王鸣秋,杨俊,常雨桐,张涛,刘丽娟.GI/GII型诺如病毒两联装甲RNA标准样品的研制[J].,2021,37(3):286-293.
GI/GII型诺如病毒两联装甲RNA标准样品的研制
Preparation of Coupled Armored RNA Reference Material for Norovirus GI/GII
投稿时间:2020-09-04  
DOI:10.13982/j.mfst.1673-9078.2021.3.0835
中文关键词:  诺如病毒  两联装甲RNA  MS2噬菌体  标准样品  实时荧光定量RT-PCR
英文关键词:Norovirus  coupled armored RNA  MS2 bacteriophage  reference material  real-time RT-PCR
作者简介:王鸣秋(1986-),女,高级工程师,研究方向:食品微生物检测 通讯作者:刘丽娟(1971-),女,博士,研究员,研究方向:病原微生物检测与检疫研究
基金项目:湖北省重点研发计划项目(2020BCA091);中国检验检疫科学研究院基本科研业务费项目(2019JK017)
作者单位
王鸣秋 (1.湖北省食品质量安全监督检验研究院,湖北武汉 430075)(2.中国检验检疫科学研究院,北京 100176) 
杨俊 (2.中国检验检疫科学研究院,北京 100176) 
常雨桐 (2.中国检验检疫科学研究院,北京 100176) 
张涛 (1.湖北省食品质量安全监督检验研究院,湖北武汉 430075) 
刘丽娟 (2.中国检验检疫科学研究院,北京 100176) 
AuthorInstitution
WANG Ming-qiu (1.Hubei Provincial Institute for Food Supervision and Test, Wuhan 430075, China) (2.Chinese Academy of Inspection and Quarantine, Beijing 100176, China) 
YANG Jun (2.Chinese Academy of Inspection and Quarantine, Beijing 100176, China) 
CHANG Yu-tong (2.Chinese Academy of Inspection and Quarantine, Beijing 100176, China) 
ZHANG Tao (1.Hubei Provincial Institute for Food Supervision and Test, Wuhan 430075, China) 
LIU Li-juan (2.Chinese Academy of Inspection and Quarantine, Beijing 100176, China) 
摘要点击次数: 15
全文下载次数: 9
中文摘要:
      针对目前缺乏适配多项检测标准、稳定、安全的诺如病毒RNA标准样品的问题,研制基于MS2噬菌体内含常见GI/GII型诺如病毒检测靶标两联装甲RNA标准参考样品。人工合成MS2噬菌体成熟酶基因、衣壳蛋白基因、包装位点及GI/GII型诺如病毒靶标基因,克隆于表达载体pET-28a(+)中,构建重组质粒pET-MS2-NoV。经大肠杆菌BL21诱导表达,先后利用PEG6000、酶处理和丙烯葡聚糖凝胶层析柱纯化表达产物。SDS-PAGE和透射电镜鉴定产物大小及结构,荧光定量PCR检测有无残留核酸。之后对纯化的病毒样颗粒(Virus-like particles,VLPs)开展定值、均匀性和短期稳定性研究。SDS-PAGE结果表明重组质粒在BL21中表达出了目的蛋白,大小在10~15 ku之间,与预期一致;纯化后的VLPs无杂蛋白和残留核酸;透射电镜下呈结构完整、大小均一的球状,直径约25 nm。纯化后AR-NoV中GI型和GII型靶标定值结果分别为(4.04±0.62)×107 copies/μL和(6.16±0.30)×107 copies/μL。单因素方差检验证实样品均一性良好,F
英文摘要:
      To provide a safe and stable reference material for Norovirus nucleic detection, the armored RNA containing target RNA of Norovirus GI and GII based on MS2 bacteriophage was developed in this work. DNA fragments including maturase coding gene, capsid protein coding gene and packing site of MS2 bacteriophage, target cDNA sequence of Norovirus GI and GII were synthesized artificially and then cloned into expression vector pET-28a(+) to construct recombinant plasmid pET-MS2-NoV. After expressed in E. coli BL21 cells by IPTG induction, the expression product was purified by PEG6000, enzyme digestion and molecular sieve chromatography. The purified product, also named AR-NoV, was identified by SDS-PAGE, transmission electron microscopy (TEM) and RT-PCR. The value, homogeneity and stability of the AR-NoV were evaluated. SDS-PAGE analysis showed that with the molecular weight of tar-get protein expressed in BL21 was 10~15 ku, which was consistent with the predicted value. There were no impure proteins and residual nucleic acids in AR-NoV after purification. The AR-NoV presented as spherical VLPs with uniform particle size (about 25 nm) and integrated structure under TEM. The values of GI and GII targets in AR-NoV were (4.04±0.62)×107 copies/μL and (6.16±0.30)×107 copies/μL, respectively. The good homogeneity of AR-NoV was confirmed by single-factor ANOVA test (F
查看全文  查看/发表评论  下载PDF阅读器
关闭