Study Of Mechanism Of Cell Entry By Porcine Epidemic Diarrhea Virus Spike Protein

Porcine epidemic diarrhea virus(PEDV),as a pathogen of porcine epidemic diarrhea,can cause piglets vomiting,dehydration,acute enteritis and watery diarrhea after being infected with piglets.The spike(S)protein mediates PEDV entry into host cells,which mediates the essential functions of receptor binding and interaction between virus and membranes during cell entry.Proliferation of PEDV in vitro cell lines need to add trypsin in the culture medium to make full use of fusion function of spike protein and promote virus proliferation.It is of great significance to study the mechanism of S protein during the process of PEDV infect the host cells,and to further elaborate the mechanism of PEDV infection from the virus and the receptor interaction level.IFA(Immunoflorescent assay)and growth curves were employed to study the characteristics of Vero cells infected by SHpd/2012 strain of PEDV(porcine epidemic diarrhea virus).The infected cells succeedingly became swell,round,shrink,gathered into syncytial body and even detached from the cell bottle.IFA showed that after inoculation,the virus began to proliferate at 12 h,reached the replication peak at 36 h,then the proliferation ability decreased and tended to be steady at 60 h.Growth curves showed that,SHpd/2012 proliferated at high speed within the first 24h-60 h and 60 hours after inoculation with a decreasing trend.Besides,we succeedingly expressed S protein,which make a foundation of PEDV interaction with receptor and studying pathogenesis of virus.Up to now,porcine aminopeptidase(pAPN)has been controversial for PEDV receptors,and the interaction between PEDV and pAPN is unclear.We expressed S protein and pAPN first,and then transfection,Western,pulldown,IFA were employed to study the the interaction between the S protein of PEDV strain SHpd / 2012 and porcine aminopeptidase(pAPN).The results showed that the functional domain of PEDV strain SHpd / 2012 interacted with pAPN protein to be amino acids 249-529,and amino acids 249 and 529 were the key amino acid sites.Compared with the S gene sequence of CV777 strain,the strain used in this study had a gene mutation at the positions of 249 aa and 529 aa.The 249 aa mutated from glycine G to isoleucine I and 529 aa mutated from aspartic D to isoleucine I.We speculate that the key amino acids(isoleucine)at both ends of the interacting domains in SHpd/2012 strain play a major role in the division of the interacting domains.The identification of the both has accumulated data for the study of the virus invasion mechanism.In order to explore the interaction between S protein and pAPN protein intuitively,the phylogenetic tree analysis and SWISS-MODEL threedimensional structure prediction were used to study the homology of S gene,S protein 3-dimensional structure and the 3-dimensional structure of the S protein after interacted with pAPN.The results showed that the S protein was highly homologous to the PEDV strain in Guangdong,and the 3-dimensional structure of S protein was a trimer structure with a top pillar.The 3-dimensional structure of S protein interacted with pAPN was a big complex dimer structure.