Preparation Of The Monoclonal Antibodies Against VP2Protein Of Bluetongue Virus Serotype8and Identification Of B-cell Epitopes

Bluetongue(BT) is a haemorrhagic disease of ruminants caused by Bluetongue virus(BTV)via an insect vector, which is an economically important orbivirus in the Reoviridae family.BTV can infect all wild and domestic ruminant, particularly sheep. The average mortality ofinfected animals is about30%, however, in sheep reach up to80%. BTV has many serotypes, atpresent,26serotypes have been described which could generate only low levels ofcross-protection. Beginning in2006, it was reported BTV8has broke out in France, Belgium,Germany and the Netherlands. After, the epidemic area has expanded uncreasingly. BTV8hashigher pathogenicity not only for sheep but also for cattle and camels. VP2is the majorserotype-specific antigen to stimulate the boby to product neutralizing antibodies, and it hashemagglutinin activity. Thus VP2becomes a study hot about BTV and it is necessary todevelop a serotype-specific diagnostic method.A pair of primers were designed and synthesized according to VP2gene sequence ofBTV8published on GenBank. The VP2gene was amplified by RT-PCR assay, and after doubledigestion it was cloned into pFastBacHTA donor plasmid. Then recombinant VP2wasexpressed by Bac-to-Bac baculovirus expression system. The result of expressed recombinantVP2protein and the protein purification effect were indicated by SDS-PAGE and Western blot.For the preparation of monoclonal antibodies (MAbs) against BTV8VP2, six weeks oldBALB/c mice were immunized with purified recombinant VP2protein of BTV8. Spleen cellsfrom sensitized BALB/c mice for three times immunization and SP2/0myeloma cells wereused to make cell fusion experiments. Positive hybridoma cells were screened by the purifiedrecombinant VP2protein and BTV8virus respectively as detecting antigen using indirectELISA method. The first screened positive hybridoma clones by recombinant VP2proteindetecting antigen were further screened by indirect ELISA using BTV8as a secondary coatingantigen, and subclone the positive clones by limited dilution method. Eventually we obtainedtwo secreting anti-BTV8VP2protein MAb cell lines, named BTV8VP2-2G4, BTV8VP2-3B7,referred to as2G4and3B7. Indirect immunofluorescence assay (IFA) results showed that2G4and3B7reacted with BTV8, and no cross reaction with BTV1-7,9-24, IBAV, chuzan virus andAKAV. Western Blot showed that the two MAbs reacted with the recombinant VP2andBHK-21cell infected with BTV8. The results showed the two MAbs were type-specific BTV8 MAbs.To identify the antigen epitope of the two MAbs,96pairs of primers were designed andsynthesized, and every pair primers encode16a.a.. Every two adjacent expressed peptidesoverlapped6a.a.. Further antigen epitope identification for the MAbs with96prokaryoticexpressed maltose-binding protein (MBP)-fused peptides covering the VP2showed that2G4’santigenic epitope was281LCRLLSTIGRKMCNTE296. The antigen epitope of2G4has highspecificity and conservativity.The study might contribute to establish serotype-specific detection method and will beuseful for analysising structure and function of VP2protein and development of Epitopepeptide vaccine.