Screening Of Mimic Epitopes Of A Monoclonal Antibody Against Classical Swine Fever Virus E2 Protein Using Phage Display

Classical swine fever(CSF),one of OIE-listed diseases,is caused by Classical swine fever virus (CSFV).CSF is a highly contagious fatal disease in pig.At present,Vaccination with the Chinese lapinized attenuated vaccine has been a general strategy in most country where CSF was epidemic.In China,a compulsive vaccination policy is pursued to prevent the disease.Extensive vaccination with C-strain(lapinized hog cholera vaccine,HCLV) makes it difficult to differentiate the animals infected with wild-type viruses from those vaccinated with C-strain vaccine using conventional assays.Epitopes,or antigenic determinants,are antigen domains with affinity to antibody.Epitopes play a very important role in the structure and function of protein antigens.Precise epitope mapping may help design more reasonable vaccines and diagnostic reagents.Phage-displayed peptide libraries technology provides a new approach to disclose protein structure and function,and has been used to map large number of epitopes.To investigate epitope difference of CSFV Shimen strain and C-strain is helpful to design reasonable epitope vaccines and clinical diagnostic reagents.One stable hybridoma cell line secreting McAb specific for Shimen strain has been established using the recombinant baculovirus-expressed E2 protein of C-strain(HCLV-E2) as tolergen and that of Shimen strain(Shimen-E2) as immunogen with cyclophosphamide-induced immunosuppression strategies.The McAb called 5B1 was shown to recognize CSFV Shimen strain,but not C-strain.The McAb was shown to a conformational epitope on the E2 protein.The McAb was identified to be IgG1 subtype.The McAb can be used to differentiate wild-type CSFV strains from C-strain.The McAb 5B1 was used as immobilized molecule to screen the phage display random 12-peptide library.After 3 rounds of biopanning,16 positive clones were obtained.From the sequencing comparison results,six clones display a consensus binding sequence YPYNLTKYYNSA.Most of those clones display a consensus binding sequence YxxxLxxYY,which show some homology with the amino acid sequence ⁹⁸⁷yAKTLKNKyy⁹⁹⁶ in CSFV E2 protein.Using synthesized short peptides,we found that the peptide could not block the reaction of Shimen-E2 with the McAb 5B1.McAb 5B1 was shown to be directed against a conformational epitope in SDS-PAGE and Western blot.So those phage clones might be mimic epitopes(mimotopes) of McAb 5B1.The mimotopes can be used for designing diagnostic tools and epitope vaccines for CSFV.