Functional objectives are to investigate genetic molecular structure of pathogens, to define the role of gene products in pathogenic mechanisms and to perform studies directed toward development of vaccines using molecular or synthetic production of immunogenic peptides from microbial agents. Our major emphasis is focused on cloning and expression of genes relevant to the toxic components of B. pertussis. Pertussis toxin is the protective component of the whooping cough vaccine; however, its toxin activity may cause the harmful side effects associated with current vaccines. The toxin is composed of five dissimilar subunits; subunit S1 contains an ADP-ribosyltransferase activity responsible for most of the toxin's biological activities. The complete structural gene for pertussis toxin has been cloned into E. coli and the nucleotide sequence as well as deduced amino acid sequences of the individual subunits have been determined. Using the sequence data, a Sau3A fragment containing DNA coding for about 75% of the mature S1 subunit was selected to be inserted into the BamH1 site of pUC18. Two clones were isolated. Clone 1 contained a Sau3A fragment extending from the second amino acid of the mature protein to amino acid number 187 of S1 in forward orientation (relative to the lac promotor) and clone 2 contains the same fragment in the reverse orientation. Expression of the partial S1 subunit in clone 1 was detected by Western blotting using an anti-S1 monoclonal antibody (Marchitto, K. et al., manuscript in preparation). Expression of S1 in clone 1 was inducible with IPTG. The molecular constructs were made in such a way that the expressed protein lacked the predicted NAD-binding site and therefore lacks toxic activities. This truncated S1 subunit may be useful for development of a safer new generation pertussis vaccine, since S1 is the immunodominant antigen in humans.
