基于Qβ噬菌体的A群轮状病毒装甲RNA标准参考样品的研制
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(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),山东 青岛 266071)

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张奇 女 硕士生 研究方向为水产品食源性病毒标准样品制备 E-mail:qixijinqianhua@foxmail.com通信作者:┣┣(中)通信作者┫┫姚琳 男 副研究员 研究方向为食源性病毒的监控与标准样品研发 E-mail:yaolin@ysfri.ac.cn

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科技部科技基础性工作专项(2013FY113300);中国水产科学研究院基本科研业务费专项(2016HY-ZD11);国家贝类产业技术体系(CARS-47)


Research on armored RNA reference material of group A Rotavirus based on Qβ bacteriophage
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(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 of P. R. China,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Shandong Qingdao 266071,China)

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    摘要:

    目的 基于Qβ噬菌体装甲RNA技术构建内含A群轮状病毒(Rotavirus,RV)检测靶标的装甲RNA(Rotavirus armored RNA,AR-RV),并开展系统的初步定值、均匀性、稳定性研究。方法 人工合成核酸片段QβSNRV,该片段5′端至3′端依次为Qβ噬菌体成熟酶编码基因、衣壳蛋白编码基因、包装位点序列、RV检测靶标cDNA序列、多克隆位点序列,并将其克隆到pET-28a(+)载体中构建重组质粒pET-QβSNRV。将pET-QβSNRV转化大肠埃希菌BL21 (DE3)并诱导表达,利用氯化铯密度梯度超速离心、丙烯葡聚糖凝胶层析纯化AR-RV后电镜观察,并参照GB/T 15000.3—2008《标准样品工作导则(3) 标准样品 定值的一般原则和统计方法》规定的方法与原则对制备的AR-RV开展定值、均匀性和稳定性研究。结果 十二烷基硫酸钠-聚丙稀酰胺凝胶电泳(SDS-PAGE)结果证实重组质粒在大肠埃希菌中有目的条带表达,大小约为14.1 kDa;经氯化铯密度梯度超速离心、丙烯葡聚糖凝胶层析纯化的AR-RV无杂蛋白干扰;电镜下可见结构完整的病毒样颗粒,大小约为25 nm;定值结果显示,AR-RV中检测靶标RNA的含量为(1.02±0.3)×107 copies/μl;均匀性分析结果为F=0.66<F0.05(9,0),表明样品均匀性良好;稳定性结果表明,AR-RV在37 ℃可保存15 d、25 ℃可保存15 d、4 ℃可保存50 d、-20 ℃至少可保存270 d、-80 ℃至少可保存360 d。结论 本研究基于Qβ噬菌体制备的AR-RV拷贝数高,均匀性和稳定性良好,可为RV分子检测提供安全、稳定的标准参考样品。

    Abstract:

    Objective To construct armored RNA reference material containing target RNA of group A Rotavirus (RV) based on Qβ bacteriophage, to determine the value and to test the homogeneity and stability of such material. Methods DNA fragment named QβSNRV which contained maturase-coding gene, capsid protein-coding gene, packing site of Qβ bacteriophage, detection target sequence of group A Rotavirus and multiple clone sites from the 5′ end to the 3′ end was synthesized, and then subcloned into pET-28a (+) expression vector. The recombinant plasmid was pET-QβSNRV that was identified by enzyme digestion and sequencing, and then transformed into Escherichia coli BL21 (DE3) competent cells and expressed. The expressed product, virus-like particles of Qβ bacteriophage containing RNA of RV, named AR-RV, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). AR-RV was centrifuged and purified by CsCl density gradient ultracentrifugation and sephacry gel chromatography. The morphology of AR-RV was observed by transmission electron microscopy. The valuation, homogeneity and stability of AR-RV were tested according to the GB/T 15000.3-2008. Results SDS-PAGE analysis showed that the molecular mass of the expressed protein product was about 14.1 kDa. The virus-like particles of AR-RV, 25 nm in diameter, with typical morphology could be observed under electron microscope. AR-RV samples prepared in this study were valued as (1.02±0.3)×107 copies/μl and behaved well in the homogeneity test, F=0.66<F0.05(9,0). The stability test indicated that the sample was stable at 37 ℃ for 15 days, 25 ℃ for 15 days, 4 ℃ for 50 days, -20 ℃ for at least 270 days, -80 ℃ for at least 360 days with no significant decrease. Conclusion The group A Rotavirus armored RNA based on Qβ bacteriophage was successfully prepared and had good uniformity, stability and high copy number. This method could supply with a good and biologically safe reference material candidate for the Rotavirus virus RNA detection.

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张奇,逄凤娇,江艳华,李风铃,姚琳,王联珠,谭志军,翟毓秀.基于Qβ噬菌体的A群轮状病毒装甲RNA标准参考样品的研制[J].中国食品卫生杂志,2018,30(4):346-352.

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  • 收稿日期:2018-04-10
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  • 在线发布日期: 2018-08-16
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