Abstract
There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.
Highlights
For rational vaccine design or development of diagnostics, it is imperative to map all functionally relevant, specific and/or conserved linear B cell epitopes (BCE) of target proteins
In order to know how many BCEs are there among three neighboring reactive 18mer-peptides of P32-34 (Fig 4A and 4B), which were blotted in first round of antigenic peptide mapping in our study on epitome decoding of huZP4 protein, we first expressed a set of 8mer-peptides (P110-120) covering the full-length sequence of reactive P32 according to procedures of steps 1 to 8 in the protocol, and they were subjected to SDS-PAGE, and transferred onto 0.2 μm nitrocellulose membrane
Peptide biosynthetic method for epitope/epitome mapping had a residue Q at its C-terminus more than that of the latter, suggesting that rabbit immune system could recognize the common antigenic site shared among homologous proteins of pig and human, no matter whether they were against native protein or not
Summary
For rational vaccine design or development of diagnostics, it is imperative to map all functionally relevant, specific and/or conserved linear (continuous) B cell epitopes (BCE) of target proteins. Peptide biosynthetic method for epitope/epitome mapping (SB/S2/JCB-040/2015) by the Science and Engineering Research Board, Department of Science and Technology, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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