弓形虫棒状体ROP16基因转染RAW264.7的实验研究

Experimental study of Toxoplasma ROP16 gene transfection to RAW264. 7

目的比较目前常用的5种基因导入技术将弓形虫棒状体ROP16基因导入RAW264.7,选择合适的方法提高转染效率。方法以绿色荧光蛋白p EGFP-ROP16融合表达构建质粒,采用Lipofectamine3000、Attractene、FugeneHD转染试剂以及电穿孔转染和慢病毒载体感染RAW264.7,利用荧光显微镜和流式细胞仪观察目的基因的表达、细胞生长状态并分析转染效率,从而确定最佳的基因导入途径。结果上述质粒载体对RAW264.7的转染效率依次为电转法>FugeneHD>Lipofectamine3000>Attractene。但是电转法转染后细胞死亡率较高,细胞贴壁不牢或死亡;而慢病毒感染效率可达61.2%,细胞生长状态佳,显著优于质粒载体(P<0.05)。慢病毒组显著优于4个质粒组(P<0.05)。结论慢病毒感染可将弓形虫棒状体ROP16成功转入RAW264.7,具有较高的转染效率,效果显著优于质粒载体,目的基因在细胞内获得高效表达。为弓形虫ROP16的功能研究提供了重要基础。

Objective Five most commonly used methods were compared to transfer Toxoplasma gondii rhoptry 16 gene into RAW264. 7 in order to select the appropriate technique for improving transfection efficiency. Methods The following five methods of Lipofectamine 3000 Transfection Reagent,Attractene Transfection Reagent,Fugene HD Transfection Reagent,electroporation transfection and lentiviral vectors were used to construct the recombinant plasmids and lentiviral vectors, which carried genes of Toxoplasma ROP16 and pEGFP fusion protein. RAW264. 7 cells were subjected to transfection with the constructs individually. Then the expressions of the target gene, cellu-lar morphology and transfection efficiency were observed with fluorescence microscopy and flow cytometry. Results The transfection efficiency of the recombinant plasmids in RAW264. 7 goes as follows：electroporation〈Fugene HD〈Lipofectamine？ 3000〈Attractene. However, the plasmids-transfected host cells became vulnerable to deaths via electroporation. Comparatively, the high efficacy of 61. 2% was achieved with lentivirus infection （P〈0. 05）. In addition, statistic analyses showed that lentivirus infection of the host cells had obvious advantages over the test-ed plasmid vectors （ P 〈0. 05 ） . Conclusion Toxoplasma gondii ROP16 has been successfully transferred to RAW264. 7 macrophages by lentivirus infection, which resultes in higher efficiency of tranfection and expression of Toxoplasma gondii ROP16 gene in the cells than those by plasmids.