A Preliminary Analysis Of Binding Site For RHDV VP60 Binding To Histo-Blood Group Antigens

Rabbit hemorrhagic disease virus(RHDV)is a member of the calicivirus family that infects rabbits and causes rabbit hemorrhagic disease(RHD).It was reported that virus-like particles of RHDV as well as Norovirus specifically bound to histo-blood group antigens contributing to RHDV infection.The capsid protein VP60,expressed in insect cells infected with a recombinant baculovirus yields,could spontaneously assemble into virus-like particles(VLPs),which were both antigenically and morphologically similar to native RHDV particles.Enzyme linked immunosorbent assay to detect the binding of RHDV VP60 to HBGAs in saliva samples and synthetic oligosaccharides has been established successfully.Several truncated fragments of VP60 P2 sub-domain(287-449aa)were determined employing saliva-VLPs binding assay and synthetic H type 2 blood group oligosaccharides-VLPs binding assay.Chimera proteins of VP60 were constructed to explore if they have influence on with the hemagglutinating character and capacity of binding to HBGAs.The research contents are as follows:1 The establishment of EIA assays detecting the binding of RHDV VP60 to HBGAsTo determine the structure-function relationship between capsid protein VP60 of RHDV and histo-blood group antigens(HBGAs),the HBGAs binding assay was established.The agglutination inhibition test was used to identify the types of HBGAs in O-type saliva sample,followed by detecting the binding of VP60 to H type HBGAs receptors.The optimal dilution of coating buffer,saliva,VP60 protein,the primary antibody 3D 11 and HRP conjugated secondary antibodies were determined by orthogonal continuous dilution method.As described previously,synthetic oligosaccharides-VLPs binding assay was established by the determination of the optimal diluent and operation temperature.Therefore,RHDV binding to HBGAs could be assessed qualitatively.2 The expression and binding points analysis of VP60 truncated fragmentsSeveral VP60 truncated fragments in P2 sub-domain were designed,followed by the construction of expression vectors pET32a(+)-250-350,pET32a(+)-330-449,pET32a(+)-330-350,pET32a(+)-330-345 and pET32a(+)-335-350.Then,VP60 truncated fragments in P2 sub-domain were expressed by Escherichia coli(DE3)expression system,named as P250-350,P330-449,P330-350、P330-345、P335-350.VP60 truncated fragments in P2 sub-domain were identified by SDS-PAGE and Western blot analysis,which were determined by saliva-VLPs binding assay and synthetic H type 2 blood group oligosaccharides-VLPs binding assay.The results showed that RHDV VP60 bound to HBGAs from saliva sample and synthetic oligosaccharides strongly.Analysis of all the VP60 truncated fragments indicates that the 335-345aa may be responsible for RHDV binding to HBGAs receptors.3 The construction and the hemagglutinating character analysis of VP60 Chimera proteinsRHDV VP60 chimera proteins were constructed with 411-417aa and 502-510aa replaced by flexible amino acids GSGGSGG and GGSGGGSGG,respectively.The recombination of eukaryotic expression vectors pFastBacTM 1-VP60-M-1 and pFastBacTM 1-VP60-M-2 was followed by the construction of shuttle vectors Bacmid-M-1 and Bacmid-M-2.The chimera protein P411-417 and P502-510 were expressed by the baculovirus expression system,and were identified by IFA,SDS-PAGE and Western blot analysis.The formation of VLPs were detected by the transmission electron microscope followed by hemagglutinating test,saliva-VLPs binding assay and synthetic H type 2 blood group oligosaccharides-VLPs binding assay.The results showed that chimera protein P411-417 was able to self-assemble into VLPs and bind to HBGAs strongly,but could not agglutinate human erythrocyte.Chimera protein P502-510 could not self-assemble into integral VLPs with missing the hemagglutinating activity.The result indicated that 502-510aa might influence the HBGAs binding assays.Therefore,411-417aa and 502-510aa may be responsible for the hemagglutinating character of RHDV.Furthermore,502-510aa may be important for the formation of RHDV VLPs.In conclusion,the determination of binding domain for RHDV binding to HBGAs and the hemagglutination analysis of VP60 may contribute to the study on the interaction between RHDV and its host.