IgE binding reactivity of peptide fragments of Bla g 4, a major German cockroach allergen

Abstract

Cockroaches have been recognized as a major cause of asthma. Bla g 4 is one of the most important German cockroach allergens. The aim of this study is to investigate IgE reactivity to the recombinant Bla g 4 (rBla g 4) in the sera of allergic patients and identify linear IgE binding epitope. For protein expression, full-length Bla g 4 (EF202172) was divided into 5 overlapping peptide fragments (E1: aa 1-100, E2: aa 34-77, E3: aa 74-117, E4: aa 114-156, and E5: aa 153-182). The full-length and 5 peptide fragments of Bla g 4 was generated by PCR and over-expressed in E. coli BL21 (DE3). The IgE binding reactivities of the full-length and peptide fragments were measured by ELISA using 32 serum samples of cockroach allergy. The sera of 8 patients (25%) reacted with rBla g 4. Four sera (100%) showed IgE-binding reactivity to full-length and peptide fragment 4, and 2 sera (50%) reacted with peptide fragment 2. One (20%) serum reacted with peptide fragment 3. The results of ELISA using overlapping recombinant fragments indicated that the epitope region was located at amino acid sequences 34-73 and 78-113, and major IgE epitope of Bla g 4 was located at amino acid sequences 118-152 of C-terminal. B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy.

Keywords: Allergen; B-cell epitope; Blattella germanica; IgE; lipocalin.

Fig. 1

(A) Schematic representation of Bla g 4 fragments for epitope analysis. (B) SDS-PAGE of full-length and peptide fragments. Proteins run on 5-20% gradient SDS-PAGE gel and stained with Coomassie brilliant blue. M, Molecular weight markers in kDa; F, full-length; E1, 1-100 amino acid; E2, 34-77; E3, 74-117; E4, 114-156; and E5, 153-182.

Fig. 2

IgE reactivity of human sera against recombinant Bla g 4. The horizontal line indicates the cut-off value. ▴, sera from healthy controls; ♦, sera from German cockroach-sensitized subjects (Uni-CAP > 0.7 kU/L).

Fig. 3

(A) Binding profiles of IgE antibodies to recombinant Bla g 4 and relevant recombinant proteins by ELISA. 1-4, allergic sera; 5, pooled sera from healthy controls; 6, bovine serum albumin. The horizontal line indicates the cut-off value. (B) IgE-binding reactivities of peptide fragments of German cockroach allergen, Bla g 4.

Fig. 4

Sequence alignment of allergenic lipocalins: Bla g 4 (Blattella germanica, EF202172), Bos d 5 (Bos domesticus, X14712), Bos d 2 (B. domesticus, L42867), Equ c 1 (Equus caballus, U70823), Can f 1 (Canis familiaris, AF027177), Can f 2 (C. familiaris, AF027178), Mus m 1 (Mus musculus, X03208), Rat n 1 (Rattus norvegicus, M26835). ^*, fully conserved residues; : , strongly conserved residues; ·, weakly conserved residues. The shaded areas indicate the IgE-binding regions.