Glycan Binding Specificity And Antibody Development Of Human Norovirus

Objective: To investigate the glycan binding specificity of norovirus capsid protein of different genotypes;To produce the monoclonal antibodies against the norovirus capsid protein and screen the antibodies with specific binding,providing the basis for establishing norovirus detection methods.Methods:(1)P particle proteins of different norovirus genotypes were obtained using the Escherichia coli protein expression system.Soluble P proteins were purified by the affinity chromatography;(2)The VP1 gene fragments of GⅡ.4 and GⅡ.6 noroviruses were ligated to the p Fast Bac1 vector to obtain the recombinant plasmids respectively.The recombinant plasmid was transfected to insect cells and the recombinant virus was harvested one week later.The recombinant virus was amplified and used to infect insect cells to express VLP protein.The VLP was purified through PEG precipitation and sucrose density gradient centrifugation;(3)The glycan binding characteristics of different P proteins and VLPs were depicted by the functional experiments such as oligosaccharide binding and saliva binding assay;(4)The polyclonal antibodies against GⅡ.4 was generated by immunizing rabbits with GⅡ.4 VLP;Mouse monoclonal antibodies against GⅡ.4 were obtained by immunizing mice with G Ⅱ.4 P protein and the antibodies with specific binding was screened.Results:(1)P particle proteins of different norovirus genotypes were obtained;Both P particles of GⅡ.4 Hong Kong strain(GⅡ.4 HK)and GⅡ.4 Sydney strain(GⅡ.4 Sydney)bind to human type A,B,AB,and O saliva and also show good binding to H disaccharides(Fucα1-2Gal)according to the functional experiments.GⅡ.8 P protein has no obvious binding to the detected oligosaccharides,but presents binding with some human A/B/O type saliva samples.GⅡ.9 P protein binds to H disaccharide(Fucα1-2Gal)and shows interactions with human A/B/O type saliva.(2)GⅡ.4 and GⅡ.6 VLP proteins were prepared using the baculovirus expression system.GⅡ.4 VLP and GⅡ.6 VLP proteins recognize H disaccharides(Fucα1-2Gal)and show no obvious binding to other detected oligosaccharides.(3)Rabbit polyclonal antibodies with high titer were obtained after immunization with GII.4 VLP in rabbits;Two mouse monoclonal antibodies were screened that presented specific binding to both GⅡ.4 P protein and GⅡ.4 VLP by immunizing mice with GⅡ.4 P particle protein.Conclusion: In this research,P particles and VLP proteins of various norovirus genotypes were successfully expressed and purified;The glycan binding characteristics of P particle proteins of different GⅡ.4,GⅡ.8,and GⅡ.9 strains were clarified;Polyclonal antibodies and monoclonal antibodies against GⅡ.4 were obtained.This study provide more understanding of the glycan receptor binding characteristics of different noroviruses and lay a foundation for establishing specific detection method of noroviruses.