Screening And Preliminary Identification Of RHDV Capisd P2 Domain Interacting Proteins In Rabbit Principal Target Cells

Rabbit hemorrhagic disease(RHD)is a devastating infectious disease,and does serious harm to the rabbit industry.Rabbit hemorrhagic disease virus(RHDV)is the pathogen of RHD,its capsid protein VP60 P2 sub-region may play an important role when RHDV adsorb and invade the host cells.It is important that P2 as bait protein was used to research pathogenesis of RHDV.Rabbit liver cells,spleen cells,kidney cells are the principal target cells of RHDV.Utilizing Co-immunoprecipitation experiment(Co-IP)and mass spectrometry to screen P2-interacting proteins in main RHDV-target cells can provide clues for further research of infection process and pathogenesis of RHDV.The main contents are as follows:1.The expression and identification of RHDV capsid P2 bait proteinWe used the gene of RHDV(FJ794180,RHDVa type)structural protein VP60 as a template to design primers and then to amplify P2 sub-region gene.P2 sub-region gene was subcloned to pET-32a(+)expression vector to constructed pET-32a-P2 recombinant vector.Then pET-32a-P2 recombinant vector was transfected into E.coli BL21 and induced by IPTG.We obtained soluble recombinant P2 protein with high expression levels.Then we utilized western blotting to identify its specificity and His TrapTM HP column to purify protein P2.2.Screening and preliminary identification of RHDV capsid P2 domain interacting proteins in rabbit principal target cellsWe isolated primary rabbit liver cells,spleen cells and kidney cells from healthy non-immunized rabbits over 2 months old.P2-interacting proteins from these cells was screened by co-immunohistochemistry(Co-IP)and mass spectrometry analysis.These screened proteins were analyzed by GO,KOG,KEGG bioinformatics analysis.The results are as follows:(1)we screened 67 P2-interacting proteins in liver cells,169 P2-interacting proteins in spleen cells and 100 P2-interacting proteins in kidney cells.Among these proteins,we found 6 proteins screened in both liver cells and spleen cells,11 proteins screened in both liver cells and kidney cells,17 proteins screened in both kidney cells and spleen cells.It's interesting that the protein Laminin receptor(LamR),which found in both liver cells and spleen cells,is a receptor for several virus.GO annotation showed the distribution of P2-interacting candidate proteins in three kinds of target cells are roughly similar.These proteins are mostly localized in the cytoplasm,organelles and cell membrane;most of them are involved in cellular processes,metabolic processes and a single organism processes;most of these proteins have catalytic activity and binding activity.KOG classification displayed that most of these proteins were grouped into lipid transport and metabolism,General function prediction only,post-translational modification,protein turnover,chaperones,amino acid transporter metabolism,energy production and conversion.KEGG pathways enrichment showed the number of proteins enriched in metabolic pathways are much more than those enriched in other pathways.3.Preliminary identification of interactions between LamR with RHDV and its proteinLaminin receptor(LamR)protein,which identified in both rabbit liver cells and spleen cells as P2-interacting candidate protein,has a wide range of physiological and pathological functions.LamR is a receptor for several virus.To further confirm the interaction between LamR and P2 proteins,we have conducted research:(1)we verified co-localization of LamR and P2 proteins in both liver cells and spleen cells by confocal laser experiment.(2)We expressed LamR fusion protein.GST pull-down tests showed the direct binding of LamR and P2 protein.Immunofluorescence experiments showed that purified GST-LamR protein was capable of blocking the binding of P2 and rabbit hepatocytes.In addition,this blocking were affected by the ratio of LamR/P2 protein concentration.(3)We bring the study back to intact RHDV.For in vitro experiment,we used confocal experiment to prove colocalization between RHDV intact particles and LamR in liver cells.For in vivo experiment,We isolated RHDV infected rabbit liver cells after infected 24 h and found co-localization of virus and LamR by confocal.We confirmed the interaction between LamR and P2 or complete RHDV and we think that LamR influence the infection of RHDV.it's worth further study to confirm the role of LamR when RHDV infected rabbits.We provide clues for the study of infection and pathogenic mechanism of RHDV.