Development Of Monoclonal Antibodies Against The Protein VP2 Of Bluetongue Virus Serotype 25 And Identification Of Antigenic Epitopes

Bluetongue?BT?is caused by the Bluetongue virus?BTV?and spread through insect vectors such as Culicoides,Aedes mosquitoes and other insects,mainly infecting a potent non-contact infectious disease.BT mainly infects ruminants,of which purebred woolly sheep are the most susceptible and the mortality rate can be as high as 80%.Office International Des Epizooties?OIE?provides that BTV is an animal disease that must be reported,and it is one of the first class of animal infectious disease in China.To date,29 serotypes have been found wordwide,with no cross-protection between different serotypes.Since the first discovery of BTV in 1979,at least ten distinct serotypes BTV in China have been found,causing serious economic losses.BTV-25 was first isolated from a serum sample of Swiss goats in 2008,yet there is no report about this disease in our country.However,with the increasingly frequent import and export trade,the disease may be introduced into our country at any time,so it is of great significance to study BTV-25 and establish an effective serological test.VP2 protein is expressed by L2 gene,and there is great difference between each serotype,which is an ideal target antigen for serotype identification.Three pairs of primers for BTV-25 L2gene sequence?EU839840?published on NCBI were designed and BTV-L2 and BTV-L2-A,B and C fragments were amplified by PCR using pET-28a?+?-VP2 plasmid as a template.The recombinant plasmid pFast-VP2,pFast-VP2-A,pFast-VP2-B and pFast-VP2-C were obtained by cloning into eukaryotic expression vector pFastBac^TMHT B.The positive recombinant plasmids were transformed into E.coli DH10bac cells,and the recombinant baculovirus BAC-VP2,BAC-VP2-A,BAC-VP2-B and BAC-VP2-C were produced.The positive recombinant plasmid BAC-VP2 was transfected into Sf9 insect cells to obtain recombinant baculovirus BACV-VP2.VP2 protein was successfully expressed on Sf9 insect cells by IFA and Western blot analysis,and could be combined with mouse anti-His antibody and mouse anti-VP2 antibody to prove its good antigenicity.His-25A,His-25BandHis-25C,whichisexpressedwiththestrains pET-28a?+?-VP2-25A/BL21,pET-28a?+?-VP2-25B/BL21,pET28a?+?-VP2-25C/BL21 as immune antigens were used to immunize BALB/c mice.The spleen cells of immunized mice were fused with myeloma cells SP2/0 by cell fusion technique.MBP-25A,MBP-25B and MBP-25C expressed by pMAL-c5X-VP2-25A/BL21,pMAL-c5X-VP2-25B/BL21 and pMAL-c5XVP2-25C/BL21 as detection antigen were used to screen and clone the fused cells.Finally,twelve hybridoma cells stably secreting anti-BTV-25 VP2 monoclonal antibody?McAb?were obtained and named as:25A2B6,25A2C4,25A3E9,25A4H2,25B2G3,25B1H7,25B2F3,25B5D2,5C4B2,25C3E6,25C5G11and 25C5E5.The McAbs were identified by using the VP2 protein expressed on Sf9 insect cells.IFA and Western blot analysis showed that eight McAbs of twelve monoclonal antibodies specifically bound to VP2 eukaryotic proteins,which were 25A2B6,25A2C4,25A4H2,25B2G3,25B1H7,25B2F3,25B5D2,25C4B2.Sf9 cells were infected with BACV-VP2 recombinant baculovirus,and the specificity and localization of VP2 eukaryotic protein was identified by immunoelectrophoresis using three specific McAbs 25A2B6,25B2G3 and 25C4B2.The results showed that the three McAbs specifically bound to the protein,and were enriched in the cytoplasm and nucleus of Sf9 cells with colloidal gold particles.It was proved that the VP2 protein was expressed both in the nucleus and in the cytoplasm.The liner epitopes recognized by 25A2B6,25B2G3 and 25C4B2 McAbs were identified by phage display technique.After 3 rounds of phage panning,single-stranded DNA was sequenced and compared with BTV-VP2 sequence to deduce that the epitope sequences were"³⁵⁹LYP³⁶¹","⁵⁸⁰NT⁵⁸¹"and"⁶²⁰TFR⁶²²".By further synthesis of the short peptide sequence,the indirect ELISA verified that the short peptide sequence was correct.The amino acid sequence of VP2 protein of 1 to 27 serotype BTV was compared with the epitope sequence,and found that the epitopes"³⁵⁹LYP³⁶¹"and"⁵⁸⁰NT⁵⁸¹"were specific to BTV-25.While the epitope"⁶²⁰TFR⁶²²"was relatively conserved.BTV-4,10,11,17,20,24 and 27 also contain this sequence,except that BTV-25 contains this sequence.In this experiment,the type-specific monoclonal antibodies against VP2 protein of BTV-25were prepared and the epitopes recognized by the antibodies were identified.The purpose of this study is to establish the material basis for establishing the type-specific immunological detection method of BTV and the functional study of VP2 protein.