Preparation Of The Monoclonal Antibodies Against VP2and VP5Protein Of Bluetongue Virus Serotype1and Identification Of B-cell Epitopes

Bluetongue (Bluetongue, BT) is a kind of animal contagious disease caused by bluetongue viruswhich belongs to Orbiviruse genus, the Reoviridae family. Goats, sheep, cattles, deers, camels and otherruminant animals can be infected through vector insects such as midge and aedes mosquitoes. Disease ischaractered as mouth, nose and gastrointestinal mucosal inflammation of ulcerative changes. BTV hasat least26serotypes now. There are no cross protection in different serotypes, so serotype diagnosis ofBTV outbreak as early as possible and the corresponding vaccine in the disease popular regions are thekey to the prevention and treatment of the disease.BTV genome is composed of10pairs of double-stranded RNA,10segments genome encoded7kinds of the structural protein (VP1~VP7) and four kinds of the nonstructural protein NS1, NS2, NS3,NS3a) respectively. VP2protein encoded by L2gene and VP5protein encoded by M5gene are BTserotype specific antigens. According to BTV1L2gene sequence in Genbank (registration number:JN848760.1) and M5gene sequence (registration number: JN848763.1) designed a pair of primersrespectively. Viral RNA was extracted from BHK-21cells infected with BTV1, L2gene and M5genewere amplified by RT-PCR. These two genes were cloned into pMAL-c4x prokaryotic expression vectorand pFastBacHTAeukaryotic expression vector respectively to express recombinant VP2and VP5.Immune4-6weeks of BALB/c mice with VP2protein and VP5protein expressed by prokaryoticexpression system. After the third immune，measured the titer of immuned mice. After the boostedimmune, spleen cells of BALB/c mice and SP2/0myeloma cell SP2/0myeloma cell were fused withPEG. VP2protein and VP5protein expressed by eukaryotic expression system were chosed as detectedantigen.4strains of hybridoma can secrete monoclonal antibodies against VP2protein stably wereselected. They are named as BTV1VP2-3E8, BTV1VP2-3A7, BTV1VP2-4E2and BTV1VP2-4B6,abbreviated as3E8,3A7,4E2,4B6.4strains of hybridoma can secrete monoclonal antibodies againstVP5protein stably were selected. They are named as BTV1VP5-1B9, BTV1VP5-1F3, BTV1VP5-3B6,BTV1VP5-4E10, abbreviated as1B9,1F3,3B6,4E10.Western blot results showed that the4strains Mabs against BTV1-VP2and4strains Mabs againstBTV1-VP5both reacted with corresponding protein of BTV1expressed in BHK-21and no responsewith BHK-21cellular debris; IFA results showed that the3E8、4E2、4B6、1B9react with BTV1and noreacted with other serotypes. This laid the foundation for establishing BTV1differential diagnosismethod. Besides reacted with BTV1,3A7,1F3,3B6,4E10have their respective response spectrum. Forexample,3A7also reacted with BTV8;1F3also reacted with BTV2, BTV9, BTV18, BTV23;3B6alsoreacted with BTV2, BTV9;4E10also reacted with BTV2, BTV18, BTV23. Identified the epitopes ofthese Mabs by using the method of pepscan and synthetic peptide. The results showed that3E8’sepitope is465YGQMINEMI473;4B6’s epitope is115AQPLKVGL122;3A7’s epitope is149SYHVEPIE156;1F3’s epitope is141EIEDEKQFDILNKAVT156;1B9’s epitope is121EKFGKELEEVYNFMNG136;3B6’sepitope is281SVVSTILEYRAKEIPD296;4E10’s epitope is ILTEEDLQMRRLATAL.Mabs and responding epitopes of this research lay the foundation for establishment of BTV1type specific test methods and research about VP2, VP5function. It’s of great significance to prevent andcontrol BT.