Expressing The P Particles Of GⅡ-4Norovirus And Their Binding To Human Histo-Blood Group Antigens

Objective:Cloned the P domains gene of GⅡ-4norovirus five strains since1991, preparedthe P particles according to expressing and purifying the P proteins. The bindingpatterns were determined by EIA experiments between the different P particles andthe histo-blood group antigens(HBGAs) in order to determine the host range ofnorovirus and prevent the infection.Methods:Selected five GⅡ-4norovirus strains and cloned the ORF2domains. ORF2domain sequences were as the templates for designing specific primers andamplifying P domains, respectively. The cDNAs encoding the P domain without thehinge were cloned into the PGEM-Teasy vector. The PGEM-Teasy-P and expressionvector PGEX-4T-1were digested by BamH I and Not I, and the P domain were clonedinto the PGEX-4T-1. Briefly, strain BL21（DE3）of E.coli was used as the host, andthe bacterial cultures were induced by IPTG. Purification of P protein from wasperformed using glutathione SepHarose4B according to the manufacturer’s protocol.The P proteins were released from GST by thrombin digestion. The saliva sampleswere collected from Shenzhen (with well-defined A, B, O, secretor).The bindingpattern of HBGAs with NVs was confirmed by EIA assay on the rabbit serum anti-NV,horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and TMB kit.Results:1. Amplified P domains of five GⅡ-4norovirus strains and constructed theexpression plasmids PGEX-4T1-P, successfully.2. Using the prokaryotic expression system, the expressed P proteins of all5 GII-4strains formed38KD P particles, which were demonstrated by SDS-PAGE.3. The binding patterns between GⅡ-4NVs and HBGAs were determined, TheP particles of Camberwell,95/96US and2006b were suggested by their strongbinding to the saliva of A, B, and O secretors but not to nonsecretors. Hunter, Sakaiwere not binded to A, B, O secretors, and O nonsecretors.Conclusions:NVs recognized HBGAs in a strain-specific manner. The P particles ofCamberwell,95/96US and2006b were suggested by their strong binding to the salivaof A, B, and O secretors but not to nonsecretors. Hunter, Sakai were not binded to A,B, O secretors, and O nonsecretors.