We report here the construction of several chimeric genes encoding the binding domains of diphtheria (DTx-B), pertussis (PT-S2), and tetanus (TT-B) toxins, as well as a modified PT-S1 enzymatic subunit, which were placed downstream and in-frame with the immunomodulatory cholera toxin B-subunit (CT-B) gene. Each chimeric gene construct was hyperexpressed in Escherichia coli, and the fusion proteins, that is, CT-B::DTx-B, CT-B::PT-S2, and CT-B::TT-B, reacted with antibodies to each component of the chimeras in an ELISA and Western blot analysis. The hyperproduced proteins induced significant antibody titers in mice against both components of the chimeric proteins. A mutagenized PT-S1 subunit gene construct was made by first using site-directed mutagenesis to modify codons encoding amino acid residues Arg9 and Glu129, which are responsible for the ADP-ribosyltransferase activity of the PT-S1 subunit. The codons for these amino acids were replaced with those encoding Lys and Gly, respectively, and the genetically inactivated PT-S1(*): gene was ligated downstream of the CT-B gene and expressed in E. coli. After hyperexpression, antibodies generated against the CT-B::PT-S1(*) construct neutralized the Chinese hamster ovary (CHO) cell clustering activity, which is a typical PT biologic response. Further, the antibodies blocked the elongation of CHO cells, which is a characteristic response of these cells to CT. These chimeric antigens may be beneficial in the development of alternative recombinant vaccines with minimal toxic side effects compared with those seen with the whole cell DPT vaccine.
|Original language||English (US)|
|Number of pages||13|
|State||Published - Jan 1 1997|
ASJC Scopus subject areas
- Microbiology (medical)