Preparation Of The Monoclonal Antibodies Against VP2 And NS3 Protein Of Bluetongue Virus Serotype 15 And Identification Of B-cell Epitopes

Bluetongue disease(BT)is a hemorrhagic disease of ruminants caused by bluetongue virus(BTV), widely transmitted by midges of the genus Culicoides. Goats, cows, deer and camels, especially sheep intend to be infected by BTV. Animals infected by BTV mainly have fever, mucous membranecyanosis,facial edema. Up to date, BTV have as much as 27 serotypes, and each serotype of BTV has no cross protection. Therefore, preparing related diagnostic reagent of specific serotype of BTV is the key to prevention and treatment of BT.BTV is the prototype member of the genus Orbivirus within the Reoviridae family. The BTV genome consists of 10 double-stranded RNA segments t hat encode seven structural proteins(VP1-VP7), and four non-structural proteins NS1, NS2, NS3/NS3 a and NS4. The VP2 protein elicits the generation of neutralizing antibodies in vivo,and NS3 mediate virion release.According to the sequences of BTV15 L2 genne(CAE51102.1) and S10 gene(AGJ83560.1)in Genbank, we designed one pair of specific primers for each gene to amplify L2 gene and S10 gene using the constructed recombinant plasmids p ZERO-B15-VP2 and PEASY-B15-NS3 which respectively contain VP2 encoding sequence and NS3 encoding sequence. The PCR products were cloned into the prokaryotic expression vector p MAL-C4 X to construct recombinant plasmid p C4X-B15-VP2 and p C4X-B15-NS3.What is more, the L2 gene was cloned into eukaryotic expression vectorp Fast BacTMHTA and S10 gene was also cloned into PET-30 a.After the four recombinant proteins were expressed and purified, the 4~6 week-old BALB/c mice were respectively immuned using the procaryotic fusion protein M BP-VP2 and HIS-NS3 to prepare monoclonal antibodys(MAbs). And the mice spleen cells were fused with SP2/0 cells. The hybridoma cells were screened by indirect-ELISA using Eukaryoticfusion protein BAC-B15-VP2 and M BP-NS3 as coating antigen. Finally, 3 strains of hybridoma cells stably secreting anti-VP2 MAbs were obtained, named 2C6,2D6,3B11; and 4 strains of hybridoma cells stably secret anti-NS3 MAbs were obtained, named 1B5,2B12,2G9 and 3D8.The MAbs were identified using western-blot(WB) assay and IFA assay. WB results showed all the MAbs recognized BHK-21 cells infected by BTV15; IFA results showed 2C6,2D6,3B11 only recognized BTV15, but 1B5, 2B12, 2G9, 3D8 recognized BTV1~24. The peptide scanning technique was used to identify the B cell epitopes of these MAbs. The results showed 2C6 recognized 62LKYRP66, 2D6 recognized 15 IAPAIIKRYP24, 3B11 recognized 99DHDVDS104, 1B5 recognized 205 YNDAVRM SF213, 2B12 recognized 204SYNDAVRM SF213, 2G9 recognized 82AEAFRDDVRLRQIK95, and 3D8 recognized 36PPRYA40. Alignment of the defined linear epitopes of different serotypes of BTV showed that these VP2 epitopes are poorly conserved in BTV serotyp es and NS3 epitopes are highly conserved in BTV serotypes.In this study, we prepared anti-BTV15 VP2 and NS3 MAbs and identified the epitopes in order to establish serotype-specific and group-specific detection and diagnostic methods.