本文關(guān)鍵詞：增強型綠色熒光蛋白（EGFP）基因在日本血吸蟲體內(nèi)及原代培養(yǎng)細胞內(nèi)的異源表達　出處：《安徽醫(yī)科大學》2006年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 轉(zhuǎn)基因血吸蟲 EGFP 電穿孔 轉(zhuǎn)染 脂質(zhì)體

【摘要】：目的 探討增強型綠色熒光蛋白(EGFP)基因在日本血吸蟲童蟲原代培養(yǎng)細胞、童蟲、成蟲體內(nèi)的異源表達，以及電穿法在血吸蟲基因轉(zhuǎn)化中的應用。為轉(zhuǎn)基因血吸蟲研究奠定基礎。 方法 制備血吸蟲童蟲原代培養(yǎng)細胞，用脂質(zhì)體將EGFP基因?qū)肱囵B(yǎng)細胞內(nèi)并觀察其表達；然后應用電穿孔技術(shù)將質(zhì)粒pEGFP-C1導入機械轉(zhuǎn)化的日本血吸蟲童蟲以及成蟲體內(nèi)，提取分離體外培養(yǎng)48h童蟲和成蟲的基因組DNA、總RNA和全蟲蛋白，分別用PCR、RT-PCR和Western blotting驗證異源基因在童蟲和成蟲體內(nèi)的轉(zhuǎn)錄和翻譯，，同時使用激光共聚焦掃描顯微鏡對EGFP在童蟲和成蟲體內(nèi)進行定位。最后，將電穿孔后的童蟲肌肉注射至小鼠體內(nèi)確定其感染力。結(jié)果成功的觀察到表達EGFP基因的血吸蟲童蟲培養(yǎng)細胞；PCR和RT-PCR分別成功擴增出760bp和276bp的預期大小的片斷；Western blotting證實了EGFP基因在童蟲及成蟲體內(nèi)的表達；激光共聚焦掃描顯微鏡觀察顯示，EGFP主要定位在童蟲和成蟲的皮層和副皮層，蟲體前端尤為明顯；成功地在試驗小鼠體內(nèi)檢獲了轉(zhuǎn)化的血吸蟲成蟲。結(jié)論 脂質(zhì)體、電穿孔分別將異源基因引入了日本血吸蟲童蟲原代培養(yǎng)細胞以及童蟲、成蟲體內(nèi)并獲得了瞬時表達；電穿孔后的童蟲可以在其終宿主體內(nèi)存活并發(fā)育成熟。本研究為建立轉(zhuǎn)基因血吸蟲和基因功能的研究打下了基礎。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R383

【共引文獻】

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