pBIFC-VN173-CXCR4和pBIFC-VC155-NT21MP真核表达质粒的构建及其在活细胞内的作用

目的:构建pBIFC-VN173-CXCR4和pBIFC-VC155-NT21MP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趋化因子受体4(chemokine receptor 4、CXCR4和CD184)与CXCR4抑制性多肽的相互作用。方法:应用化学合成法获得巨噬细胞炎症蛋白Ⅱ(viralmacrophage inflammatory protein-Ⅱ,vMIP-Ⅱ)N端21肽(N-terminal 21-mer peptide,NT21MP)编码的基因序列,克隆至经Kpn I和EcoRⅠ酶切的pBiFC-VC155中,筛选含有目标基因的正确克隆。利用RT-PCR扩增人乳腺癌细胞株SKBR3的CXCR4全长基因后,T-A亚克隆至经Kpn I和EcoRⅠ酶切的pBiFC-VN173载体中。然后,经酶切鉴定及DNA测序分析2个基因是否正确连接至真核表达载体中。应用脂质体转染的方法共转染pBiFC-VC155-NT21MP和pBiFC-VC155-CXCR4至细胞株COS-7中,在荧光显微镜下观察NT21MP与CXCR4在胞内的相互作用。结果:经DNA测序及同源性对比,证实pBiFC-VC155-NT21MP和pBiFC-VN173-CXCR4重组载体构建成功;基因片段与NCBI基因库vMIP-Ⅱ和CXCR4基因CDS序列同源性达99.9%。BiFC法观察NT21MP与CXCR4在细胞内结合出现的荧光信号,该信号分布细胞内。结论:本实验成功构建了应用BiFC技术的真核表达载体,并且在活细胞内检测到NT21MP与CXCR4的互相结合。

Construction of pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and their interaction in living cells

Objective: To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4(CXCR4) and viral macrophage inflammatory protein-Ⅱ(vMIP-Ⅱ) N terminal 21 peptides(NT21MP) in living cells.Methods: DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155.The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR3 cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173.Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing.The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000.The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation(BiFC) assay.Results: The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design.The BiFC plasmids were successfully cotransfected into the target cells and expressed.The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm.Conclusions: The eukaryotic expression plasmids for BiFC assay are successfully constructed.The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.