Expression of recombinant preenzyme Blo t 1 allergen in Escherichia coli and its IgE reactivity

Blomia tropicalis is a predominant house dust mite in tropical-subtropical climates, and its allergen Blo t 1 is recognized as a key trigger of allergic responses. This study aimed to express, purify recombinant Blo t 1 from B. tropicalis collected in Vietnam and assess its IgE-binding capacity using Vietnamese allergic sera. The cDNA encoding Blo t 1 was amplified from local samples, cloned into pET32b (+), and expressed in Escherichia coli BL21 (DE3). IgE-binding properties were evaluated through dot blot and Western blot assays. The recombinant Blo t 1 showed anomalous SDS-PAGE migration at ∼63 kDa, comprising ∼38.11 kDa from 333 amino acids of the pre-enzyme form of Blo t 1 and ∼19.47 kDa derived from vector-associated sequences, including His-tag. The protein was successfully expressed and purified using His-tag affinity chromatography. Dot blot analysis showed that 19/30 sera (63.33 %) from B. tropicalis-sensitized patients reacted with the recombinant protein, whereas none of the 24 control sera did. Western blot confirmed IgE-binding specificity. The recombinant Blo t 1 was purified under denaturing conditions, so the native three-dimensional structure is unlikely to be retained, and the observed IgE reactivity mainly reflects linear epitope recognition. Even so, the protein represents a useful research material corresponding to a Blo t 1 variant circulating in Vietnam. Along with expanding the limited molecular and IgE-reactivity data available for B. tropicalis in Southeast Asia, it also provides a basis for studies on allergen structure, epitope features, and early exploratory work related to allergen-specific immunotherapy and SIT-oriented approaches.