Identification of antigenic epitopes of monoclonal antibodies against the VP2 protein of the 25 serotype of bluetongue virus

Serious outbreaks of bluetongue, an arbovirus of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), have occurred around the world. More than 27 distinct serotypes are recognized throughout the world. A new virus, BTV-25 (Toggenburg orbivirus [TOV]), was first detected in Switzerland, and has not yet been found in China. VP2 is an important outer shell protein that defines BTV serotypes and is, therefore, an ideal target antigen for serotype identification. To produce a monoclonal antibody against VP2 of BTV-25, the segment 2 gene was divided into three segments, cloned into pET-28a (+) and pMAL-c5X vectors, and the protein was expressed in E. coli BL21 with different tags. Monoclonal antibodies (mAbs) were prepared by using the purified His-25A, 25B, 25C proteins as the immunogen and the purified MBP-25A, 25B, 25C proteins as the detection antigen. Twelve hybridoma cell lines stably secreting mAbs against different VP2 segments of BTV-25 were produced. The segment 2 gene was cloned into pFastBac™HT B vector and a positive recombinant plasmid pFastBac-VP2 was used to identify mAbs. The recombinant baculovirus BACV-VP2 and eukaryotic expression of protein VP2 were obtained by the recombinant bacmid BAC-VP2 transfected Sf9 insect cells; western blotting showed that only eight mAbs were reactive. Finally, we identified the epitopes of VP2 recognized by three specific mAbs (25A-2B6, 25B-2G3, 25C-4B2) using phage display technology. The linear epitopes of VP2 protein were “359LYP361”, “580NT581”, “620TFR622”. The preparation of mAbs and identification of the epitopes provided a foundation to analyze VP2, and may assist in the serological diagnosis of BTV-25.