Notch1胞内结构域真核表达质粒构建及在H9c2心肌样细胞的表达和生物学功能

Construction of eukaryotic expression plasmid for Notch1 intracellular domain and its expression and function in H9c2 cells

目的构建Notch l胞内结构域(N1ICD)真核表达质粒,确定能在H9c2心肌样细胞内表达,并发挥生物学效应,为后续Notch1信号通路的研究提供实验依据。方法设计并合成1对含有BamH I和EcoR I酶切位点的特异性引物,以小鼠cDNA文库为模板,经PCR扩增得到了1 122 bp的目的片段,定向克隆至pcDNA 3.1/myc-His质粒,构建pcDNA3.1/N1ICD-myc-His真核表达质粒。通过双酶切及基因测序鉴定,经Lipofectamine 2000转染至H9c2心肌样细胞,48 h后提总蛋白,Western-blotting检测myc和Hes1,确定N1ICD能在H9c2细胞表达,并启动下游基因表达。结果表达质粒pcDNA3.1/N1ICD-myc-His双酶切及基因测序正确,N1ICD可在H9c2心肌样细胞内高效表达,具有Notch1生物学效应。结论真核表达质粒pcDNA3.1/N1ICD-myc-His构建成功,为Notch1信号通路在心血管领域的功能研究奠定了基础。

[Objective] To construct an eukaryotic expression plasmid for Notchl intracellular domain （NIICD）, determine its expression and function in H9c2 cells, which provides an experimental basis for further study on Notchl signaling. [Methods] A pair of specific primers with BamH I and EcoR I restriction enzyme sites were de- signed and synthesized. With the mouse cDNA as a template, the 1 122 bp target fragment was amplified by PCR, then directly cloned into pcDNA 3.1/myc-His plasmid to construct eukaryotic expression plasmid pcDNA3.1/ NlICD-myc-His. pcDNA3.1/NlICD-myc-His plasmid was verified by double restriction enzyme analysis and DNA gene sequencing. Afterwards, the recombinant pcDNA3.1/NlICD-myc-His was transfected into H9c2 cell by Lipo- fectamine 2000. Total protein was extracted after 48h, the expression of NIlCD and Hesl were identified by West- ern-blotting analysis with myc and Hesl antibodies. [Results] Restriction analysis and sequencing proved that eu- karyotic expression plasmid pcDNA3.1/NIICD-myc-His was constructed successfully. NIICD was expressed in H9c2 cells and had biological effects of Notch l. [ Conclusion ] The eukaryotic expression plasmid for N IICD was successfully constructed and expressed quantatively in H9c2 cells, which lays a foundation of function study of Notchl signaling pathway in the cardiovascular field.