Preparation of Coupled Armored RNA Reference Material for Norovirus GI/GII
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Abstract:
To provide a safe and stable reference material for Norovirus nucleic detection, the armored RNA containing target RNA of Norovirus GI and GII based on MS2 bacteriophage was developed in this work. DNA fragments including maturase coding gene, capsid protein coding gene and packing site of MS2 bacteriophage, target cDNA sequence of Norovirus GI and GII were synthesized artificially and then cloned into expression vector pET-28a(+) to construct recombinant plasmid pET-MS2-NoV. After expressed in E. coli BL21 cells by IPTG induction, the expression product was purified by PEG6000, enzyme digestion and molecular sieve chromatography. The purified product, also named AR-NoV, was identified by SDS-PAGE, transmission electron microscopy (TEM) and RT-PCR. The value, homogeneity and stability of the AR-NoV were evaluated. SDS-PAGE analysis showed that with the molecular weight of tar-get protein expressed in BL21 was 10~15 ku, which was consistent with the predicted value. There were no impure proteins and residual nucleic acids in AR-NoV after purification. The AR-NoV presented as spherical VLPs with uniform particle size (about 25 nm) and integrated structure under TEM. The values of GI and GII targets in AR-NoV were (4.04±0.62)×107 copies/μL and (6.16±0.30)×107 copies/μL, respectively. The good homogeneity of AR-NoV was confirmed by single-factor ANOVA test (F
