Characterization of recombinant per a 10 from Periplaneta americana.

摘要

Cockroach allergen is a major risk factor for IgE-mediated allergic response and asthma in sensitized individuals. Serine proteases have been identified from various sources and characterized as major allergens. The present study was aimed to express and characterize recombinant allergen Per a 10 (rPer a 10) from Periplaneta americana. rPer a 10 was expressed in Escherichia coli and purified in soluble form, yielding 0.75 mg/liter of culture. Homology of the Per a 10 protein sequence exhibited 27 to 38% similarity to the mite serine protease and 41 to 52% similarity to other insect trypsins. The purified rPer a 10 protein resolved at 28 kDa on SDS-PAGE and was recognized by cockroach-hypersensitive patients' sera by immunoblotting and enzyme-linked immunosorbent assay (ELISA). In competitive ELISA, rPer a 10 required 96 ng of purified protein for 50% inhibition of IgE binding, whereas 34 ng of native protein (nPer a 10) was required for the same inhibition. rPer a 10 and nPer a 10 induced basophil histamine release in the range of 47 to 64% and 60 to 85%, respectively, when sensitized with cockroach-hypersensitive patients' sera. In conclusion, Per a 10 was subcloned, and the protein was purified to homogeneity. rPer a 10 showed reduced IgE binding and histamine release and showed no proteolytic activity. These data suggest that rPer a 10 has potential for immunotherapy.