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

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

目的：针对目前检测领域缺乏诺如病毒（Norovirus,No V）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No V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No V检测靶标对应的c DNA序列及辅助多克隆位点的DNA片段QINVGII,将其克隆到p ET-28a（＋）载体中,构建重组质粒p ET-QINVGII。将p ET-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14.1k Da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No V检测靶标RNA的含量为（1.06±0.06）×107copies/μl;均匀性分析结果表明样品均匀性良好,即F=2.24< F0.05 ( 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。

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/p,l and behaved well in the homogeneity test, F = 2.24 〈 F0.05 （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.