Identification of three novel linear B-cell epitopes located on the glycoprotein of Micropterus salmoides rhabdovirus using monoclonal antibodies.

Abstract

Micropterus salmoides rhabdovirus (MSRV) is one of the most devastating pathogens in largemouth bass (Micropterus salmoides) aquaculture, causing fatal epidemics in farmed populations and leading to significant economic losses. Glycoprotein (G) is located on the surface of MSRV virion and interacts with the receptor on the surface of host cell to initiate MSRV adsorption and invasion, serving as an ideal target for vaccine development and serological diagnosis. However, the antigenic epitopes of G protein remain unclear. Here, high purity of recombinant G protein was obtained through prokaryotic expression system and used to immunize Balb/c mice for the preparation of monoclonal antibodies (mAbs). After cell fusion and three rounds of subclone screening, three strains of hybridoma cells secreting antibody against G protein of MSRV were obtained, named 1B3, 4A1, and 2D3, respectively. Western blot and indirect immunofluorescence assay (IFA) showed that these three mAbs reacted specifically with the G protein of MSRV and MSRV-infected cells. To identify the epitopes recognized by these mAbs, a series of overlapped truncated peptides of G protein were designed and expressed as GFP tag fusion proteins. Subsequently, the fusion proteins were probed with these three mAbs separately using Western blot, and three novel linear B-cell epitopes on G protein were identified. The minimal epitope recognized by mAb 1B3 was identified asQGWHDV, the minimal epitope recognized by mAb 4A1 was identified asLEMHGGIWIPSEA, and the minimal epitope recognized by mAb 2D3 was identified asRPVGAQGDIIHAVGE. Alignment of amino acids of G protein revealed that the three linear epitopes are highly conserved among different MSRV isolates. These findings provide new insights into the structure and function of the MSRV G protein and lay the experimental foundation for the development of serological diagnosis and vaccines.