首页 > 核心刊物 > 中国生物工程杂志 2018.1 Vol 38, No.1 > 基于Qbeta噬菌体装甲RNA技术的诺如病毒RNA标准参考样品的研制

基于Qbeta噬菌体装甲RNA技术的诺如病毒RNA标准参考样品的研制

张奇,姚琳,江艳华,李风铃,张媛,许东勤,朱文嘉,郭莹莹,王联珠,翟毓秀

1 上海海洋大学 上海 201306 2 中国水产科学研究院黄海水产研究所 农业部水产品质量安全检测与评价重点实验室 农业部水产品质量安全风险评估实验室(青岛) 青岛 266071 3 獐子岛集团股份有限公司 大连 116001

 
Key Words:  诺如病毒; 装甲RNA; Qbeta噬菌体; 标准参考样品; 实时荧光定量RT-PCR
 

Abstract

针对目前检测领域缺乏诺如病毒(Norovirus, NoV)核酸标准样品这一瓶颈,基于Qbeta噬菌体装甲RNA技术构建内含GII型NoV检测靶标RNA的病毒样颗粒(virus like particles, VLPs)标准参考样品。 人工合成包含Qbeta噬菌体成熟酶编码基因、衣壳蛋白编码基因、包装位点、ISO/T15216-2 2012中规定的GII型NoV检测靶标对应的cDNA序列及辅助多克隆位点的DNA片段QINVGII,将其克隆到pET-28a(+)载体中,构建重组质粒pET-QINVGII。将pET-QINVGII转化大肠杆菌BL21(DE3)感受态细胞并诱导表达。表达产物经SDS-PAGE和透射电镜分析后,利用氯化铯密度梯度离心,制备纯化VLPs,并对纯化后的VLPs开展均匀性、稳定性及定值研究。 SDS-PAGE结果证实重组大肠杆菌在14.1kDa左右有目的条带表达;透射电镜下可观察到大量结构完整、直径约为25nm的VLPs。定值结果显示,制备的VLPs样品中GII型NoV检测靶标RNA的含量为(1.06±0.06)×107 copies/μl;均匀性分析结果表明样品均匀性良好,即F=2.24


 

Abstract

To develop armored RNA reference material containing target RNA of norovirus based on Qbeta bacteriophage for norovirus nucleic acid detection. Synthesize DNA fragment named QINVGII containing maturase coding gene, capsid protein coding gene and packing site of Qbeta bacteriophage, cDNA corresponding detection target sequence of gene group II norovirus in ISO/T15216-2 2012. QINVGII fragment was cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was identified by enzyme digestion and sequencing, and then expressed in E. coli BL21 (DE3) cells through isopropyl-β-thiogalactopyranoside (IPTG) induction. The expression product, VLPs was analyzed by SDS-PAGE and the electron microscope. The VLPs was centrifuged and purified by CsCl density gradient after digestion with DNase I and RNase A. The homogeneity and stability of the reference material were tested according to the GB/T15000.3—2008 [directives for the work of reference materials (3) - reference material -general and statistical principles for certification]. SDS-PAGE analysis showed that the molecular mass of the expressed protein product was about 14.1kDa, which was consistent with the prediction. The 25nm VLPs could be observed under electron microscope. The VLPs samples were valued as (1.06±0.06)×107 copies/μl and behaved well in the homogeneity test, F=2.24


期刊简介
 
主办单位:
中国生物工程学会(CSBT)、中国生物技术发展中心(CNCBD)、中国科学院文献情报中心。
编委会:
最新一届编辑委员会由国家生物技术研究开发计划的评审专家及有关院士组成,代表中国生物技术的最高学术水平。
编委会主任:陈志南
通信地址:北京市中关村北四环西路33号《中国生物工程杂志》编辑部(100190)
国内刊号:CN11-4816/Q
国际刊号:ISSN1671-8135
邮发代号:82-673