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

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

Key words:Hepatitis A virus, Qβ Bacteriophage, Armored RNA, Reference material, Real-time fluorescence RT-PCR