摘要
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 rat acid sensing ion channel 1 （ ASIC1 ） promoter recombined luciferase reporter gene plasmid, and then identify its function. Methods Primers were designed and synthetised, ASIC1 promoter fragment from rat genome DNA was replicated by polymerase chain reaction （PCR）. The luciferase report gene pGL3-Basie reporter vector and ASIC1 promoter were digested with restriction enzymes NheI and XhoI separately, and then ASIC1 promoter was connected to pGL3-Basie reporter vector. 293T cells were transiently co-transfected with the constructed pGL3-ASICl-promoter plasmid and pRL-TK control plasmid, and then detected for lueiferase activity after 48 hours. Results Rat ASIC1 gene promoter was amplified by PCR and pGL3-ASICl-promoter re- porter vector was successfully constructed, and the result of colony PCR and sequencing analysis of recombined plasmid were correct. The transcriptional activity of pGL3-ASICl-promoter plasmid group was significantly in- creased compared to that of pGL3-Basie plasmid group （P 〈 0.01 ）. Conclusion The rat ASIC1 promoter lucifer- ase reporter gene vector can be successfully constructed, ry mechanism of ASIC1 gene in transcription. which provides a pivotal basis for further study of regulato-
出处
《安徽医科大学学报》
CAS
北大核心
2016年第5期620-624,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81271949)
