重组质粒pEGFP-C3-caveolin-1构建及在小鼠足细胞中的表达

Construction and Expression of Recombinant Plasmid pEGFP-C3-Caveolin-1 in Cultured Podocytes from Mouse Kidney

目的:构建真核表达重组质粒pEGFP-C3-caveolin-1及稳定转染小鼠足细胞。方法:用RT-PCR法扩增小鼠肾脏caveolin-1的开放阅读框(ORF),构建TA克隆,用EcoRⅠ、BamHⅠ酶切位点亚克隆至真核表达质粒pEGFP-C3中,酶切和测序鉴定。脂质体转染法转染小鼠足细胞,荧光显微镜下动态观察转染后荧光蛋白的表达,培养72 h后,加入选择性抗生素G418(400 mg/L)筛选稳定转染细胞株。RT-PCR及Western-blot检测caveolin-1mRNA及蛋白表达。结果:重组质粒pEGFP-C3-caveolin-1经用EcoRⅠ、BamHⅠ双酶切,产生0.56 kb的目的插入片段及4.7 kb的载体片段,经DNA测序鉴定证实0.56 kb的插入片段与小鼠来源的caveolin-1 ORF序列一致;重组质粒转染小鼠足细胞后8 h在荧光显微镜下观察到绿色荧光,12 h后荧光表达逐渐增多;RT-PCR及Western-blot检测稳定转染细胞的caveolin-1 mRNA及蛋白表达水平为对照细胞的约2-3倍(P<0.05)。结论:重组pEG-FP-C3-caveolin-1真核表达载体包含小鼠caveolin-1完整ORF,稳定转染小鼠足细胞为后续相关研究奠定基础。

Objective： To construct an eukaryotic expression recombinant plasmid named pEGFP-C3-caveolin-1 and transfect it into podocytes derived from mouse kidney.Methods： Mouse caveolin-1 gene base sequence was inserted to multiple clone sites of TA clone vector by DNA recombinant technique.Then the recombinant vector was identified by incision enzyme EcoRⅠ and BamHⅠ and DNA sequencing.Abundant caveolin-1 gene base sequence was acquired and inserted to multiple clone sites of pEGFP-C3 by DNA recombinant technique.Then a new eukaryotic expression recombinant plasmid named pEGFP-C3-caveolin-1 was generated and identified by incision enzyme EcoRⅠ and BamHⅠ and DNA sequencing.pEGFP-C3-caveolin-1 was transfected into mouse podocytes in vitro with Lipofectamine 2000.The treated cells were continuously traced by fluorescence microscope.After 72 hours cultivation,400 mg/L G418 was added to select the stable transfected cells,from which total RNA and proteins were extracted respectively to detect the expression of caveolin-1 by RT-PCR and Western-blot analysis respectively.Results： Fragments of 0.56 kb and 4.7 kb were generated from the pEGFP-C3-caveolin-1 cut by EcoRⅠ and BamHⅠ.DNA sequence of the 0.56 kb fragment was identical with mouse caveolin-1 mRNA in GenBank.Green fluorescence could be seen by fluorescence microscrope after 8 h and a peak emerged within 24 h to 48 h.RT-PCR and Western-blot analysis revealed that the expression of caveolin-1 in the stable transfected cells was twice or three times more than that in the untransfected podocytes.Conclusion： The eukaryotic expression recombinant plasmid and the stable caveolin-1-transfected podocytes were successfully constructed,which will be a useful tool for our further research.