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

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (3): 213-217.

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

赵楠¹, 李青¹, 胡薇^1,^2,^*()

1 复旦大学生命科学学院微生物学与微生物工程系,上海200438
2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,上海200025

收稿日期:2017-01-16 出版日期:2017-03-30 发布日期:2017-09-07
通讯作者: 胡薇
基金资助:
国家自然科学基金（No. 81271867）

Lentivirus-mediated expression of green fluorescent protein in Schistosoma japonicum

Nan ZHAO¹, Qing LI¹, Wei HU^1,^2,^*()

1 Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China

Received:2017-01-16 Online:2017-03-30 Published:2017-09-07
Contact: Wei HU
Supported by:
Supported by National Natural Science Foundation of China(No. 81271867)

摘要/Abstract

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pEGFP-LacZ-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 × 10⁸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 pEGFP-LacZ-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.

关键词: 日本血吸虫, 绿色荧光蛋白, 慢病毒载体

Abstract: Objective To investigate whether exogenous green fluorescence protein(GFP) could be expressed in Schistosoma japonicum and generate green fluorescence. Methods The fusion protein-expressing plasmid pEGFP-LacZ-C1 was constructed and used to transfect 293T cells and S. japonicum schistosomula at 14 days after infection, respectively, using PEI as the transfection reagent. The GFP fluorescent signal was then observed under a fluorescence microscope at 48 h after transfection; β-galactosidase staining was performed in these cells to observe the presence of blue products under an optical microscope. Meanwhile, the schistosomula were added to 12-well plates of 293T cells and observed by optical microscopy and fluorescence microscopy. Finally, a high concentration of lentivirus(titer: 3 × 10⁸ colony-forming units/ml) was used to infect schistosomula and 293T cells, respectively, and GFP fluorescent signal was observed at 96 h after infection. Results Both GFP fluorescent signal and blue spots were seen in 293T cells transfected with pEGFP-LacZ-C1. However, neither was observed in schistosomula. In addition, the GFP fluorescence luminance in 293T cells was higher than that of spontaneous fluorescence of S. japonicum, and differential colors were seen: the color of the former was bright green, while the later was yellow-green. In the experiment of lentivirus infection, the fluorescent spots were clearly seen around the intestine of schistosomula under the fluorescent microscope. Besides, the movements of fluorescent spots were consistent with muscle movement. Conclusion The spontaneous fluorescence of S. japonicum does not influence the detection of GFP fluorescent signal under sufficient expression of exogenous GFP. Lentivirus infection at a high concentration can improve the transfection efficiency and increase GFP expression at 96 h after infection. In this way, the fluorescent signal of GFP transfected into schistosomula has been observed.

Key words: Schistosoma japonicum, Green fluorescence protein, Lentiviral vector

中图分类号:

R383.24

赵楠, 李青, 胡薇. 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[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 213-217.

Nan ZHAO, Qing LI, Wei HU. Lentivirus-mediated expression of green fluorescent protein in Schistosoma japonicum[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(3): 213-217.

图/表 4

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