Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8

Abstract

Dermatophagoides farinae, a domestic mite species, produces some of the most potent allergens that contribute to allergy in China and worldwide. We sought to clone and express the group 8 allergen of D. farinae (Der f 8) to investigate its IgE-binding reactivity. The full-length cDNA encoding Der f 8 was generated by using RT-PCR and 5' RACE, cloned into pCold-TF expression vector, confirmed by nucleotide sequencing, sub-cloned into pET-28b (+), and transfected into E. coli BL21 cells for expression. After purification by nickel affinity chromatography and identified by SDS-PAGE, the recombinant Der f 8 bound with sera from 40.9 % (9/22) of mite-allergic patients according to ELISA testing. Analysis of the recombinant DNA sequence revealed a 231 amino acid open reading frame encoding a protein with a derived molecular mass of 26.4 kDa and an isoelectric point of 6.84. The deduced amino acid sequence has nine phosphorylation sites, displaying strong homology with glutathione S-transferase, and its secondary structure comprises alpha helix (45.5 %), extended strand (11.3 %), and random coils (43.3 %). BLAST through the National Center for Biotechnology Information database and alignment identified similarity with group 8 allergens or glutathione S-transferases of Dermatophagoides pteronyssinus, Suidasia medanensis, Lepidoglyphus destructor, Glycyphagus domesticus, and Aleuroglyphus ovatus (64, 65, 53, 53, and 50 %, respectively). The first recombinant Der f 8 protein produced in full length successfully bound with patient IgE, demonstrating the importance of Der f 8 in mite allergy.

Dust mite allergen, glutathione S‐transferase, induces T cell immunoglobulin mucin domain‐4 in dendritic cells to facilitate initiation of airway allergy

“…We found that the Derf8 gene sequence was similar to glutathione S-transferase; the latter has a close relation to allergic asthma [7]. Published data show that 40.9% of mite allergic patients had Derf8specific IgE in the sera [8,9]. It suggests that Derf8 may play an important role in the induction of allergy.…”

Dust Mite Allergen Der F 8 Promotes Th2 Polarization via Modulating Chromatin Structure at the TIM4 Gene Locus in Dendritic Cells

“…The group 8 allergen (Der f 8) of D. farina was cloned and characterized by us; the DNA sequence is listed in the NCBI database (http://www.ncbi.nlm.nih.gov/nuccore/KM010011.1), from which we found that the gene sequence of Der f 8 was quite similar to the gene sequence of glutathione S-transferase; the latter has a close relation with the pathogenesis of allergic asthma [9]. Previous studies have shown that 40.9 % of mite allergic patients with asthma exhibited IgE reactivity to Der f 8 [10,11]. It reveals that Der f 8 is a significant allergen of mite allergy, but the role of Der f 8 leading to allergic disorders remains to be further investigated.…”

“…We and others previously reported that TIM4 played a critical role in the expansion of Th2 response and allergic disorders [12][13][14]. Clinical data show that Der f 8-specific IgE can be detected in the patients with mite allergy [10]. Yet, the mechanism of the over expression of TIM4 by DCs in allergic diseases is unclear.…”

Transcriptomic/proteomic identification of allergens in the mite Tyrophagus putrescentiae

“…PCR products were inserted into expression plasmid pET‐28a(+) vector and transformed into Escherichia coli BL21 (DE3). Expressed recombinant proteins were identified with SDS‐PAGE . Recombinant proteins were purified using HiTrap TALON (5 ml TALON Superflow, 28‐9537‐66; GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) on AKTA Prime Plus (GE) and dialyzed with PBS buffer using SnakeSkin Dialysis Tubing (3500 MWCO, 68035, Lot No.…”