Development Of Armored RNA Reference Material Of Rotavirus And Astrovirus Based On Q? Bacteriophage

To construct Armored RNA reference material containing target RNA of rotavirus or astrovirus food borne virus based on Q? bacteriophage,for food borne virus nucleic acid detection.DNA fragment named Q?SNASTV,Q?SNRV containing matures coding gene,capsid protein coding gene and packing site of Q? bacteriophage,and c DNA corresponding to detection target RNA sequences of RV or Ast V was synthesized and subcloned into p ET-28a(+)expression vector.The recombinant plasmid were named p ET-Q?SNRV?p ET-Q?SNASTV.The p ET-Q?SNRV,p ET-Q?SNASTV were successfully constructed through enzyme digestion reaction and sequencing identification respectively.The p ET-Q?SNRV,p ET-Q?SNASTVwere transformed into E.coli BL21(DE3)competent cells and expressed.The best expression condition of rotavirus was 0.2 m M of final IPTG concentration and 10 h induction in the temperature of 37 ?.The best expression condition of astrovirus was 0.2 m M of final IPTG concentration and 10 h induction in the temperature of 37??The expression protein Virus Like Particles(VLPs)was purified by Cs Cl density gradient ultracentrifugation and Sephacryl molecular sieve chromatography after analysis by SDS-PAGE.The VLPs was efficiently expressed in E.coli with molecular mass of 14.1 k D shown on SDS-PAGE.The VLPs,25 nm in diameter,with typical morphology could be observed under electron microscope.The purified product named AR-RV,AR-ASTV respectively.The AR-RV was preliminary quantified as(1.02±0.3)×107 copies/?Land behaved well in the homogeneity test,p=0.73>0.05.The stability test indicated that the sample was stable at 37? for 12 days,at 25?for 15 days,at 4? for at least 50 days,-20? for at least 270 days,at-80? for at least360 days with no significant decrease.With the standard of GB/T 1500.3-2008,the AR-ASTVwas preliminary quantified as(5.33±0.44)×106 copies/?L and behaved well in the homogeneity test,p=0.08>0.05.The stability test indicated that the sample was stable at 37? for 15 days,at 25?for 20 days,at 4? for at least 40 days,-20? for at least 270 days,at-80? for at least 360 days with no significant decrease.DNA fragment named Q?Mul V containing matures coding gene,capsid protein coding gene and packing site of Q? bacteriophage,and c DNA corresponding to detection target RNA sequences of No V(genotype GI and GII),RV,Ast V,and HAV was synthesized and subcloned into p ET-28a(+)expression vector.The recombinant plasmid were named p ETQ?Mul V.The p ET-Q?Mul V were successfully constructed through enzyme digestion reaction and sequencing identification respectively.p ETQ?Mul V were transformed into E.coli BL21(DE3)competent cells and expressed.The best expression condition of multiplex foodborne viruses was 0.2 m M of final IPTG concentration and 11 h induction in the temperature of 37??The expression protein Virus Like Particles(VLPs)was purified by Cs Cl density gradient ultracentrifugation and Sephacryl molecular sieve chromatography after analysis by SDS-PAGE.The VLPs was efficiently expressed in E.coli with molecular mass of 14.1 k D shown on SDS-PAGE.The VLPs,25 nm in diameter,with typical morphology could be observed under electron microscope.The AR-Mul V was preliminary quantified as(1.24±0.2)×107,(2.54±0.6)×107,(2.24±0.3)×107,(2.96±0.5)×107and(3.19±0.4)×107 copies/?L for No V(GI and GII),RV,Ast V and HAV respectively.The AR-Mul V behaved well in the homogeneity test,p=0.45 < 0.05.The stability test indicated that the sample was stable at 37? for 12 days,at 25?for 15 days,at 4? for at least 150 days,-20? for at least 150 days,at-80? for at least15 0days with no significant decrease.The reference material of GII for No V(AR-GII)and ar-Mul V were added lyophilization reagent than tested the stability.The AR-GII of lyophilization was stable at 37? for 15 days,at 25?for 80 days,at 4? for at least 90 days,-20? for at least 90 days,at-80? for at least90 days with no significant decrease.The AR-Mul V of lyophilization was stable at 37? for 15 days,at 25?for 50 days,at 4? for at least 90 days,-20? for at least 90 days,at-80? for at least90 days with no significant decrease.The armored RNA based on Q? bacteriophage was successfully prepared in this study,which could serve as a could supply with a good and security reference material candidate for the foodborne virus RNA detection.