Expression of Orf Virus B2L Protein and Monoclonal Antibody Preparation

Currently, there is no effective ELISA detection methods for orf virus (ORFV) based on recombinant proteins and monoclonal antibodies(mAbs). This research aims to express recombinant B2L protein (rB2L) of ORFV and prepare high specificity monoclonal antibodies targeting rB2L. After codon optimization for ORFV B2L gene, the optimized gene fragment was chemically synthesized and ligated into a prokaryotic pET-30a(+) vector for expression and purification of rB2L. Taking the purified rB2L as immunogen, three stable hybridoma cell lines (1F2, 2B3 and 3H6) secreting anti-ORFV mAbs were obtained through indirect ELISA combined with immunofluorescence. Subtype identification results indicated all three antibodies carried κ light chains with heavy chain subtypes IgG1, IgG2b and IgG2a, respectively. Indirect ELISA titer detection showed all three mAbs possessed titers higher than 1:6400. Among them, mAb 2B3 achieved the highest titer up to 1:25,600. Western blot verified that mAb 2B3 could specifically bind both rB2L and ORFV. Indirect immunofluorescence assay also confirmed mAb 2B3 could recognize viral antigens inside ORFV-infected primary goat testicular cells. These data suggest that rB2L and its specific monoclonal antibody 2B3 provide core experimental materials for developing an effective ORFV detection method.