基于Qβ噬菌体的甲肝病毒装甲RNA标准参考样品的研制

Armored RNA reference material of hepatitis A virus based on Qβ bacteriophage

【背景】目前食品中甲肝病毒分子的检测缺乏安全、稳定的RNA标准参考样品,影响了检测结果的科学性与准确性。【目的】基于Qβ噬菌体装甲RNA技术构建内含甲肝病毒检测靶标的装甲RNA (Hepatitis A virus armored RNA,AR-HAV),并开展初步定值、均匀性、稳定性研究,为HAV分子检测提供标准参考样品。【方法】人工合成包含Qβ噬菌体成熟酶编码基因、衣壳蛋白编码基因、包装位点、HAV检测靶标cDNA序列的核酸片段QGBHAV,并亚克隆到pET-28a(+)中构建重组质粒pET-QGBHAV,转入大肠杆菌BL21(DE3)感受态细胞进行原核表达,利用超速离心、丙烯葡聚糖凝胶层析柱纯化AR-HAV后电镜观察。通过实时荧光RT-PCR对AR-HAV进行初步定值及均匀性和稳定性研究。【结果】SDS-PAGE结果表明重组质粒在大肠杆菌中有约14.1 kD的目的蛋白表达;纯化后的AR-HAV无杂蛋白和残留质粒;电镜下可见结构完整、大小约为25nm的病毒样颗粒;定值结果显示,AR-HAV中检测靶标RNA的含量为(2.57±0.12)×107 copies/μL;均匀性分析结果为F=1.23<F0.05 (9,20),证实AR-HAV的均匀性良好;稳定性结果表明,AR-HAV在可37°C保存9 d,25°C保存25 d,4°C保存40 d,-20°C保存180 d,-80°C至少保存360 d。【结论】基于Qβ噬菌体制备的AR-HAV拷贝数高,具有良好的均匀性和稳定性,可为HAV的分子检测提供安全、稳定的标准参考样品。

[Background]The stable RNA reference material without biohazard to improve the accuracy and reliability of the detection result,is urgently needed for hepatitis A virus(HAV)detection in food.[Objective]To construct armored RNA reference material containing target RNA of HAV(HAV armored RNA,AR-HAV)based on Qβbacteriophage,and to test its homogeneity,valuation and stability.[Methods]DNA fragment named QGBHAV containing matures coding gene,capsid protein coding gene,packing site of Qβbacteriophage,and detection target sequence of HAV in GB/T 22287-2008 was synthesized,and subcloned into pET-28 a(+)expression vector to construct the recombinant plasmid pET-QGBHAV,and then transformed into Escherichia coli BL21(DE3)competent cells and expressed with isopropyl-β-thiogalactopyranoside(IPTG)induction.The expression product,virus like particles of Qβbacteriophage containing RNA of HAV,named AR-HAV,was analyzed by SDS-PAGE.AR-HAV was centrifuged and purified by CsCl density gradient ultracentrifugation and Sephacry molecular sieve chromatography.The morphology of AR-HAV was observed by transmission electron microscopy.The valuation,homogeneity and stability of AR-HAV were tested according to the GB/T 15000.3-2008.[Results]SDS-PAGE analysis showed that the molecular mass of the expressed protein was about 14.1 kD.The virus like particles of AR-HAV,25 nm in diameter,with typical morphology could be observed under electron microscope.AR-HAV samples prepared in this study had no other proteins nor recombinant plasmid DNA residual contamination,were valued as(2.57±0.12)×107 copies/μL and behaved well in the homogeneity test,F=1.23<F0.05(9,20).The sample was stable at 37°C for 9 days,at 25°C for 25 days,at 4°C for 40 days,-20°C for 180 days,at-80°C for at least 360 days without significant decrease.[Conclusion]The HAV armored RNA based on Qβbacteriophage was successfully prepared and had good uniformity,stability and high copy number,could be supplied as a good and secure reference material candidate for HAV nucleic acid detection.