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目的利用pmirGLO Dual-Luciferase miRNA Target Expression Vector(简称为pmirGLO报告基因载体)构建并鉴定含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区报告基因载体(pmir-Insr-3′UTR及pmirmutant-Insr-3′UTR)。方法以大鼠肝脏cDNA为模板,PCR获取目的片段(即含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区);用PmeI、XbaI双酶切pmirGLO报告基因载体和含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区,用T4 DNA连接酶连接纯化后的酶切产物;连接产物转化DH5α大肠杆菌感受态细胞并挑选阳性克隆,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒。结果 PCR和双酶切证实pmir-Insr-3′UTR及pmir-mutant-Insr-3′UTR重组载体中均插入目的片段;DNA测序结果进一步证实pmir-Insr-3UTR重组载体中成功插入了胰岛素受体mRNA 3′UTR区,pmir-mutant-Insr-3′UTR重组载体中成功插入了在miR-497结合位点含有3个突变碱基的胰岛素受体mRNA 3′UTR片段。结论成功构建了含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR报告基因载体pmir-Insr-3′UTR及pmir-mutant-Insr-3′UTR。
Objective To construct and identify pmir-Insr-3 ’reporter gene vector of pmRGLO Dual-Luciferase miRNA Target Expression Vector (pmirGLO reporter vector) for insulin receptor mRNA with miR-497 wild- UTR and pmirmutant-Insr-3’UTR). Methods The rat liver cDNA was used as a template to obtain the target fragment (ie, the 3’UTR region of insulin receptor mRNA with wild-type and mutation-binding site of miR-497). The pmirGLO reporter vector and the miR- -497 wild-type and mutant binding sites of insulin receptor mRNA 3’UTR region, the use of T4 DNA ligase purified digestion products; ligation products were transformed DH5α E. coli competent cells and positive clones selected and by PCR, bis Restriction enzyme digestion, DNA sequencing identified the constructed recombinant plasmid. Results PCR and double enzyme digestion confirmed that both pmir-Insr-3’UTR and pmir-mutant-Insr-3’UTR recombination vector were inserted into the target fragment; DNA sequencing results further confirmed the pmir-Insr-3UTR recombinant vector successfully inserted into the insulin receptor The 3’UTR fragment of insulin receptor mRNA containing 3 mutant bases at the miR-497 binding site was successfully inserted into the pmir-mutant-Insr-3’UTR recombinant vector. Conclusion The pmir-Insr-3’UTR and pmir-mutant-Insr-3’UTR of 3’UTR reporter gene vector of insulin receptor mRNA with miR-497 wild-type and mutation binding sites were successfully constructed.