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目的构建脆性X智力低下一号基因启动子双荧光素酶报告基因,并检测启动子活性。方法从K562细胞中钓取启动子片段MSE,然后与pGL-4载体连接,鉴定后转染hela细胞,检测其启动子的活性。结果从K562细胞中成功钓取了启动子片段MSE,将MSE与PGL4载体连接成重组质粒pGL-4/MSE位,重组质粒经过PCR、酶切鉴定后测序,测序结果与GeneBank报道序列一致。重组质粒pGL-4/MSE位转染hela细胞的相对荧光素酶值与阴性对照组相比有明显区别(P<0.05)。结论成功构建了脆性X智力低下一号基因启动子双荧光素酶报告基因,并具有满意的活性,为后续研究发病机制及作用位点提供依据。
Objective To construct a dual luciferase reporter gene encoding fragile X mental retardation 1 gene and test its promoter activity. Methods The promoter fragment of MSE was obtained from K562 cells and then ligated with pGL-4 vector. After identification, hela cells were transfected and the activity of the promoter was detected. Results The promoter fragment MSE was successfully obtained from K562 cells. MSE and PGL4 vector were ligated into pGL-4 / MSE recombinant plasmid. The recombinant plasmids were identified by PCR and identified by restriction enzyme digestion. The sequencing results were consistent with those reported by GeneBank. Compared with the negative control group, the relative luciferase value of recombinant plasmid pGL-4 / MSE transfected hela cells was significantly different (P <0.05). Conclusion The dual luciferase reporter gene of fragile X mental retardation 1 gene was successfully constructed and had satisfactory activity, which provided the basis for further study on the pathogenesis and site of action.