增强型绿色荧光蛋白（EGFP）基因在日本血吸虫体内及原代培养细胞内的异源表达

发布时间：2018-01-04 09:16

本文关键词：增强型绿色荧光蛋白（EGFP）基因在日本血吸虫体内及原代培养细胞内的异源表达　出处：《安徽医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的探讨增强型绿色荧光蛋白(EGFP)基因在日本血吸虫童虫原代培养细胞、童虫、成虫体内的异源表达，以及电穿法在血吸虫基因转化中的应用。为转基因血吸虫研究奠定基础。方法制备血吸虫童虫原代培养细胞，用脂质体将EGFP基因导入培养细胞内并观察其表达；然后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以及成虫体内，提取分离体外培养48h童虫和成虫的基因组DNA、总RNA和全虫蛋白，分别用PCR、RT-PCR和Western blotting验证异源基因在童虫和成虫体内的转录和翻译，，同时使用激光共聚焦扫描显微镜对EGFP在童虫和成虫体内进行定位。最后，将电穿孔后的童虫肌肉注射至小鼠体内确定其感染力。结果成功的观察到表达EGFP基因的血吸虫童虫培养细胞；PCR和RT-PCR分别成功扩增出760bp和276bp的预期大小的片断；Western blotting证实了EGFP基因在童虫及成虫体内的表达；激光共聚焦扫描显微镜观察显示，EGFP主要定位在童虫和成虫的皮层和副皮层，虫体前端尤为明显；成功地在试验小鼠体内检获了转化的血吸虫成虫。结论脂质体、电穿孔分别将异源基因引入了日本血吸虫童虫原代培养细胞以及童虫、成虫体内并获得了瞬时表达；电穿孔后的童虫可以在其终宿主体内存活并发育成熟。本研究为建立转基因血吸虫和基因功能的研究打下了基础。
[Abstract]:Objective to investigate the heterologous expression of EGFP gene in primary cultured cells of Schistosoma japonicum and adults. And the application of electroporation method in gene transformation of Schistosoma japonicum lay a foundation for the study of transgenic Schistosoma japonicum. Methods the primary cultured cells of Schistosoma japonicum were prepared. The EGFP gene was introduced into the cultured cells by liposome and its expression was observed. Then the plasmid pEGFP-C1 was introduced into the mechanically transformed Schistosoma japonicum and adult by electroporation, and the genomic DNA of the juvenile and adult worms were isolated and isolated for 48 h in vitro. Total RNA and whole insect protein were analyzed by PCR and Western blotting, respectively. The transcription and translation of allogenes in juvenile and adult worms were confirmed by PCR and Western blotting, respectively. At the same time, we use confocal laser scanning microscope to locate EGFP in children and adults. Finally. After electroporation, the infective cells of Schistosoma japonicum expressing EGFP gene were successfully observed by intramuscular injection into mice to determine its infectivity. The expected size fragments of PCR and RT-PCR were 760bp and 276bp, respectively. Western blotting confirmed the expression of EGFP gene in juvenile and adult worms. Laser confocal scanning microscopy showed that EGFP was mainly located in the cortical and paracortex of juvenile and adult worms, especially in the front end of the parasite. The transformed adult Schistosoma japonicum was successfully seized in mice. Conclusion Liposome and electroporation have introduced allogenic genes into the primary culture cells of Schistosoma japonicum and juvenile worm, respectively. The transient expression was obtained in adult. After electroporation, the child worm could survive and mature in its final host. This study laid a foundation for the establishment of transgenic Schistosoma japonicum and gene function.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R383

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