Preparation Of Monoclonal Antibodies Against VP2Protein Of Bluetongue Virus Serotype8and Identification Of The Antigenic Epitopes

Bluetongue（BT） is a non-contagious disease caused by bluetongue virus（BTV）,which belongs tothe Orbiviruse genus,the Reoviridae family.BTV can infect all ruminant species including sheep,cattleand other wild rumiants and the virus is an arbovirus which is transmitted between ruminant hosts bysome species of the Culicoides genus.The average mortality of infected animals is30%,however,insheep as high as80%.BTV has many serotypes and up to now,at least24distinctserotypes（BTV1-BTV24） have been reported around the world which could generate only low levels ofcross-protection.In addition,two new serotypes,BTV25and BTV26have also arised.Since2006,BTV8has broken out in many European countries,causing serious economic losses.However,at the momentBTV8has not yet been reported in China,but with international trade BTV8may happen in China at anytime.And therefore we must strengthen the study of BTV,particularly BTV8,and make the necessarytechnical reserves.VP2protein encoded by L2gene is the main determinant of BTV serotypes.A pair of PCR primerswas designed according to BTV8L2gene sequence（GenBank accession No. AJ585129）.The viralgenomic RNA was isolated from supernatant of BHK-21cells infected by BTV8using trizol reagentand the open reading frame （ORF） of L2gene was amplified by RT-PCR assay.The amplicons werechecked,purified and cloned between two restriction enzymes sites of the prokaryotic expression vectorpMAL-C4X to construct the recombinant plasmid pC4X-VP2.The E.coli BL21（DE3） competent cellswere transformed with the recombinant plasmid pC4X-VP2to express the recombinant VP2proteininduced by addition of IPTG at37°C.Samples were analysed by SDS-PAGE and the results indicatedthat the recombinant VP2protein had been expressed a lot.And the results analysed by Western Blotshowed that the fusion expression of recombinant VP2protein with MBP tag was completed.Becausethe protein expressed mostly as inclusion bodies,the recombinant VP2protein was purified by cuttingthe gel slices which contain the right bands in order to prepare the immunogen.The L2cDNA was cloned into donor plasmid pFastBacHTA vector of eukaryotic expressionsystem by restriction enzyme digestion to construct the recombinant donor plasmid pFast-VP2.DH10Bac competent cells containing baculovirus shuttle vector （Bacmid） were transformed with therecombinant donor plasmid pFast-VP2.Arecombinant Bacmid BAC-VP2containing the gene of L2wasgenerated following transposition of the recombinant donor plasmid pFast-VP2.The recombinantBacmid BAC-VP2DNA was transfected into Sf9insect cells to generate a recombinant baculovirusBACV-VP2.Samples were analysed by SDS-PAGE and the results indicated that the recombinant VP2protein had been expressed in Sf9insect cells.Because the protein expressed mostly as inclusion bodies,the recombinant VP2protein was purified by the purification assay of inclusion bodies to prepare thescreening antigen.To prepare monoclonal antibody（Mab） against BTV8VP2protein,SP2/0myeloma cells were fusedwith spleen cells of BALB/c mice immunized with the recombinant VP2protein expressed by theprokaryotic expression system.Two hybridomas named BTV8VP2-3E11and BTV8VP2-4D9（3E11 and4D9for short）,were developed and determined by indirect ELISA coated with the recombinant VP2protein expressed by the eukaryotic expression system.The results of Western Blot indicated that3E11and4D9can specially react with VP2protein expressed in BHK-21cells infected by BTV8.The resultsof IFA demonstrated that3E11and4D9were able to react with BTV8positively,whereas nocross-reactivity were found with other BTV seretypes.Consequently,3E11and4D9play an importantrole in developing differential diagnostic method of BTV8.24pairs of PCR primers was designed according to the recombinant plasmid pC4X-VP2genesequence.The products of PCR,respectively,were cloned into the prokaryotic expression vectorpMAL-C4X by restriction enzyme digestion to construct the recombinant plasmids pMAL-VP2-1﹣pMAL-VP2-24.The E.coli BL21（DE3） competent cells were transformed with the recombinantplasmids to express24short peptides overlapping10-13amino acids between every two adjacent shortpeptides.Initially identified the range of3E11and4D9epitope by peptide scanning,continuousoverlapping peptides were synthesized to identify that the epitope of3E11is⁹⁹HSEFHTKSNWVQW¹¹¹and the epitope of4D9is⁶²²AQYVFEKTCLYVLE⁶³⁵.The amino acid sequences of3E11and4D9antigenic epitopes are conservered between BTV8strains by the alignment of Blast.These results cancontribute to know more of antigenic structure and function in immune response of VP2protein as wellas the development of synthetic peptide vaccine.