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

doi:10.13523/j.cb.20180105

中国生物工程杂志

2018, Vol. 38

Issue (1): 42-50 DOI: 10.13523/j.cb.20180105

技术与方法

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¹,姚琳²,江艳华²,李风铃²,张媛³,许东勤¹,朱文嘉²,郭莹莹²,王联珠²(

),翟毓秀²

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

Development of Armored RNA Reference Material of Norovirus Based on Qbeta Bacteriophage

Qi ZHANG¹,Lin YAO²,Yan-hua JIANG²,Feng-ling LI²,Yuan ZHANG³,Dong-qin XU¹,Wen-jia ZHU²,Ying-ying GUO²,Lian-zhu WANG²(

),Yu-xiu ZHAI²

1 Shanghai Ocean University, Shanghai 201306,China
2 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality,Ministry of Agriculture, P. R. China, Laboratory of Quality & Safety Risk Assessment for Aquatic Products (Qingdao), Ministry of Agriculture, P. R. China, Qingdao 266071, China;
3 Zhangzidao Group Co., LTD, Dalian 116001, China;

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摘要： 目的

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。

方法

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。

结果

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<F_0.05(9,20);稳定性结果表明,制备的VLPs在37℃可保存12天、室温(20~25℃)可保存24天、4℃至少可保存90天、-20℃至少可保存200天、-80℃至少可保存300天。

结论

基于Qbeta噬菌体制备的NoV装甲RNA均匀性和稳定性良好,拷贝数高,为NoV分子检测提供了良好的标准参考样品。

关键词： 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: Objective:

To develop armored RNA reference material containing target RNA of norovirus based on Qbeta bacteriophage for norovirus nucleic acid detection.

Methods:

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].

Results:

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<F_0.05(9,20). The stability test indicated that the sample was stable at 37℃ for 12 days, at room temperature (20~25℃) for 24 days, at 4℃ for at least 120 days, -20℃ for at least 150 days, at -80℃ for at least 300 days with no significant decrease.

Conclusion:

The armored RNA based on Qbeta bacteriophage prepared, which had good uniformity, stability and high copy number, could be a good reference material candidate for the norovirus RNA detection.

Key words: Norovirus Armored RNA Qbeta bacteriophage Reference material Real-time RT-PCR

收稿日期: 2017-05-31 出版日期: 2018-01-31

ZTFLH:

Q33

基金资助: * 中国水产科学研究院基本科研业务费重点项目(2016HY-ZD11);科技部科技基础性工作专项(2013FY113300);国家科技支撑计划资助项目(2015BAD17B03)

作者简介: 通讯作者王联珠。E-mail: wanglz@ysfri.ac.cn

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	张奇
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	王联珠
	翟毓秀

引用本文:

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. 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[J]. 中国生物工程杂志, 2018, 38(1): 42-50.

Qi ZHANG,Lin YAO,Yan-hua JIANG,Feng-ling LI,Yuan ZHANG,Dong-qin XU,Wen-jia ZHU,Ying-ying GUO,Lian-zhu WANG,Yu-xiu ZHAI. Development of Armored RNA Reference Material of Norovirus Based on Qbeta Bacteriophage. China Biotechnology, 2018, 38(1): 42-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180105 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I1/42

表1 引物、探针信息

图1 QINVGII片段PCR扩增

图2 pET-QINVGII的酶切鉴定图

图3 表达产物的SDS-PAGE分析

图4 VLPs电镜照片 400 000×

图5 VLPs中残留质粒检测电泳图

表2 VLPs定值结果分析

表3 均匀性实验的方差分析

表4 VLPs稳定性实验结果

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