Production and characterization of mouse monoclonal antibodies against lysis protein E of phiX174

• Purified mAbs were screened against a Ph.D.-12 library.
• One epitope showed a good match with protein E at 57–63aa (KPLN--R).
• Epep (VRLKPLNCSR) strongly inhibited combination of E-A5 with E fusion proteins.
• E-A5 indicated specific immunoreactivity with E. coli-expressed native protein E.

Bacterial ghosts offer a new avenue for the study of inactivated vaccines. However, for many years the mechanism of genetic inactivation was controversial. To obtain mouse monoclonal antibodies (mAbs) against protein E will allow the observation of its location and dynamic expression to expand understanding of the lysis mechanism. In this study, a His-tagged ΔE fusion protein expressed in bacteria was used as the immunogen, and mAbs targeting protein E were produced by the hybridoma technique and selected by enzyme-linked immunosorbent assay using GST-E and GST-ΔE as coating proteins. Purified mAbs from mouse ascites were screened against a phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 30 phage clones were randomly selected and sequenced, and their amino acids were deduced. One epitope showed a good match with protein E at 57–63aa (KPLN--R) and the synthetic decapeptide Epep (VRLKPLNCSR) strongly inhibited combination of E-A5 with E fusion proteins. Immunofluorescence microscopy indicated specific immunoreactivity of E-A5 with Escherichia coli-expressed native protein E. The novel mAbs may be of great potential value in analysis of the function and lysis pathway of protein E and other relative phages and in evaluation of E as potential marker of bacterial ghosts.