Preparation Of Monoclonal Antibodies Against NS1 Protein Of Bluetongue Virus 12 And Identification Of B-cell Epitopes

Bluetongue disease(BT) is an insect-borne infectious disease caused by the bluetongue virus(BTV),causing haemorrhagic fevers mainly in sheep. BTV is transmitted to ruminant hosts mainly by the bite of midges of the genus Culicoides, occasionally by seminal and transplacental transmission. The diversity and multi-segmented genomic organization of BTV genome increases the likelihood of genetic recombination during viral replication and assembly, and 27 serotypes have been reported to date. In addition, global climate change raises the possibility that the distribution of Culicoides insect vectors may spread and expand the scope of BT outbreaks. Like other members of the genus Orbivirus, vaccines are the most effective way to prevent BT infection and limit viral spread. But because of the antigen diversity between BTV different serotypes, little or no immunity protection is effective among BTV serotypes. Therefore, the preparation of type-specific MAbs and group-specific MAbs could serve as a basis for BTV diagnostic and research approaches and play important role in BT prevention and control.The BTV genome consists of 10 segments of double-stranded RNA( Seg-1~Seg-10), and the NS1 protein encoded by S5 gene is highly conserved among different BTV serotypes. A pair of PCR primers was designed according to BTV12 S5 gene sequence carried in recombinant plasmid p EASY-NS1-BTV12 template preserved in our lab(Gen Bank accession No. KM485310). The coding sequence of S5 gene was amplified by PCR. The S5 gene after the digestation was then insereted into prokaryotic expression vector p ET-30 a, Bac-to-Bac baculovirus expression vector p Fast BacTMHT A to construct recombinant plasmid p ET-NS1-BTV12 and p FAST-NS1-BTV12, respectively.The recombinant plasmid p ET-NS1-BTV12 was transformed into E. coli BL21 competent cells and express recombinant NS1 protein by 0.5m M IPTG induction. SDS-PAGE analysis showed that prokaryotic recombination NS1 protein was expressed in inclusion body and purified by the Ni2+NTA column. Western blot(WB) demonstrated that recombination NS1 protein could react with BTV-positive polyclonal antiserum preserved in our lab. The recombinant plasmid p FAST-NS1-BTV12 was transformed into E. coli DH10 Bac TM competent cells, generating recombination bacmid(r Bacmid DNA). r Bacmid DNA was then transfected with sf21 insect cells and acquire recombination baculovirus BACV-NS1 with high expression of recombinant NS1 protein. SDS-PAGE analysis showed that recombinant NS1 protein in sf21 cells was expressed in inclusion body as well and purified by the electrophoretic method for elution. Western blot(WB) demonstrated that recombinant NS1 protein could also react with BTV-positive polyclonal antiserum.Hybridoma lines generated using splenocytes from immunized mice fused with sp2/0 myeloma cell were screened by indirect ELISA to identify lines producing NS1-reactive MAbs. Twenty-five hybridoma cell lines were identified which stably secreted NS1-specific MAbs. WB analysis demonstrated that these 25 MAbs showed a strong reactivity with r P-NS1-BTV12 and r E-NS1-BTV12, while no reactivity was noted against p ET-30 a empty vector or wild-type baculovirus protein. IFA showed that Twenty-two of 25 MAbs demonstrated strong reactivity with BTV12, whereas 3 of 25(2B7, 3E9, and 4B9) failed to bind detectably to BTV12. Six MAbs(1F11, 2F2, 2C11, 3C2, 3G6, and 4E5) among 25 MAbs exhibited strong cross-reactivity with all BTV serotypes tested(BTV1-24). In contrast, MAb 1F8 reacted only with BTV12 and not with any other BTV serotypes. The remaining 15 MAbs recognized a set of additional BTV serotypes.We next mapped the BTV12 NS1 epitopes recognized by a NS1-reactive MAbs using a set of 55 overlapping peptides covering the entire NS1 protein sequence. NS1-derived peptides were prokaryotically expressed as MBP fusion products and were used as targets in an ELISA screen to define the epitopes recognized by MAbs. Seven unique linear peptide epitopes in the BTV12 NS1 protein were identified. Four MAbs(1B2, 2E8, 3E9, and 3D10) failed to react with any peptide in the series, suggesting that they may recognize conformational epitopes. We next analyzed the degree of conservation of each identified NS1 epitope among various members of the genus Orbivirus in the family Reoviridae. We found that all seven linear peptide epitopes are conserved among BTV12 serotypes. Meanwhile, the seven linear peptide epitopes are relatively conserved among BTV1–26, but are not conserved among AHSV, EHDV, and CV.