pBiFC-VN173-Olig2和pBiFC-VC155-Id4真核表达质粒的构建及其在活细胞内的相互作用

Construction of pBiFC-VN173-Olig2 and pBiFC-VC155-Id4 Eukaryotic Expression Vectors and Their Interaction in Living Cells

目的构建pBiFC-VN173-Olig2和pBiFC-VC155-Id4真核表达质粒,利用双分子荧光互补(BiFC)技术,在活细胞内直接观察Olig2与Id4的相互作用。方法利用逆转录聚合酶链反应(RT-PCR),以新生大鼠脊髓RNA为模板,扩增Olig2和Id4基因,分别定向克隆到pBiFC-VN173和pBiFC-VC155载体中,获得pBiFC-VN173-Olig2和pBiFC-VC155-Id4真核表达质粒。对此2种质粒进行酶切鉴定、测序;用脂质体方法共转染pBiFC-VN173-Olig2和pBiFC-VC155-Id4质粒到人结肠癌细胞系SW-1116细胞中,在荧光显微镜下观察Olig2与Id4之间的相互作用。结果通过酶切鉴定和测序表明成功构建了pBiFC-VN173-Olig2和pBiFC-VC155-Id4真核表达质粒;该质粒能够有效地共同转染到靶细胞,Olig2和Id4在靶细胞中能够高效表达,并可以相互结合出现BiFC荧光信号,且该信号主要分布于胞质中。结论成功构建了用于BiFC技术的真核表达载体,并在活细胞内检测到Olig2和Id4分子的相互结合。

Objective To construct pBiFC-VN173-Olig2 and pBiFC-VC155-Id4 eukaryotic expression plasmids for detecting the interaction of Olig2 and Id4 in living cells by bimolecular fluorescence complementation（BiFC）assay.Methods The RT-PCR was used to amplify rat Olig2 and Id4 genes from RNA of neonatal rat spinal cord.The Olig2 and Id4 genes were inserted into BiFC eukaryotic expression vectors pBiFC-VN173 and pBiFC-VC155 to construct recombinant expression vectors pBiFC-VN173-Olig2 and pBiFC-VC155-Id4,respectively.The recombinant vectors were identified by restriction enzyme digestion and DNA sequencing.The recombinant expression vectors were co-transfected into human colon cancer SW-1116 cells by using Lipofectamine 2000.The interaction of Olig2 and Id4 was detected by fluorescence inverted microscope.Results The restriction enzyme digestion and DNA sequencing showed that the sequences and open read frames of the two vectors were completely concordant with experiment design.The BiFC plasmids could be successfully co-transfected into the target cells and expressed.The strong BiFC signals could be detected in pBiFC-VN173-Olig2 and pBiFC-VC155-Id4 co-transfected cells and the fluorescence signal was located in the cytoplasm.Conclusion The eukaryotic expression plasmids for BiFC assay are successfully constructed.The interaction of Olig2 and Id4 in living cells can be detected using this technology.