Expression of norovirus G Ⅱ.4 GZ121 strain P protein and analysis of its binding activity with HBGAs receptor / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct a prokaryotic expression system of norovirus (NoⅤ) G Ⅱ-4 strain P protein (P particle and P dimer) and to explore its binding activity and patterns with HBGAs receptor.Methods P domain sequence of GZ121 NoⅤ ORF2 gene was cloned and its phylogenic tree was constructed to identify the gene cluster.The pGEX-4T-1-based expression plasmids were constructed respectively by inserting P domain gene fragments with hinge and P-CDCRGDCFC without hinge,and then transformed into BL21 to express fusion proteins,which was induced with 0.6 mmol/L IPTG at 22℃ overnight.P proteins were purified by thrombin cutting and characterized by FPLC.The binding patterns of NoⅤ P protein to HBGAs antigens were analyzed by EIA.Results P region gene of GZ121 belonged to genotype G Ⅱ.4/2004 cluster.SDS-PAGE analysis showed the relative molecular weight of P particle and P dimer was about 36×103,which was consistent with other reports.The peak appeared at 830×103 confirmed the formation of P particle by FPLC.The expression of P protein was further confirmed by Western blot.The EIA results showed that GZ121 P protein could bind to saliva of A-group,B-group and O-group secretors,but not to nonsecretor.The binding affinity of P particle was 80-100 times higher than that of P dimer.Compared with VA387 P particle,it showed stronger binding affinity to O-group,but weaker to A-group.Conclusion The NoⅤ GⅡ-4 GZ121 P proteins including P particle and P dimer were successfully expressed and HBGAs receptor binding assays were established.This pave the way for further studies on the evolution dynamics of NoⅤ G Ⅱ.4 strains and the development of NoⅤ vaccines.