The two DNA fragments encoding predicted main antigenic regions, aa 20-160 and aa 257-344 on gD protein of bovine herpesvirus-1 (BoHV-1) were tandem-cloned into the vector pET28a. The recombinant His-tagged Δ gD1-Δ gD2 was expressed in Escherichia coli BL21(DE3)pLysS by induction with 0.5 mM isopropyl-1-thio-β-D-galactoside (IPTG) and Western blot analysis demonstrated that the recombinant His-tagged Δ gD1-Δ gD2 reacted with the positive serum against BoHV-1. A monoclonal antibody (MAb), designated as 2B6, was prepared by fusion of SP2/0 myeloma cells with splenocytes from a female 8-week-old BALB/c mouse immunized with purified His-tagged Δ gD1-Δ gD2 protein with the addition of Freund's adjuvant. The titer of the ascitic fluid triggered by hybridoma cells secreting MAb 2B6 was 1:2.5 × 108 and the subtype of MAb 2B6 was IgG 2a/κ. MAb 2B6 positively recognized with BoHV-1 infected Madin-Darby bovine kidney cells by an indirect fluorescent antibody assay. Moreover, MAb 2B6 showed 1:160 viral neutralizing activity using 60% plaque reduction assay. Therefore, this work suggested that MAb 2B6 has potential in the diagnosis of bovine respiratory disease complex associated with BoHV-1 and function analysis.
Keywords: bovine herpesvirus-1; gD protein; indirect fluorescent antibody assay; monoclonal antibody; neutralizing.