Analysis Of Interaction Proteins Of Porcine Epidemic Diarrhea Virus Infecting Cells

PEDis a highly contagious and infectious disease of piglets with diarrhea caused by PEDV.Its pathogen is single stranded positive strand RNA viruses with a high mutation rate,which makes the diagnosis and prevention more difficult.In this research,we collect diarrheal diseases samples from several farms in Shanghai,the Vero cells are inoculated with the new strain SHpd/2012 of PEDV that isolated by our laboratory.Finally we find that the new strain can infected Vero cells after the cytopathic effect experiment and the indirect immunofloresence assay.We get the gene sequence of S1 and S2 gene by sequence alignment,then design primers and add two enzyme loci and a flag label of these two genes by oligo6.0.The eukaryotic expression plasmidpCAGGS-S1 and pCAGGS-S2 are constructed by cloning S1 and S2 gene in eukaryotic expression vectors pCAGGS respectively.The recombinant plasmids are transfected into Vero cells respectively by liposome transfection reagent,and after the transfection of 24h/48h/72 h an immunofluorescence assay was performed to verify whether the recombinant plasmids could express in Vero cells.Once we confirmed that the expression was successful,we obtain large amount of fusion protein by extract the cellular protein after transfection of 48 h.The fusion protein that extracted from cells is used for flag-pulldown experiment with anti-flag affinity gel.After that we collect the gel for protein electrophoresis and then measure whether we have pull down the interacting protein by silver staining.Cut the silver stain protein strip belt and then analysis it by mass spectrum analyzer,and then we get 268 proteins of S1 group and 291 proteins of S2 group after filter.We use the proteins for Gene Ontology analysis and KEGG Pathway analysis,and get the protein interaction net work diagram from string datebase,preliminarily determined the range of protein interaction that may be related to protein S when PEDV infectVero cells.The identification of protein enriched the Vero cell proteomics databases,laid the important foundation for further study for the new strains infect cells and the mechanism of pathogenesis,as well as an indispensable part of the diagnostic work stepfor disease prevention and control.