慢病毒介导的绿色荧光蛋白基因在日本血吸虫体内的成功表达

发布时间：2018-02-28 12:08

本文关键词： 日本血吸虫绿色荧光蛋白慢病毒载体　出处：《中国寄生虫学与寄生虫病杂志》2017年03期 　论文类型：期刊论文

【摘要】：目的了解日本血吸虫(Schistosoma japonicum)体内能否表达外源绿色荧光蛋白(green fluorescent protein,GFP),并产生绿色荧光。方法构建p EGFP-Lac Z-C1融合蛋白表达质粒,以聚乙烯亚胺(polyethylenimine,PEI)为转染试剂,分别转染哺乳动物293T细胞和感染小鼠后14 d的日本血吸虫童虫,未转染阴性对照组不做任何处理;转染后48 h,在荧光显微镜下观察被转染的293T细胞和日本血吸虫童虫是否有GFP绿色荧光产生;对被转染的293T细胞和日本血吸虫童虫进行β-半乳糖苷酶原位染色,在光学显微镜下观察是否有蓝色产物产生。同时,将日本血吸虫童虫放入培养293T细胞的12孔板中,分别在光学显微镜和荧光显微镜下观察。用未稀释的、滴度为3×10~8菌落形成单位(CFU)/ml的慢病毒体外感染日本血吸虫童虫和293T细胞96 h后,在荧光显微镜下观察是否有GFP绿色荧光产生。结果p EGFP-Lac Z-C1转染293T细胞后,可以观察到GFP绿色荧光,β-半乳糖苷酶原位染色后也可以观察到蓝色的斑点,而在对照组和日本血吸虫童虫中,均未观察到GFP荧光和蓝色斑点。日本血吸虫童虫和293T细胞GFP荧光的比较发现,293T细胞发出的GFP荧光亮度明显高于日本血吸虫童虫的自发荧光,且293T细胞GFP荧光为艳绿色,而日本血吸虫童虫的自发荧光为黄绿色。在慢病毒感染实验中,未稀释的慢病毒感染96 h后,在荧光显微镜下可以清晰地看到血吸虫肠道外围组织中有许多艳绿色的荧光亮点,日本血吸虫童虫GFP的颜色及亮度与293T细胞中的基本一致,且荧光亮点的移动与虫体的肌肉运动基本保持一致。结论日本血吸虫童虫的背景荧光在外源GFP表达充足的情况下并不会影响荧光的观察。未稀释的慢病毒感染日本血吸虫童虫96 h后可提高转染效率,增加GFP表达量,在体内观察到GFP的绿色荧光。
[Abstract]:Objective to investigate whether the exogenous green fluorescent protein can be expressed in Schistosoma japonicum and produce green fluorescence. Methods the expression plasmid of p EGFP-Lac Z-C1 fusion protein was constructed and polyethylenimine (PEI) was used as transfection reagent. The transfected mammalian 293T cells and Schistosoma japonicum infecting mice were treated without any treatment in the negative control group. 48 h after transfection, the GFP green fluorescence was observed in 293T cells and Schistosoma japonicum, and 尾 -galactosidase was used to stain the transfected 293T cells and Schistosoma japonicum in situ. At the same time, Schistosoma japonicum was placed in a 12-well plate cultured with 293T cells, observed under optical microscope and fluorescence microscope, respectively. Lentiviruses with a titer of 3 脳 10 ~ (8) colony forming unit (CFU / ml) were infected with Schistosoma japonicum and 293T cells in vitro for 96 h, and the GFP green fluorescence was observed under fluorescence microscope. Results after transfection of p EGFP-Lac Z-C1 into 293T cells, GFP green fluorescence was observed, and blue spots were observed after in situ staining of 尾 -galactosidase, while in the control group and schistosomiasis japonicum, GFP fluorescence and blue spots were not observed. The GFP fluorescence of Schistosoma japonicum and 293T cells was significantly higher than that of Schistosoma japonicum and 293T cells, and the GFP fluorescence of 293T cells was bright green. However, the autofluorescence of Schistosoma japonicum was yellowish green. In the experiment of lentivirus infection, after 96 h of undiluted lentivirus infection, there were many bright green fluorescent spots in the peripheral tissues of intestinal tract of Schistosoma japonicum under fluorescence microscope. The color and brightness of Schistosoma japonicum GFP are basically the same as those in 293T cells. Conclusion the background fluorescence of Schistosoma japonicum does not affect the observation of fluorescence when the expression of exogenous GFP is sufficient. The transfection efficiency of Schistosoma japonicum was improved after 96 h. The green fluorescence of GFP was observed in vivo by increasing the expression of GFP.
【作者单位】：复旦大学生命科学学院微生物学与微生物工程系;中国疾病预防控制中心寄生虫病预防控制所世界卫生组织热带病合作中心科技部国家级热带病国际联合研究中心卫生部寄生虫病原与媒介生物学重点实验室;
【基金】：国家自然科学基金(No.81271867)~~
【分类号】：R383.24

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