Construction And Functional Identification Of Bacterial Surface Displayed Human Noroviruses Capsid Proteins

Human noroviruses(HuNoVs)are major contributors to acute nonbacterial gastroenteritis outbreaks.Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs,and lack of efficient small animal models.Surrogates for HuNoVs,such as recombinant viral like particles(VLPs)expressed in eukaryotic system or P particles expressed in prokaryotic system,have been used for studies in immunology and interaction between the virus and its receptors.However,it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins.To solve this problem,a new system was constructed through the use of ice nucleation protein(INP)to display recombinant capsid proteins of HuNoVs on bacterial surfaces.In this system,the VP1 capsid encoding gene(ORF2)and the protruding domain(P domain)encoding gene(3' terminal fragment of ORF2)of HuNoVs GI.1 and GII.4 were fused with 5' terminal fragment of ice nucleation protein encoding gene(inaQn).The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids.Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors after testing by whole cell immunoassay,measuring the HBGAs binding abilities and membrane proteins-HBGAs-ELISA,and all of them showed significantly higher signals than recombinant VP1 and P domain of HuNoVs without protein InaQN(p<0.01).In both cases,displayed P domains had significant different binding abilities than VP1(p<0.01).This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples,to investigate interaction between the virus and its receptors,as well as to develop an oral vaccine for HuNoVs.