用电穿孔法将绿色荧光蛋白基因导入日本血吸虫童虫并表达

Transformation of Schistosomulae by Electroporation and Transient Expression of the Enhanced Green Fluorescent Protein (EGFP) Gene

目的探讨增强型绿色荧光蛋白(EGFP)基因在日本血吸虫童虫体内异源表达,以及电穿孔技术在血吸虫基因转化中应用的可能性。方法应用电穿孔技术将质粒pEGFP-C1导入机械转化的日本血吸虫童虫体内,提取分离体外培养48 h童虫的基因组DNA、总RNA和全虫蛋白,分别用PCR、逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blotting)验证转基因在童虫体内的存在、转录和翻译。同时,使用激光共聚焦扫描显微镜对EGFP在童虫体内进行定位。结果PCR和RT-PCR分别成功扩增出760 bp和276 bp的预期大小的片段,Western blotting证实了EGFP基因在童虫体内的表达;激光共聚焦显微镜观察表明EGFP主要定位在童虫的皮层和副皮层,虫体前端尤为明显。结论电穿孔技术成功地将异源基因引入日本血吸虫童虫体内并获得表达。

Objective To explore the possibility of heterogenous gene to express in juvenile Sckistosoma japonicum and the application of electroporation in transformation of schistosomulae. Methods The plasmids of pEGFP- C1 were introduced into mechanically transformed schsitosomula with electroporation. The presence, transcription and translation of the transgene in electroporated schistosomula were confirmed by PCR, RT-PCR and Western blotting analysis respectively using the genomic DNA, total RNA and protein extracted and isolated from schistosomula cultured in vitro for 48 hours. Meanwhile, localization of EGFP within electroporated schistosomula was performed with confocal laser scanning microscope. Results 760 bp and 276 bp amplified products by PCR and RT -PCR were found coincident with the expected size and expression of EGFP gene in elctroporated schistosomula was confirmed by Western blotting. Fluorescence of EGFP was localized in tegument and subtegument of the electroporated schistosomula with confocal microscopy, especially in the anterior part of the worm. Conclusion The heterogenous gene of EGFP has been successfully introduced into juvenile S. japonicum by electroporation and the expression of transgene was confirmed with molecular and microscopical methods.