Abstract
Previously, we successfully prepared a monoclonal antibody (mAb) named 2E4, that directly recognizes the major envelope protein B2L of the orf virus (ORFV), but there is little information about its epitope. Here, we meticulously mapped the 2E4 epitope through combinatorial programs and identified the functional binding domain and a key amino acid residue. Briefly, the simulated epitope peptide closely resembles 84VDVQSKDKDADELR97 located at the N-terminus of B2L, strongly suggesting that the epitope is conformationally or spatially structure-dependent. Subsequently, we combined these findings with the results from the antigenicity prediction of B2L to design three truncated fragments of B2L (F1, F2 and F3) selected using 2E4, and only the F1 fragment was found to be eligible for the advanced stage. Alanine-scanning mutagenesis suggested that the D94 residue is structurally crucial for the 2E4 epitope. The other participating residues, including K61, E62, and D92, together with D94 were responsible for enabling 2E4 binding and served as factors that synergistically enabled binding to the whole 2E4 epitope. In this paper, we describe, for the first time, the architecture of an ORFV conformational epitope, and it is also expected that mAb 2E4 and its epitope can be used for applications relating to orf control.
Highlights
We successfully prepared a monoclonal antibody named 2E4, that directly recognizes the major envelope protein B2L of the orf virus (ORFV), but there is little information about its epitope
We describe, for the first time, the architecture of an ORFV conformational epitope, and it is expected that monoclonal antibody (mAb) 2E4 and its epitope can be used for applications relating to orf control
ORFV, a species from the family Poxviridae and genus Parapoxvirus (PPV), is responsible for contagious ecthyma, mainly leading to a partial epitheliotropic effect via broken or scarified skin and gives rise to pustular lesions[2,3]. The aetiology of this condition is shown to be the presence of poxvirus, which can be identified in clinical samples through the use of electron microscopy, serological methods, polymerase chain reaction (PCR), and genetic analysis of unique genes, such as B2L, which is encoded by ORFV4,5
Summary
We successfully prepared a monoclonal antibody (mAb) named 2E4, that directly recognizes the major envelope protein B2L of the orf virus (ORFV), but there is little information about its epitope. Based on the B2L gene, a real-time quantitative PCR assay has been developed for ORFV DNA quantification in infected cells or organic cultures[9] Another helpful tool for orf detection is using serological methods to assess antigen-antibody interactions. By screening peptide mimics from a random biological protein molecule library, researchers have broken through many barriers in the methodology of epitope mapping This process facilitates the discovery of antibody-binding fractions or epitopes[18,19,20]. Detailed information related to the 2E4 epitope will assist us by providing with a better ability to combat this viral disease in the future
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