ISOLATION AND PRELIMINARY CHARACTERIZATION OF CDNA-ENCODING AMERICAN COCKROACH ALLERGENS

Background: Two prominent proteins of 78 and 72 kd in Cr-PI have been found to be the major allergens of American cockroach (Periplaneta americana). Methods: A lambda gt22A cDNA library generated from messenger RNA of American cockroach was packaged into Escherichia coli Y1090(r(-)) and initially screened with rabbit polyclonal antiserum raised to crude extract of American cockroach (CRa-A). Results: Twenty-nine anti-CRa-A-positive clones were isolated and 11 clones were recognized by rabbit anti-Cr-PI and reactive with IgE antibodies of atopic serum pool. Among these 11 clones, eight were recognized by murine anti-Cr-PI monoclonal antibodies Four clones (C7, C8, C12 and C29) were found to contain inserts of 26 kilobases (kb), and clones C5 and C20 were found to contain inserts of 24 kb. The remaining clones (C13, C23, C25, C28, and C35) were found to contain inserts of 1.8, 1.6, 2.5, 1.7, and 0.9 kb, respectively. Clones C12, C20, C13, and C28 were selected, subcloned into the expression pET vectors, and used to transform E. coli BL21(DE3). Immunoblot analyses of clones C12, C20, C13, and C28 with anti-Cr-PI monoclonal antibodies revealed fusion proteins with molecular weights of 78 and 50 kd 72 and 43 kd, 54 kd, and 46 kd respectively. However, among those fusion proteins only those with molecular weights of 78, 72, 54, and 46 kd were able to bind human specific IgE antibodies. Conclusions: The cDNA clones are expected to code for the major and principal allergens of American cockroach, and recombinant allergens may therefore be valuable for diagnostic and therapeutic purposes.