Preliminary Identification Of Receptor Binding Domain Of Porcine Hemagglutinating Encephalomyelitis Virus S Protein

Porcine hemagglutinating encephalomyelitis coronavirus (PHEV) is a member ofthe Coronaviridae family, which causes porcine encephalomyelitis. PHEVpredominantly affects piglets. When PHEV infects the piglets, its transmission speedwill be quick and with a high mortality rate. The major structural proteins of the virusnamed small membrance(E), membrance(M),spike(S),nucleocapsid (N) protein andhemagglutinin-esterase (HE).Base on the study of other coronavirus, the Coronavirus Spike (S) protein isdivided into S1functional domain and S2functional domain.The S1functionaldomain initiates entry of viruses into cells by binding to cell surface receptors, and theS2functional domain helps viral fusion with cellular membranes. S1function domaincontains the receptor binding domain (RBD). Given this, it is necessary that screeningand identification of the RBD to provide some valuable information for developmentof diagnosis technology and novel vaccine of PHEV.According to the presence of conserved nonamer and the GxCx motifs at theproteolytic cleavage site of S protein in other members of coronavirus, PHEV Sprotein was divided into S1and S2function domain, The1-794aa of PHEV S proteinis the S1and the795-1349aa is the S2.Therefore, based on standard of coronavirus Sprotein, and compared with some other coronavirus S protein amino acid sequence, Sprotein is with strong hydrophilic ability, and there are10domains maybe combinewith the receptor accessibility on the S1.Considering to accessibility domains predicted, the S1functional domains ofPHEV S protein was divided into three truncated fusion proteins, S1-291、S277-794andS548-868.The plasmids pGEXS1-291, pGEXS277-794, pGEXS548-868were individuallytransformed in E. coli BL21cells separately and three truncated fusion proteins wereexpressed successfully.In the preliminary work of our project group, it has found thatthe neural cell adhesion molecule (NCAM) interacts with S1functional domain of PHEV S protein.And it was confirmed that NCAM participates in the process bywhich PHEV infects neurons.NACM may be a receptor for PHEV in N2acells.Therefore, pET-NC was transformed in E. coli BL21cells and the NCAM genewas expressed successfully,too.To screened the domains that could interact with NCAM,this study used GSTpull down and the interaction was further confirmed by a yeast wo-hybrid systemassay. The results showed that S277-794were identified to interact with NCAM,whileS1-291and S548-868not.S277-794is located in S1subunit. In addition,because S1-291orS548-868could not interact with NCAM, and there are overlapping region betweenS277-794and S1-291as well as S277-794and S548-868, we conclude that the S1subunit maycontain a minimal cellular receptor binding region. These findings suggested that the258-amino-acid fragment, residues291to548, may be the NCAM RBD of the Sprotein.Finally, a small fragment (258-amino-acid fragment, residues291to548) onthe PHEV S protein was posited to be the minimum number of amino acids necessaryto interact with NCAM, and it is very likely to the RBD that mediates PHEV bindingto NCAM.In order to further analysis for fragment, His fusion gene of the S291-548wasexpressed in BL21. The mixture contained S291-548and NCAM was incubated withS277-794-GST immobilized Glutathione Sepharose4B.The inhibiting protein interactionGST pull-down experiment shows that S291-548completely blocked the bindingbetween S277-794and NCAM，further proved that S291-548is able to interact with theNCAM.The polyclonal antibody of S291-548was prepared with immunized rabbit. Aftermixing the prepared polyclonal antibody and PHEV, and then was inoculated to theneural cells with the HEV. At48h after the inoculation, the hemagglutination titer ofthe HEV in the supernatant fluid of the cell culture was1:22test group,and thehemagglutination titer of HEV in the supernatant fluid of the cell culture was1:24inthe control group. A difference of two gradients existed between the hemagglutinationtiters of the two groups, thus the difference was significant.Results showed thatS291-548protein antibodies could inhibit the proliferation of PHEV in the cell. Theseresults provided evidences that S291-548may be a potential of NCAM receptor binding domain. Meanwhile,these data could provide the basis for the development ofidentifying the effective target epitope for a S protein-based vaccine and furtherstructure and function analysis of S protein.