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(SDSPAGE)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)×107copies/μl;均匀性分析结果为F=0.66<F0.05(9,20),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。
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 Qβbacteriophage,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-28 a（ ＋） 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 productwas 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（ 9,20）. 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.
作者
张奇
逄凤娇
江艳华
李风铃
姚琳
王联珠
谭志军
翟毓秀
ZHANG Qi;PANG Feng-jiao;JIANG Yan-hua;LI Feng-ling;YAO Lin;WANG Lian-zhu;TAN Zhi-jun;ZHAI Yu-xiu(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)
出处
《中国食品卫生杂志》
北大核心
2018年第4期346-352,共7页
Chinese Journal of Food Hygiene
基金
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)
