC1-promoter recombinant plasmid and pRLTK reference plasmid were transiently co-transfected into 293T cells to detect ASIC1 promoter activity. Results The promoter fragment of rat ASIC1 gene was amplified by PCR. The pGL3-ASIC1-promoter reporter vector was successfully constructed. The colony PCR and sequencing results showed that the promoter DNA sequence was correct. The luciferase activity of pGL3-ASIC1-promoter plasmid transfection group was significantly higher than that of empty plasmid pGL3-Basic transfection group (P <0.01). Conclusion The rat gene promoter reporter gene ASIC1 was successfully constructed, which laid the foundation for exploring the regulatory mechanism of ASIC1 transcription.