Epitope Identification Of Classical Swine Fever Virus E2 Protein By 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 pigs with a mortality around 80ï¼…to 90ï¼…, and it isepidemic or endemic worldwide (not including North America and Oceania).Epitopes, or antigenic determinants, are antigen domains with affinity to antibody. Epitopes play avery important role in the structure and function of protein antigens. Precise epitope mapping may helpdesign more reasonable vaccines and diagnostic reagents. To facilitate understanding the structure andfunction of CSFV E2 protein, we mapped the epitopes in the E2 protein of CSFV Shimen strain byphage display.A consensus sequence, LFDGSNP, was screened from a random 12-peptide library by biopanningusing a monoclonal antibody (McAb) HQ06 against CSFV E2 protein. The motif showed highhomology with the amino acid sequence ⁷⁷²LFDGTNP⁷⁷⁸ in CSFV E2 protein. An oligo DNA fragmentcoding for LFDGTNP was expressed as a GST-fusion product in E. coli. The fusion protein was shownto be reactive with HQ06 in Western blot and ELISA. We conclude that ⁷⁷²LFDGTNP⁷⁷⁸ represents alinear B-cell epitope of CSFV E2 protein, and can be used for designing diagnostic tools for CSFV.The E2 genes of CSFV Shimen and CSFV HCLV strains were expressed in insect/baculovirus. Therecombinant proteins Shimen-E2 and HCLV-E2 expressed in secreted dimmer forms in culture mediadiffered in molecular weight (MW), but both were recognized by CSFV antisera and McAb HQ06.Site-directional mutagenesis showed that the different MW between Shimen-E2 and HCLV-E2 wasresulted from the ⁹⁸⁶N glycosylation site unique to HCLV. The purified Shimen-E2 protein was used toimmunize BALB/c mice, and a hybridoma cell line stably secreting McAb (named 6El0) directedagainst the E2 protein was screened. It seems that the McAb 6E10 is directed against a conformationalepitope as demonstrated by its good reaction with CSFV but poor reaction with denatured E2 protein.The McAb 6E10 was subjected to three rounds of biopanning using a phage-displayed 12-peptidelibrary, resulting in 9 phage clones showing strong binding activity with 6E10. The 9 clones display aconsensus YWH, which is not found in the E2 protein. This implies that the phage displayed 912-pepides might be mimic epitopes of McAb 6E10.In summary, a linear B-cell epitope ⁷⁷²LFDGTNP⁷⁷⁵was identified in E2 protein and a hybridomacell line secreting McAb 6E10 directed against CSFV E2 protein was developed. This study is helpfulfor further structural and functional analyses of E2 protein and diagnosis development for CSFV.