Preparation Of The Monoclonal Antibodies Against VP2 Protein Of Bluetongue Virus Serotype 17 And The VP2 B-cell Epitopes Identification

Bluetongue virus (BTV), an orbivirus of the Reoviridae family encompassing 24 known serotypes, causes a haemorrhagic disease mainly in sheep and occasionally in cattle. BTV is transmitted between its mammalian hosts by certain species of biting midges and it can infect all ruminant species. BT represents a major barrier to international trade in animals and some animal products, causing worldwide economic losses. So, it is important to establish BTV serotype-specific detection method for using vaccines of the homologous BTV serotype to control BT disease.In this study, a pair of primers was designed based on the published GenBank BTV17 L2 gene sequence (Accession No. M17438.1). The viral genome dsRNA was extracted from BHK-21 cell infected with BTV17 by trizol method. The L2 gene amplified by RT-PCR was cloned into donor vector pFastBac HTA of Bac-to-Bac baculovirus expression system. The results of enzyme digestion analysis and sequencing showed that the recombinant plasmid pFast-VP2 was constructed successfully. The recombinant donor vector pFast-VP2 was transformed into DH10Bac competent cells which contained Bacmid. Through the homologous recombination between donor vector and Bacmid, a recombinant Bacmid Bac-VP2 containing the interest gene was obtained. The Bac-VP2 was transfected into Sf9 insect cell, the recombinant Baculovirus BacV-VP2 was generated finally. SDS-PAGE showed that Sf9 insect cells infected with BacV-VP2 would successfully express recombinant VP2 protein as inclusion body form. Western blot indicated that the recombinant VP2 protein could react with anti-BTV17 positive serum, retained good antigenicity.According to the published GenBank BTV17 L2 gene sequence (Accession No. M17438.1), two pairs of primers were designed to amplified A and B gene sequence of L2 from the recombinant donor vector pFast-VP2. Using prokaryotic expression system to express two partial overlapping VP2 protein. The A and B gene were cloned into prokaryotic expression vector pET-30a respectively. The results of enzyme digestion analysis and sequencing showed that the recombinant expression vectors pET-A and pET-B were constructed successfully. The recombinant vectors pET-A and pET-B were transformed into E. coli BL21 competent cells and induced BL21 with IPTG at 28℃. SDS-PAGE showed that recombinant protein VP2-A and VP2-B as inclusion body form could be expressed successfully. The inclusion body was degenerated and dissolved by 8M urea, then it was purified with the Ni+NTA column including His-Bind Resin. Western blot indicated that the purified recombinant protein VP2-A and VP2-B retained good antigenicity.In this study, monoclonal antibodies (MAbs) against bluetongue virus (BTV) serotype 17 were developed by fusion the myeloma cells with spleen cells from BALB/c mice immunized with purified two partial overlapping recombinant proteins VP2-A and VP2-B of BTV17 expressed in E. coli cell. Two hybridoma cell lines stably secreting MAbs against VP2 of BTV17, designated BTV17 VP2-3F4 and BTV17 VP2-4H10 (3F4 and 4H10 for short) , were identified by indirect ELISA using the recombinant protein VP2-A and VP2-B as coating antigen. In order to exclude false positive clones, the first screening of positive clones were further screened by indirect ELISA using the eukaryotic expression of the recombinant protein VP2 as coating antigen. Both of MAbs could react with recombinant protein VP2-A and VP2-B by western blot. Indirect immunofluorescence assay indicated that both of MAbs could react positively with BTV17, but MAb 3F4 was showed weakly positive reactions with BTV10 and BTV24, whereas no cross reaction was found for BTV1~9, BTV11~23, Ibaraki virus, Chuzan virus, Akabane virus, bovine rotavirus and bovine reovirus. Further epitope mapping of BTV17 VP2 with a set of synthetic peptides showed that MAb 3F4 recognized the linear antigenic determinants of 540DPWNNR545 and 4H10 reacted against 540DPWNNRA546 on the VP2 of BTV17, respectively.The preparation of MAbs and identification of VP2 epitopes might have potential applications in BTV serotype-specific detection method establishment, and structural and functional analysis of VP2 of BTV17.