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.

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Background It is crucial to reveal entire epitomes of more target homologous proteins and specificity of each mapped B cell epitope (BCE) within them for the development of high-risk (hr-) human papillomavirus (HPV) type-specific diagnostic reagents. Methods Recombinant E6 and -E7 oncoproteins from HPV16/18/45 were immunized mice to prepare Rabbit antisera. Overlapping 16mer/8mer-peptides for two rounds of antigenic peptide and fine BCE motif mapping were expressed as GST188 fusion proteins. Fine BCEs were delineated by Western blot and sequence alignment. Results In this work, we decoded six epitomes of E6 and E7 oncoproteins from three HPV types 16, 18 and 45 that are the most common hr-HPVs in cervical cancer patients worldwide, in which total 35 fine BCEs (8, 6 and 4 for E6; 7, 6 and 4 for E7) were mapped using rabbit antisera to respective recombinant proteins. The specificity of each mapped BCE among 20 defined or possible hr-HPVs was delineated by sequence alignment based on BCE minimal motif. According to similarities of immune responses to E6/E7 existed among rabbit and humans, 7 human-recognizing (HR) BCE motifs in HR-peptides of HPV16/18-E6 and E7 proteins were delimitated by comparing with corresponding rabbit-recognizing BCEs. Also, the unique BCE distribution within three delineated E7 epitomes was confirmed, in which almost mapped BCEs were clustered at the first half of the molecules, suggesting that it may be a common characteristic of hr-HPV E7 proteins. Conclusions The results would form the basis for identifying HR-BCEs and developing serodiagnostic reagents used in HPV-based cervical cancer screening and HPV-positive women managing.

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