Preparation Of Monoclonal Antibodies Against Feline Calicivirus And Epitope Mapping

Feline Caliciviral Disease?FCVD?is an oral and respiratory disease caused by Feline Calicivirus?FCV?.FCV is a member of the genus Vesivirus of the family Caliciviridae.The infection of FCV cause seriously oral ulcers,sneezing,conjunctivitis and chronic gastroenteritis,Although there is only one serotype,sequence analysis of different FCV isolates suggest its mutation with high frequency.Infection can cause severely secondary infection.Therefore,the study on diagnosis and control of the FCV is very necessary.The main structual protein VP1 is the capsid protein and main protective antigen of FCV.VP1 is divided into six regions from A to F based on sequence conservation.The B region which is located in the 125-397aa of VP1 is highly homologous among various FCV strains.What's more,non neutralizing monoclonal antibodies?MAbs?can be induced by B region.The E region is located between 427-524aa,which is further divided into hypervariable regions and conserved regions.Previous reporters have shown that neutralizing monoclonal antibodies can be induced by the E region.In this study,the truncated VP1,namely VP1-B and VP1-E,was expressed and purified.Next,the purified VP1-B and VP1-E protein were respectively mixed with Freund's adjuvant to immunize BALB/c mice.Then,the spleen of immuneed mice and SP2/0 cells were fused by cell fusion technology.The purified FCV2280virus particles were used to screen mAb-secreting hybaidoma lines by ELISA method.After three rounds of screening and subcloning,4 hybridoma cells secreting antibodies against VP1-B were screened out and designated as 4G2,B1E5,B4G4 and B4G5.2 strains of hybridoma clones against VP1-E protein were screened out and designated E2F1 and E2F6.To further analysis the characterization of these MAbs,positive clones were further confirmed by western blotting and IFA.All MAbs were reactive with four FCV strains including FCV2280,F9,TIG-1 and HRB-SS.What's more,the subtype,neutralizing activity of monoclonal antibody and the titer of the ascites were analyzed.The result suggest that all these MAbs were IgG1 subtybe with?chain,but had no neutralizing activity.Titers of VP1-B and VP1-E MAbs in the ascites were 1:6553600,1:12800 and 1:25600 respectively.To map the epitope of monoclonal antibodies?MAbs?to VP1-B and VP1-E protein,a panel of overlapping peptides representing VP1-B and VP1-E protein was expressed in prokaryotic expression system.A panel of peptides of VP1-E was synthesized.The results suggest that a B-cell linear epitope DDGSITA at amino acids 126–132 of B region was identified by western blotting.The minimal of the B-cell linear epitope recognized was DDGSITA.All VP1-B MAbs can recognize DDGSITA.A B-cell linear epitope YICGSLQRAWG at amino acids 467–477 of E region was identified.All VP1-E MAbs can recognize YICGSLQRAWG.Furthermore,alignment of the sequence suggested that the epitope recognized by VP1-B MAbs is not conserved among FCV strains.The epitope recognized by VP1-E MAbs is relatively conserved among FCV strains.The change of the epitope was induced by the substitutions and insertion of amino acids.To define the amino acids required for antibody binding,a series of mutational shorter peptides was expressed and evaluated for 4G2 and E2F1.The other mutational shorter peptides were identified except for I130V and A132T.The study suggested that ¹³⁰I and ¹³²A of the epitope play a pivotal role in the antigenicity of VP1.The monoclonal antibody prepared in this study has important value for basic and epidemiological investigation of FCV.