Monoclonal antibody and B-cell epitope mapping of the VP7 protein in bluetongue virus

Abstract

Bluetongue virus (BTV) VP7 is a group-specific protein that is highly conserved in different serotypes. In this study, BALB/c mice were immunized with purified recombinant BTV-1 VP7 protein expressed in E. coli. Then six monoclonal antibodies (mAbs), 2A7, 2B2, 2B3, 2D3, 2D7, and 2H2, against the BTV-1 VP7 protein were produced using hybridoma technology. The reactivity of the mAbs was identified using western blotting, enzyme-linked immunosorbent assay, and immunofluorescence assay. A series of truncated peptides derived from VP7 expressed as glutathione S-transferase fusion proteins were mapped with mAbs by western blotting. The results indicated that 2A7 recognized the epitope ⁷¹SAAGINVGPI⁸⁰, 2B3 recognized ¹¹⁰ARVTGETSTWG¹²⁰, 2B2 and 2D3 recognized ¹²⁵PYGFFLETEET¹³⁵, and 2D7 and 2H2 recognized ³³²VNPMPGPLTRA³⁴². Amino acid sequence analysis showed that these four epitopes were conserved in 24 typical BTV serotypes. Cross-reaction results showed that mAb 2A7 could recognize the recombinant VP7 protein of BTV-1, African horse sickness virus serotype 1 (AHSV-1), and epidemic hemorrhagic disease virus serotype 1 (EHDV-1). The mAbs 2B2, 2B3, and 2D3 could recognize the recombinant VP7 protein of BTV-1 and EHDV-1, and the mAbs 2D7 and 2H2 specifically recognized the BTV-1 VP7 protein. These specific mAbs and identified B-cell epitopes provided key insights into the structure and function of VP7, while facilitating the development of BTV diagnostics and the design of epitope-based vaccines.

Keywords: B-cell epitope mapping; Bluetongue virus; Monoclonal antibodies; VP7 protein.

Fig. 1

Schematic diagram of VP7 epitope mapping strategy. The fragments recognized by mAbs 2A7, 2B2, 2B3, 2D3, 2D7, and 2H2 are represented by orange lines, and the fragments that are not recognized by any mAb are represented by blue lines. A, B, and C represent three rounds of truncation

Fig. 2

Expression, purification, and identification of recombinant BTV VP7. A SDS-PAGE identification of the expression of VP7. Lane 1: Uninduced bacteria; lanes 2–5: Bacteria induced for 2, 4, 6, and 8 h. B Solubility analysis of recombinant VP7. Lane 1: VP7 in supernatant after ultra-sonic treatment; lane 2: VP7 in precipitate after ultra-sonic treatment. C Purified VP7 verified by SDS-PAGE. Recombinant VP7 was identified by anti-6 × His tag mAb (D) and rabbit anti-BTV-1 positive serum (E)

Fig. 3

Characterization of the mAbs. A The reactivity of mAbs was analyzed with indirect ELISA. The dotted line is 2.1-fold that of the negative control. B The reactivity of monoclonal antibodies was analyzed using western blotting. Lane1: BHK-21 cells infected with BTV-1; Lane 2: Recombinant VP7. C Immunofluorescence analysis of the mAbs. BHK-21 cells infected with BTV-1 were subjected to immunofluorescence assay at 24 h. Alexa Fluor488-conjugated goat anti-mouse IgG was used as a secondary antibody. D Isotype identification of mAbs

Fig. 4

Fine-mapping of the epitopes with mAbs. Primary (A), secondary (B), and tertiary (C) VP7 fusion proteins truncated by the prokaryotic expression were screened to show the antigenic epitopes of the six mAbs by western blotting. The empty vector pGEX-6p-1-GST and full-length VP7 were used as the negative and positive controls, respectively

Fig. 5

Sequence alignment of VP7 of three orbiviruses. The epitope ⁷¹SAAGINVGPI⁸⁰ (Box 1) identified by 2A7, the epitope ¹¹⁰ARVTGETSTWG¹²⁰ (Box 2) identified by 2B3, the epitope ¹²⁵PYGFFLETEET¹³⁵ (Box 3) identified by 2B2 and 2D3, and the epitope ³³²VNPMPGPLTRA³⁴² (Box 4) identified by 2D7 and 2H2 are circled with red. Black dots indicate the same amino acids, and the dotted lines indicate the gap regions

Fig. 6

Cross-reactivity assays between mAb and AHSV and EHDV VP7 proteins. Reaction of mAbs 2A7, 2B2, 2B3, 2D3, 2D7, and 2H2 with the VP7 of three orbiviruses. Lane 1: rec-BTV VP7, lane 2: rec-AHSV VP7, lane 3: rec-EHDV VP7

Fig. 7

Structural analyses of VP7. A VP7 structure and four epitopes recognized by mAbs. Red, yellow, and blue represent monomer structures of VP7. The epitopes ⁷¹SAAGINVGPI⁸⁰, ¹¹⁰ARVTGETSTWG¹²⁰, ¹²⁵PYGFFLETEET¹³⁵, and ³³²VNPMPGPLTRA³⁴² are colored in green, magenta, cyan, and orange, respectively. B The VP7 models of BTV (blue), AHSV (green), and EHDV (magenta) have been superimposed on to each other to show structural similarity