| Human noroviruses（HuNoVs）are the worldwide principal cause of acute non-bacterial gastroenteritis in human beings.They have a significant impact on food safety and public health.The receptors of HuNoVs are poorly understood due to the lack of stable cell lines for culturing HuNoVs and the efficient small animal models.The current research on the interaction between HuNoVs and their receptors/co-receptors relies mainly on in vitro binding experiments using the huNoVs alternatives,including recombinant HuNoVs capsid proteins,such as virus-like particles（VLPs）and P particles（P particles）,and cultivable Caliciviruses,such as Tulane virus（TV）.The TV can be cultured in vitro and,like HuNoVs,is an enterovirus that causes gastrointestinal diseases.It recognizes human tissue blood group antigens（HBGAs）and sialic acid（SA）as receptors or ligands for invading viruses.Because of those advantages,TV becomes one of the best surrogates in studying the interaction between HuNoVs and their receptors.Based on the previous studies,the P domain of the TV capsid protein（TV-P）was anchored to the surface of Escherichia coli by gene recombination and bacterial cell surface display technology,using the protein encoding gene（ina Qn）as an anchoring motif.A sequence which can be hydrolyzed by bovine thrombin was inserted between the Ina QN and the TV-P as a ligation fragment to form a recombinant plasmid.The recombinant plasmid was transferred into E.coli BL21 cells to construct the Tulane virus capsid protein bacterial surface display system.This system was about a hundred times volume magnification compared with the virus size.The system was induced to express and anchor the TV-P to the surface of the bacterial cells.After treatment with thrombin,the surface displayed TV-P could be separated from the E.coli cells by simple centrifugation.The TV-P was purified to prepare the rabbit anti-TV-P antiserum.Results of Western-blot experiments showed that TV-P was displayed on the surface of E.coli bacteria,and the surface-displayed TV-P could be separated from E.coli by thrombin treatment.Furthermore,the results of ELISA experiments indicated that the isolated TV-P had the ability to recognize and bind to TV-specific receptors,HBGAs and SA.These results demonstrated that the TV-P,prepared by this system maintained the antigenicity and binding ability with its receptors.The system of Tulane virus capsid protein bacterial surface display was further incubated with the rhesus monkey kidney cell line（LLC-MK2）lysate which was treated by boiling（denaturing the proteins）and NaIO4（oxidizing the sugars）,respectively.Then,an improved ELISA was performed.The receptors of TV capsid protein were polysaccharides（p<0.01）rather than proteins（p>0.05）.Moreover,The TV receptors in LLC-MK2 were further captured by this system.The complex of the TV-P-viral ligands were obtained after digestion by thrombin.The results indicated that the monosaccharides binding to TV in LLC-MK2 cells were probably ribose,galactose and glucose by UPLC-MS/MS analysis.In summary,the Tulane virus capsid protein bacterial surface display system can be used for the discovery and analysis of viral receptors in host cells,which provides theoretical basis for further exploration and understanding of HuNoVs receptors.The establishment of this system can also provide new ideas and tools for the industrial preparation of viral vaccines. |
