Production And Epitope Mapping Of Monoclonal Antibodies Against Canine Distemper Virus P Protein

Canine distemper?CD?is a highly contact infectious disease caused by Canine distemper virus?CDV?,which provokes a variety of animals and multi-systems infections.In order to obtain the canine distemper virus wild type strain?CDV-PS?truncated phosphoprotein?aa 232-507?.In this study,the P protein gene?aa 232-507?of genotype Aisa-1 CDV-PS strain was amplified by PCR and introduced into the prokaryotic expression vector pEASY^?R-Blunt E1.Then the constructed E1-CDV-P was moved into Escherichia coli BL21 and optimized the induction condition.Induce products were analyzed solubility after ultrasonication.Subsequently,His-truncated recombinant P protein was purified by Ni-NTA affinity chromatography columns and identified by SDS-PAGE and Western blot.The results showed that the His-turncated P protein was about 37 kDa,at the final concentration of 0.1 mmol/L IPTG and the induction time 8 h,the highest expression of recombinant P protein existed in the form of inclusion body.Truncated recombinant P protein was successfully purified by SDS-PAGE and Western blot identification.And recombinant P protein had good immunoreactivity.In a word,the P protein gene?aa 232-507?of genotype Aisa-1 CDV-PS strain was successfully prepared,which had potential applications in the developement of CDV antibody diagnosis methods and provided a basis for futher study to screen and identify epitope.To obtain monoclonal antibodies?MAbs?against P?aa 232-507?,BALB/c mice were vaccinated with purified P?aa 232-507?protein.Then we obtained four MAbs against CDV-PS strain P?aa233-507?,named Pc7,Pc8,Pc11 and Pc25,were prepared by cell fusion technique and indirect ELISA antibody screening method.Western blot and IFA showed that these could react specifically with P?aa232-507?of CDV-PS strain.However,Pc8 MAb does not specifically recognize CDV3 vaccine strain,indicating that there are key mutations in the antigenic epitopes of Pc8 MAb between CDV-PS strain and CDV3 vaccine strain.Pc25 MAb does not specifically recognize CDV-Rockborn vaccine strain,indicating that there are key mutations in the antigenic epitopes of Pc25 MAb are between CDV-PS strain and CDV-Rockborn vaccine strain.Monoclonal antibodies against CDV-PS strain P?aa232-507?protein were successfully prepared in this study,which laid a foundation for further screening the linear epitopes of four monoclonal antibodies.To identify the linear antigenic epitopes of four monoclonal antibodies,Pc7,Pc8,Pc11 and Pc25,the antigenic epitopes were identified by indirect ELISA and peptide scanning technology.The results showedthat²³⁸SHGMGIVAGSTN²⁴⁹,²⁶⁴GPSVSAENVRQ²⁷⁴,³⁹⁰INPELRPIIGR⁴⁰⁰and²⁵²TQSALKSTG²⁶⁰,are minimal linear epitopes recognized by the Pc7,Pc8,Pc11 and Pc25 MAbs,respectively.Conservative analysis of antigen epitopes showed that the antigenic epitope of Pc7 MAb has mutations in many different strains;the antigenic epitope of Pc25 MAb has no mutation in the corresponding antigenic epitope regions of Asia-1?except CDV-ZC;140816?,America-1 and Europe strains,but has mutations in Asia-2 and most of America-2 strains;the antigenic epitope of Pc8 MAb has mutations in many strains;the linear B-cell epitope of the Pc11 MAb is highly conserved among different CDV strains.In this study,the linear epitopes of four monoclonal antibodies were screened successfully,which laid the foundation for the diagnosis of CDV.