Production Of Monoclonal Antibodies And Epitope Mapping Of Canine Distemper Virus H Protein And Canine Parvovirus VP2 Protein

Canine distemper virus(CDV)and Canine parvovirus(CPV)are the main viral diseases affecting the pet industry and fur animal industry at present.Canine distemper hemagglutinin protein is a critical protein in CDV tropism,which induces the production of neutralizing antibodies in the host.Most non-synonymous mutations in the VP2 gene of canine parvovirus affect the antigenicity and host range of CPV.According to the amino acid mutation of VP2 protein,the virus can be divided into CPV-2A,CPV-2B,CPV-2C,New CPV-2A and New CPV-2B genotypes.Mutations in key sites may affect existing diagnostic methods.Therefore,the preparation of monoclonal antibodies against CDV H protein and CPV VP2 protein and the identification of linear epitopes are helpful for the development of antibody diagnostic reagents for related diseases.In this study,the H gene of Asian-I CDV strain was used as a template to amplify the 5'end of612bp,and the fragment was ligated into a prokaryotic expression vector.The correct positive plasmid was sequenced to induce expression,and the purified target protein was identified by protein electrophoresis and Coomassie brilliant blue staining.The results showed that the recombinant H protein was about 30k Da.The specific bands of Western blot further verified that the truncated H protein has good immunoreactivity.BALB/c mice were then immunized with purified H(aa 1-204).Two monoclonal antibodies 1A5 and 2B8 against CDV-PS strain H were prepared by cell fusion technique and indirect ELISA antibody screening method.Western blot and IFA confirmed that the two CDV monoclonal antibodies could react specifically with CDV-PS and CDV-R strains.However,the monoclonal antibody 1A5 could not specifically identify the CDV3 vaccine strain,which indicated that there were mutations at key sites between CDV-PS and CDV3 vaccine strains in the epitope identified by monoclonal antibody 1A5.The epitope identified by m Ab 1A5 is ¹⁷⁸ARGDIFPPY¹⁸⁶,and the epitope identified by m Ab 2B8 is ¹²⁰QKTNFFNPNREFDFR¹³⁴.The results of epitope comparison showed that there were mutations in the epitope of monoclonal antibody 1A5 in many different strains.There was no mutation in the epitope region of Asia-1,Asia-2,America-1,America-2,Europe and Arctic.Serological experiments and protein structural analysis showed that both epitopes had high practical value.The same strategy was used to express and purify CPV VP2,and the monoclonal antibody MAb5B18 against VP2 protein was prepared.Monoclonal antibody 5B18 can react specifically with CPV-2A,CPV-2B,CPV-2C and FPV strains isolated from different regions in China.After VP2expression,MAb 5B18 was detected to identify the subregion of epitope,and then the linear epitope¹⁴⁰SFEQEIFNVVLKTV¹⁵³was identified by indirect ELISA,which was highly conserved in different canine parvovirus subtypes,this epitope has great potential in cross-species parvovirus detection.In this study,two monoclonal antibodies against CDV H protein and one monoclonal antibody against CPV VP2 protein were obtained,and two canine distemper H protein epitopes and one canine parvovirus VP2 protein epitope were identified.It provides a tool for further study on the function of protein and detection and purification of disease.Highly conserved epitopes have great potential in detecting samples of different subtypes and natural reservoir.