Construction And Application Of The Surface Displayed Human Noroviruses System

Human noroviruses(HuNoVs)are the dominant cause of food-borne outbreaks of acute gastroenteritis.GII.4 is epidemic strain.Lettuce and oysters are the main carriers of HuNoVs.However,fundamental research on HuNoVs has been limited by the currently immature system to culture HuNoVs and the lack of efficient small animal models.Therefore,the discovery of receptor / ligand of HuNoVs is very important to overcome the bottleneck.In this study,an upgraded bacterial surface displayed system was developed as a new platform to discover candidate receptors of HuNoV(GII.4).A thrombin-susceptible “linker” sequence was added between the sequences of bacterial transmembrane anchor protein and P domain of HuNoV(GII.4)capsid protein in a plasmid that displays the functional P proteins on the surface of bacteria.In this new system,the surface-displayed HuNoV P proteins could be released by thrombin treatment.The released P proteins self-assembled into small particles.The P proteins could bind and isolate the histo-blood group antigens(HBGAs),which have always been considered as specific receptors of HuNoVs.The bacteria with the surface-displayed P proteins were incubated with pig stomach mucin which contained HBGAs,The HuNoV P proteins-HBGAs complex was then separated from the recombinant bacterial surface by thrombin treatment.ELISA results showed the system could capture and easily isolate type A HBGA.We used this upgraded bacterial surface displayed system to mine the receptors or ligands of HuNoV(GII.4)in romain lettuce.Receptor-P-protein complex was released by the thrombin.ELISA analysis showed that the ligand in lettuce could be recognized by antibodies against type A?H1 and Lewis a HBGA.The results of oxidation experiment showed that the leaves and veins of the lettuce contain HuNoV(GII.4)polysaccharide ligands.The HPLC results showed that the polysaccharide ligand of HuNoV(GII.4)in lettuce was composed of mannose,rhamnose,galactose,glucose,Arabia sugar and xylan.In conclusion,the upgraded bacterial surface displayed system can be used for the discovery and analysis of viral receptors in HuNoVs host cells,and providing technical support for the construction of in vitro proliferation model in the future.