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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。
Objective To investigate whether Schistosoma japonicum can express green fluorescent protein (GFP) in vivo and produce green fluorescence. Methods The p EGFP-Lac Z-C1 fusion protein expression plasmid was constructed and transfected into 293T cells with polyethylenimine (PEI) as transfection reagent and infected with Schistosoma japonicum Schistosoma japonicum Transfected 293T cells and Schistosoma japonicum Schistosoma japonicum whether GFP green fluorescence was observed 48 hours after transfection, the transfected 293T cells and Schistosoma japonicum Insect β-galactosidase in situ staining, observed under a light microscope whether there is a blue product. Meanwhile, the Schistosoma japonicum squirrel was placed in a 12-well plate of 293T cells and observed under a light microscope and a fluorescence microscope. After in vitro infection of Schistosoma japonicum and 293T cells with undiluted lentivirus with a titer of 3 × 10-8 CFU / ml for 96 h, GFP green fluorescence was observed under a fluorescence microscope. Results EGFP-Lac Z-C1 transfected 293T cells can be observed GFP green fluorescence, β-galactosidase in situ staining can also be observed after the blue spots, and in the control group and Schistosoma japonicum , No GFP fluorescence and blue spots were observed. Comparison of GFP fluorescence between Schistosoma japonicum and 293T cells showed that GFP fluorescence of 293T cells was significantly higher than autofluorescence of Schistosoma japonicum, GFP fluorescence of 293T cells was bright green, while that of Schistosoma japonicum was yellow green. In lentivirus infection experiment, after 96 h of undiluted lentivirus infection, there are many bright green fluorescent spots in peripheral tissues of Schistosoma japonicum under a fluorescence microscope. The color and brightness of GFP of Schistosoma japonicum 293T cells are basically the same, and the movement of fluorescent highlights and the parasites of muscle movement basically the same. Conclusion The background fluorescence of Schistosoma japonicum Schistosoma japonicum does not affect the fluorescence observation when the expression of exogenous GFP is sufficient. The undigested lentivirus infection of Schistosoma japonicum Schistosoma japonicum could increase the transfection efficiency, increase the expression of GFP, and observe the green fluorescence of GFP in vivo.