应用Tet-On基因表达系统实现Mac-1-FP在CHO细胞中的可调控性表达

Inducible expression of Mac-1-FP in Chinese hamster ovary cells using Tet-On gene expression system

目的:应用Tet-On基因表达系统构建可调控性真核表达质粒,实现Mac-1-FP在CHO细胞中的可调控性表达。方法:采用DNA重组技术构建真核表达质粒pTRE-Tight-CFP-CD11b和pTRE-Tight-YFP-CD18(CD11b和CD18是Mac-1的两个亚基),通过脂质体介导共转染CHO细胞,用荧光显微镜观察转染后阳性细胞;应用RT-PCR和荧光强度分析观察不同浓度(0、0.01、0.1、0.5、1、2μg/ml)强力霉素(Dox)对细胞内CD11b和CD18表达水平的影响;检测PMA(1μg/ml)刺激前后CHO-Mac-1-FP细胞与其配基ICAM-1黏附活性的变化。结果:酶切鉴定和测序证实,已成功构建真核表达质粒pTRE-Tight-CFP-CD11b和pTRE-Tight-YFP-CD18,在荧光显微镜下观察到转染成功的CHO细胞发出青色荧光和黄色荧光。通过荧光显微镜观察到随着培养液中Dox浓度的增加,CHO细胞内的荧光强度相应增强,同时CD11b和CD18在mRNA水平的表达也随Dox浓度的增加而增加。PMA刺激后,CHO-Mac-1-FP细胞与其配基ICAM-1黏附率增高(2 h和4 h时最高,此后逐渐下降)。结论:本研究成功实现了Mac-1-FP在CHO细胞中的可调控性表达并具有野生型Mac-1的黏附活性,为进一步开展单分子光谱(SMS)和荧光共振能量转移(FRET)技术研究活细胞内Mac-1分子的2个亚基的二聚化、群集、构象及与配基亲和力变化创造条件。

Objective： To construct eukaryotic inducible expression plasmids pTRE-Tight-CFP-CDllb and pTRE-Tight- YFP-CD18 using Tet-On gene expression system and co-transfect them into Chinese hamster ovary （CHO） cells, so as to achieve the inducible expression of Mac-1-FP. Methods： The eukaryotic expression vectors pTRE-Tight-CFP-CN11 b and pTRE- Tight-YFP-CD18 were constructed by recombinant DNA technique. The 2 vectors were co-transfected into CHO cells using liposome to fuse CD11b and CD18： the 2 subunits of Mac-1. Fluorescence microscope was used to observe the cyan fluorescence and the yellow fluorescence of Mac-1-FP. The influence of different levels of Dox （0, 0.01, 0. 1, 0.5, 1, 2μg/ml） on expres- sion levels of CD11b and CD18 in CHO cells was analyzed by RT-PCR and fluorescence intensity analysis. The adhesive rate of CHO-Mac-I-FP cells with ligand ICAM-1 was analyzed before and after PMA （1 μg/ml） stimulation. Results： The recombinant plasmids of pTRE-Tight CFP-CDllb and pTRE-Tight-YFP-CD18 were successfully constructed. The cyan and yellow fluorescences were observed in co-transfected CHO cells under fluorescence microscope. The fluorescence intensity of the cells was increased with the increase of Dox concentration. RT-PCR analysis demonstrated that the CD11b and CD18 mRNA increased with the increase of Dox concentration. The adhesive rate of CHO-Mac-1-FP cells with ICAM-1 was increased after PMA stimulation （peaked at 2 h and 4 h after stimulation and decreased afterwards）. Conclusion： This study achieves the inducible expression of Mac-1-FP in CHO cells. And Mac-1-FP, like widetype Mac-1, exhibits adhesive activity with ligand ICAM-1, which lays a foundation for studying the consisting dimmer, clustering, conformation and affinity of the ligands of Mac-1 using single mole cule spectroscopy and fluorescence resonance energy transfer technique in living cells.