生物学杂志

生物学杂志 ›› 2019, Vol. 36 ›› Issue (6): 12-.doi: 10.3969/j.issn.2095-1736.2019.06.012

• 研究报告 • 上一篇    下一篇

轮状病毒NSP5真核表达载体的构建、鉴定及功能研究

  

  1. 中国医学科学院 北京协和医学院 医学生物学研究所，云南省重大传染病疫苗研发重点实验室， 昆明 650118
  • 出版日期:2019-12-18 发布日期:2019-12-12
  • 通讯作者: 李鸿钧，研究员，长期从事病毒学及分子生物学相关研究，E-mail：lihj6912@hotmail.com
  • 作者简介:陈林林，硕士，专业方向为生物化学及分子生物学相关研究，E-mail：380951945@qq.com
  • 基金资助:

    中国医学科学院与健康科技创新工程协同创新团队项目（2016-I2M-3-026）；国家自然科学基金项目（31700154）；云南省重大科技专项（生物医药）（2018ZF006）；云南省科技计划项目（2016FB034）


Construction, identification and functional study of the eukaryotic expression vector of rotavirus NSP5 

  1. Institute of Medical Biology, Chinese Academy of Medical Science, Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease, Kunming 650118, China
  • Online:2019-12-18 Published:2019-12-12

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摘要：

研究轮状病毒（Rotavirus，RV）非结构蛋白5（NSP5）在体外对RV复制的作用。以质粒pEGFP-N2为载体构建出可特异性表达重组蛋白NSP5的真核表达载体后，将重组质粒转染MA104细胞并分别通过免疫荧光和Western Blot检测NSP5在转染后不同时间的表达差异。随后，重组质粒转染组及对照组细胞分别接种相同剂量的RV，一定时间后根据GFP分布来确认重组蛋白NSP5在细胞内的迁移变化情况，并比较实验组和对照组之间的RV复制能力差异。结果发现，NSP5蛋白在重组质粒转染细胞48 h后表达达到顶峰。此外，GFP也显示NSP5蛋白随RV的感染从弥散状转变为点状聚集，且转染该重组质粒的细胞内RV复制水平明显高于对照细胞。说明了轮状病毒NSP5对病毒的复制具有明显促进作用，这也为深入了解RV的感染、复制及致病机理提供理论基础。


关键词: 轮状病毒, NSP5, 真核表达载体, 病毒复制

Abstract:

This study focused on the effect of Rotavirus (RV) non-structural protein 5 (NSP5) on RV replication in vitro. After the construction of eukaryotic expression vector for expressing recombinant protein NSP5 specifically based on pEGFP-N2, the recombinant plasmid was transfected into MA104 cells. The expression of NSP5 at different time was detected by Immunofluorescence and Western Blot respectively. Subsequently, the RV replication and the migration of NSP5 based on the distribution of GFP were confirmed by fluorescence observation when MA104 cells were transfected with recombinant plasmid and empty plasmid infected with the same dose of RV. The recombinant plasmid expressed the strongest at 48 h post transfection in MA104 cells. Moreover, the location of recombinant protein NSP5 changed from dispersion to aggregation because of RV infection, and RV replication in the cells transfected with pEGFP-N2-NSP5 was significantly higher than in the control cells. In this study, the NSP5 could promote RV replication, which provides theoretical basis for further study on RV infection, replication and pathogenesis.


Key words: rotavirus, NSP5, eukaryotic expression vector, virus replication

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