增强型绿色荧光蛋白基因在日本血吸虫成虫体内的瞬时表达  

Transient expression of enhanced green fluorescent protein(EGFP) gene in Schistosoma japonicum

作　　者：袁小松[1] 沈继龙[1] 汪学龙[1] 董慧芬[2] 蒋明森[2] 

机构地区：[1]安徽医科大学病原生物学教研室安徽省基因研究重点实验室,合肥230032 [2]武汉大学医学院病原生物学教研室

出　　处：《中国人兽共患病学报》2006年第1期18-21,共4页Chinese Journal of Zoonoses

基　　金：国家863计划资助项目(2004AAZZ3570)

摘　　要：目的探讨异源性增强型绿色荧光蛋白(EGFP)基因在日本血吸虫成虫体内表达的可能性。方法运用电穿孔技术将质粒pEGFP-C1导入日本血吸虫成虫体内,提取分离体外培养48小时成虫的基因组DNA、总RNA和全虫蛋白,分别用PCR、RT-PCR和Western blot验证转基因在成虫体内的存在、转录和翻译。同时,使用激光共聚焦扫描显微镜对EGFP在成虫体内进行定位。结果PCR和RT-PCR分别成功的扩增出760bp和276bp的预期大小的片断,Western-blot证实了EGFP基因在成虫体内的表达;激光共聚焦显微镜观察到,EGFP主要定位在成虫的皮层,口吸盘和尾部尤为明显。结论电穿孔技术成功地将异源基因引入日本血吸虫成虫体内并获得表达,为转基因血吸虫和基因功能的研究打下基础。To approach for the possibility of heterogenous EGFP gene to express in Schlstosoma japonicum, after plasmids of pEGFP-C1 had been introduced into Schistosoma with electroporation, the presence, transcription, and translation of the transgene in electroporated schistosoma were confirmed by PCR, RT-PCR, and Western blotting, respectively by using the genomic DNA , total RNA, and protein from schistosome cultured in vitro for 48 hours. Meanwhile, localization of EGFP within electroporated schistosome was performed with confocal laser scanning microscope. Consistent with the expected size, 760 bp and 276 bp of amplified products of PCR and RT-PCR were obtained and expression of EGFP gene in elctroporated schistosome was confirmed by Western blotting. Fluorescence of EGFP was mainly localized in tegument of the adults with corffocal microscopy especially predominateing around the head sucker and at the posterior part of the parasites. The heterogenous gene of EGFP has been successfully introduced into the S .japonicum by electroporation and the expression of transgene was confirmed by molecular and microscopical methods, which laid a foundation for the further study of transgenic schistosoma and gene function.

关 键 词：电穿孔技术 增强型绿色荧光蛋白 转基因血吸虫 

分 类 号：R383.2[医药卫生—医学寄生虫学]