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

摘要:

【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25 nm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

Abstract:

[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 Qβ bacteriophage, and to test its homogeneity, valuation and stability. [Methods] DNA fragment named QGBHAV containing matures coding gene, capsid protein coding gene, packing site of Qβ bacteriophage, and detection target sequence of HAV in GB/T 22287-2008 was synthesized, and subcloned into pET-28a(+) 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 Qβ bacteriophage 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