摘要
【目的】构建小鼠DVL3基因野生型及突变型重组荧光素酶报告质粒,验证miR-204与DVL3的靶向关系。【方法】采用生物信息学方法预测miR-204与DVL3基因的结合位点。采用PCR法扩增小鼠DVL3基因3’UTR片段,克隆至pGL3-control载体,构建野生型及突变型重组荧光素酶报告质粒。293T细胞分为6组,分别将野生型与突变型荧光素酶质粒与miR-204过表达质粒以及对照质粒共转染,检测6组细胞中荧光素酶的活性。【结果】电泳和测序结果证实成功构建小鼠野生型和突变型DVL3基因重组荧光素酶报告质粒;双荧光素酶活性检测结果显示,野生实验组相对荧光素酶活性比野生对照组和NC组均降低,且差异具有统计学差异(P<0.05)。【结论】成功构建小鼠野生型和突变型DVL3基因重组荧光素酶报告质粒,miR-204与DVL3存在靶向关系。
【Objective】To construct mouse DVL3 gene wild-type and mutant recombinant luciferase reporter plasmids to verify the relationship between miR-204 and DVL3.【Methods】Bioinformatics methods were used to predict the binding sites of miR-204 and DVL3 genes.The 3'UTR fragment of mouse DVL3 gene fragment were amplified by PCR and cloned into pGL3-control vector and the control plasmid respectively,and the luciferase activities of 6 groups of cells were detected.【Results】The results of electrophoresis and sequencing confirmed the successful construction of mouse DVL3 gene wild-type and mutant recombinant luciferase reporter plasmids.The results of dual luciferase assay showed that compared with wild-type control group and NC group,the relative luciferase activity in wild-type experimental group was decreased,and the difference was statistically significant(P<0.05).【Conclusion】The wild-type and mutant recombinant luciferase reporter plasmids ofDVL3n gene and the miR-204 overexpression plasmid were successfully constructed.Targeting the relationship between miR-204 and of DVL3 was achieved.
作者
王欣
曾强
高雅
李培
娄贺仁
WANG Xin;ZENG Qiang;GAO Ya;LI Pei;LOU Her-en(TianjinCenter for Disease Control and Prevention,Tianjin 300011,China)
出处
《武警后勤学院学报（医学版）》
CAS
2021年第8期28-30,33,共4页
Journal of Logistics University of PAP（Medical Sciences）
基金
中国疾病预防控制中心化学污染与健康安全重点实验室开放基金(2021CDCKL01)
天津市青年医学新锐人才项目(2018)。
