Screening And Identification Of Binding Domain Of RHDV VP60 To Histo Blood Group Antigens

Rabbit Hemorrhagic Disease Virus(RHDV)belongs to the family Caliciviridae and is the etiological agent of the Rabbit Hemorrhagic Disease(RHD)which also known as rabbit plague.RHDV was shown to bind to the HBGA H type 2,A type 2 and B type 2 oligosaccharides.These structures were shown to be present on the surface of the epithelial cells of the upper respiratory and digestive tracts that the virus firstly encounters when infecting the host.RHDV VLP strongly agglutinated human adult erythrocytes as the result of binding to glycolipid ligands present on the erythrocyte surfaces.Subsequent studies revealed that several caliciviruses use the carbohydrate moiety of host-cell histo-blood group antigens(HBGA)for attachment(e.g.ABH/O and Lewisantigens)initiating their replication cycle.In this study,we investigated the relationship between the binding of RHDV VP60 to HBGAs and immunological protection by synthetic H type 2 blood group oligosaccharides-VLPs blocking assay.The specific phage display peptide library of Rabbit Hemorrhagic Disease Virus VP60 gene has been established successfully.Several binding domains of VP60 to HBGAs were identified employing the specific phage display peptide library.This provides clues for further explanation of RHDV infection,pathogenicity and immunological protection.The main research contents are as follows:1.Blocking effect of immune serum on VP60 VLPs binding to receptor HBGAsThe VP60 VLPs were expressed in insect cells and purified by red blood cell adsorption release test.The purified VP60 VLPs were identified by western-blot.The enzyme linked immunosorbent assay was used to detect the binding of VP60 VLPs to HBGAs in Synthetic H type 2 histo-blood group antigens.The determination of the blocking effect of VP60 VLPs binding to HBGAs by convalescent sera from type-specific rabbit antisera shall be determined.The results showed that serum samples from day 7 to day 240 post-immunization efficiently blocked the binding of H type 2 to RHDV VLPs;for each sample,only 1%(1:100)of pre-incubated serum was used.The OD values of wells containing serum samples from the vaccinated groups,from day 7 to day 240 post-immunization,significantly differed from those of the PBS-inoculated group(p<0.05).Serum samples obtained at day 7 post-immunization blocked binding of H type 2 to RHDV VLPs,with a blocking rate of almost 60%,while serum samples from day 14 and day 30 post-immunization exhibited a blocking rate of almost 75%and 85%,respectively.Serum samples from days 60 to 240 post-immunization were demonstrated to block the binding of H type 2 to RHDV VLPs at a blocking rate of almost 100%.So the immune serum blocking test can be used to replace the virus neutralization test.2.Construction and identification of specific phage peptide display librarie of rabbit hemorrhagic disease virus VP60 geneThe VP60 gene of RHDV WF/China/2007 strain was amplified by PCR and the PCR products were digested into random segments around 100 bp by DNase I random digestion method.The digested segments were blunted by T4 DNA polymerase,and then ligated with phosphorylated EcoR I linker followed by being digested with EcoR I.The digested DNA were cloned into pC89 phagemid vector which had been digested by EcoR I and treated with alkaline phosphatase.The recombinants were transformed into competent E.coli XLI-Blue cells,and co-infection with phage VCSM13.Finally,gene-targeted phage random peptide library of VP60 protein was constructed.At the same time,the randomness and titer of the peptide library were identified.Finally monoclonal antibody(MAbs)1B8,1D4,5H3 and A3C were used to screen the epitopes in VP60 library by biopanning and phage in situ hybridization.The results showed that the titer of peptide library was about 4.3×1012 PFU/mL,and the size and position of the inserted fragments had a good randomness and diversity.Results indicated that the richness and specificity of the library were well.It lays the foundation for further study of screening and enriching epitopes of VP60 protein.And furthermore the library could be used to identify the receptor domains bind to VP60.And it plays a significant role in expounding the infection,pathogenesis and mechanism of immune protection of RHDV.3.Screening and identification of binding sites of RHDV VP60 and receptor HBGAs.The binding domains of RHDV VP60 to HBGAs were screened by the VP60 gene-specific phage display peptide library of rabbit hemorrhagic disease virus with good specificity and richness.The binding domains were further identified by prokaryotic expression and synthetic H type 2 blood group oligosaccharides-VLPs blocking assay.The results showed that two binding sites of RHDV VP60 to HBGAs were identified,which were RFADIDHR(291-298),VLQFWY(312-317).It lays the foundation for further revealing the molecular mechanism of binding of virus to HBGAs,which is of great significance to elucidate the infection and pathogenesis of RHDV.