Serum samples from 30 individuals with HDM allergy (13 males and 17 females; age range, 18-55 years) and 24 non-allergic individuals (10 males and 14 females; age range, 18-55 years) were obtained from the First Affiliated Hospital of Guangzhou Medical College (Guangdong, China). The allergic patient sample consisted entirely of individuals who had experienced anaphylaxis caused by HDM exposure and who had IgE levels >3, measured using a clinical ImmunoCAP allergen detection system (Phadia; Thermo Fisher Scientific, Inc.). Patient clinicopathological information, including sex, age, ImmunoCAP HDM-specific IgE levels and previous diagnoses are presented in Table I. Ethics approval for the current study was obtained from the First Affiliated Hospital of Guangzhou Medical College (approval no. 2012-51). Informed consent was obtained from all individual participants included in the study. All procedures involving human participants were in accordance with the ethical standards of the committee. Sera pooled from 10 patients with HDM allergy were used to detect the ability of IgE-binding to recombinant Der (rDer) f 24 and hybrid proteins.

Female BALB/c mice (4-5 weeks old) were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China), and housed in a specific pathogen-free environment with a relatively stable temperature (24 ± 1°C) and humidity (55 ± 10%) for >1 week before experimentation. All studies involving mice were approved by the Animal Ethical and Welfare Committee of the School of Medicine of Shenzhen University (Guangdong, China).

The amino acid (aa) sequences of Der f 24 (GenBank no. AJK91617.1), Der p 24 (GenBank no. KP893174.1), human UQCRB (GenBank no. EAW91749) and mouse UQCRB (GenBank no. NM_026219) were obtained from the National Center for Biotechnology Information library (NCBI; https://www.ncbi.nlm.nih.gov/protein/) and imported into DNAMAN 8 software (version 8; Lynnon LLC) for homology comparison. FASTA-format sequences were obtained for further analysis.

Human UQCRB has not been reported to have IgE epitopes that induce an autoimmune response. Hybrid Der f and human UQCRB protein sequences (Table II) were designed and the resultant cDNA sequences were synthesized by GenScript (Nanjing) Co. Ltd. The following synthetic genes were registered in the GenBank database: Der f 24-Hyb1 [mite, 1-59 aa; human, 60-111 aa (accession no. KP939229)]; Der f 24-Hyb2 [human, 1-59 aa; mite, 60-118 aa (accession no. KP939230)]; Der f 24-Hyb3 [mite, 1-78 aa; human, 79-111 aa (accession no. KP939231)]; and Der f 24-Hyb4 [mite, 1-39 aa; human, 40-111 aa (accession no. KP939232)]. These cDNAs were cloned into pET-His vectors (Miaoling Bioscience and Technology Co., Ltd.). The recombinant plasmids were confirmed by DNA sequencing, and for protein expression, 100 ng DNA was transformed into Escherichia coli BL21 (DE3) pLysS competent cells (Novagen; Merck KGaA) by heat shock. The expression and purification of recombinant protein was performed as described previously (28). The rDer f 24 protein and the hybrid proteins were isolated in the form of inclusions. They were purified by Ni-NTA gel affinity chromatography (GE Healthcare) and subjected to IgE-western blotting or IgE-dot blotting. rDer f 24 protein quality was evaluated by determining the activity of IgE-binding with HDM-allergic sera, and required a positive rate of ~50% using 10 individual HDM-allergic sera in IgE-ELISA (9). The recombinant protein concentrations were determined using the Bradford method (Bio-Rad Laboratories, Inc.). The recombinant proteins (40 μg/well) were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel was stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories, Inc.).

Five overlapping Der f 24 polypeptides (31-33 aa) were designed and synthesized by Shanghai Qiang Yao Biotechnology Co. Ltd. The overlapping regions were 10 aa long. The polypeptides were coupled with bovine serum albumin (BSA; Amresco, LLC) or ovalbumin (OVA; Sigma-Aldrich; Merck KGaA) at the cysteine residue of the N- or C-terminus using a 3-maleimidobenzoic acid N-hydroxysuccinimide method, which was performed by Shanghai Qiang Yao Biotechnology Co. Ltd.

rDer f 24 and hybrid proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore) for western blotting. For dot blotting, 2 μl rDer f 24 (concentration 1 μg/μl) and synthetic polypeptides (BSA-1-32 aa, BSA-23-54 aa, BSA-45-76 aa, BSA-67-98 aa and BSA-89-118 aa) were dotted onto PVDF membranes. The membranes were blocked with 5% Difco™ skim milk (DSM; BD Biosciences) diluted in Tris buffered saline containing 0.05% Tween-20 at 4°C overnight. Western blot membranes were incubated with a mixture of allergic sera from different 10 patients with HDM allergy (1:10 dilution) for 2 h at 37°C. For dot blotting, a mix of 10 serum samples from HDM-allergic patients with high IgE-binding to rDer f 24 and a mix of 3 non-allergic sera samples (1:10 dilution) were incubated for 2 h at 37°C. Following washing, the membranes were incubated with horse radish peroxidase-conjugated mouse anti-human IgE antibody (1:2,000 dilution; cat. no. 9160-05; SouthernBiotech) for 1.5 h at 37°C. Antigen-antibody complexes on membranes were visualized using a Pierce™ 3'-diaminobenzidine substrate kit (Thermo Fisher Scientific, Inc.).

rDer f 24 protein and conjugated polypeptides (200 ng/well; diluted in carbonate buffer; pH 9.6) were coated onto 96-well plates, and incubated at 4°C overnight. The plates were blocked with 5% DSM in phosphate-buffered saline containing 0.05% Tween-20 (PBST) for 3 h at 37°C. Washed plates were incubated with sera (1:10 dilution) from 15 HDM-allergic patients and 15 non-allergic individuals for 2 h at 37°C. Serum IgEs were then detected by incubating with a mouse anti-human IgE horseradish peroxidase-conjugated antibody (1:2,000 dilution; SouthernBiotech) for 1.5 h at 37°C. The plates were washed with PBST buffer. The reaction was detected by adding 100 μl 3,3,5,5′-tetramethylbenzidine substrate (1 mM; Invitrogen; Thermo Fisher Scientific, Inc.), and absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). After screening, the 15 HDM allergic sera all displayed positive reaction to allergen Der f 24.

Female BALB/c mice (6-8 weeks of age; 17-20 g in weight) were purchased from Guangzhou Experimental Animal Center (Guangdong, China). The mice (n=3) were sensitized by intraperitoneal injection of 50 μg OVA or OVA-1-32 aa conjugate with Imject™ Alum adjuvant (2:1 v/v in total volume 200 μl; Thermo Fisher Scientific, Inc.). On day 21 after injection, the mice were euthanized by cervical dislocation under deep isoflurane anesthesia and the sera were collected. Serum levels of specific IgE and IgG targeting the BSA-1-32 conjugate were measured by ELISA as described previously (29). The health status of the experimental mice was monitored twice daily and humane endpoints were used to determine if the mice met criteria to be euthanized. These criteria included weight loss >10-15%, lethargy, inability to stand and anorexia. Mice that met the designated criteria were euthanized by cervical dislocation under deep isoflurane anesthesia.

The rat mast cell leukemia cell line, RBL-2H3 (Guangzhou Cellcook Biotech Co., Ltd.) was cultured in complete Dulbecco's modified eagle medium with 4.0 mM L-glutamine, sodium pyruvate penicillin (100 U/ml), 100 μg/ml streptomycin, non-essential amino acids and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C and 5% CO₂. A cell degranulation assay was performed as previously described (30). The RBL-2H3 mast cell cells were seeded in 96-well plates for 18 h. After washing three times using Tyrode's buffer (Beijing Solarbio Science & Technology, Co., Ltd.), cells were exposed to sera of sensitized mice for 2 h and then stimulated with 10 μg/ml BSA-1-32aa conjugate for 45 min. Degranulation was assessed by measurement of β-hexosaminidase release into the supernatant and of unreleased enzyme in the respective cell lysate.

The structure of cytochrome b-c1 complex subunit 7 (QCR7, also known as UQCRB) template from Bos Taurus was obtained from the RCSB Protein Data Bank (http://www.rcsb.org/; PDB code, 5klv.1), and was used as template for 3D modeling of the Der f 24 protein. The 3D structure of Der f 24 was modeled in SWISS-MODEL online software (http://swissmodel.expasy.org/) based on the bovine QCR7 template. Ramachandran plots and root mean square deviation (RMSD) structure assessment methods were used to assess the reliability of the predicted 3D model using the PDBsum database (http://www.ebi.ac.uk/pdbsum/) which specializes in structural analyses and pictorial representations of models. The loci of 5 synthetic polypeptides in the Der f 24 protein were indicated with color-coding for IgE-binding activity analysis. The key residues in the 1-32 aa domain region of Der f 24 were evaluated using the Immune epitope database (IEDB) prediction online tool (https://www.iedb.org/) (31). Amino acid property scales for predicting antigenic determinants were developed using the BepiPred Linear Epitope Prediction tool (version 1.0; http://www.cbs.dtu.dk/services/bepipred/).

Experimental data are presented as mean ± SD. Statistical significance was calculated using GraphPad Prism 7 (GraphPad Software, Inc.). Group differences were determined by analysis of variance followed by Dunnett's test for multiple comparisons. P<0.05 were considered to indicate a statistically significant difference.

The Der f 24 gene encodes a protein sequence of 118 aa and is registered in the NCBI library. As presented in Fig. 1, a homology comparison in DNAMAN 8 showed Der f 24 was highly homologous to Der p 24 (96.61%). Human UQCRB protein was highly homologous to mouse UQCRB (86.61%). However, low aa homology was observed between Der f 24 and the human UQCRB protein (39.34%).

Pooled samples of serum from patients with HDM allergy were found to have improved IgE specificity compared to individual samples in preliminary experiments. Therefore, pooled serum samples were used for screening the immunodominant IgE epitope of the allergen. The Der f and human components of purified rDer f 24, hybrid Der f proteins and human UQCRB protein are presented in Fig. 2A. IgE-western blotting was performed on these proteins by probing with sera pooled from HDM-allergic patients. All of the recombinant proteins except for Der f 24-Hyb2, which lacked the N-terminal 59-residue region of Der f 24, showed positive IgE-binding (Fig. 2B and C).

The IgE epitopes of Der f 24 were further investigated using overlapping synthetic polypeptides coupled with BSA at the N- or C-terminus (Fig. 3A; Table II). IgE-ELISA results revealed that the 1-32 aa peptide group had a stronger binding to HDM allergic-serum IgE compared with that for the other four groups (Fig. 3B). Likewise, IgE-dot blotting showed that the BSA-1-32 aa group had a robust positive reaction to pooled sera from 10 allergic individuals, while the other groups did not have a strong positive reaction (Fig. 3C and D). The rDer f 24 protein was subjected to SDS-PAGE and transferred to PVDF membrane for competitive IgE western blotting. With the addition of increasing concentrations of the BSA-1-32aa conjugate, the capacity of rDer f 24 binding to IgE from HDM allergic sera became weaker (Fig. 3E). These results suggested that the B cell epitope of Der f 24 may be located mainly in the N-terminal 32-residue region.

To further explore B cell epitope immunogenicity, the titers of IgGs or IgEs targeting the BSA-1-32 aa conjugate were detected mice sensitized with OVA- or OVA-1-32aa conjugate, as OVA induces a Th2 response in vivo. At 3 weeks post-sensitization, the OVA-1-32 aa group produced high specific IgG and IgE antibody titers against the BSA-1-32 aa (Fig. 4A and B), confirming a role for the N-terminal 1-32 region of Der f 24 in IgE-binding activity. Importantly, serum from mice immunized with OVA-1-32aa, but not OVA-injected mice, promoted significant release of β-hexosaminidase from the mast cell line, RBL-2H3 with the addition of BSA-1-32 aa conjugate (Fig. 4C). Additionally, a competitive IgE-ELISA showed that pooled serum samples of the OVA-1-32 aa-sensitized mice reduced the ability of IgE in serum pooled from 10 HDM allergic patients to bind to the BSA-1-32 aa antigen (Fig. 4D). These data indicated that the 1-32 aa domain of Der f 24 contains the immunodominant IgE epitopes of Der f 24.

Superimposing the 3D structural model of Der f 24 on a bovine QCR7 template revealed sequence identity between the template and Der f 24 of 46.60%. Ramachandran plots and RMSD assessment showed that none of the residues were present in the disallowed region, 88 residues were present in the most favored regions, and the overall G-factor of the Der f 24 structure model was -0.01 (Fig. S1). In addition, structural analysis of homology modelling using the SWISS-MODEL system showed that the 3D structure of Der f 24 was highly similar to that of the QCR7 template (Fig. S2). The RMSD assessment indicated that there were 118 aligned residues in the superimposition (RMSD value, 0.207). These results indicate that the backbone dihedral angles, phi and psi, of the 3D model were within the acceptable range.

Homology modeling in the SWISS-MODEL software revealed that the secondary structure of Der f 24 consisted of three α-helices and a free loop (Fig. 5). The structure was positioned on the outside of whole folded protein. This external location may facilitate IgE-binding. According to the results of IEDB prediction, lysine (Lys) 28 and tyrosine (Tyr) 29 residues were located on the α-helices and were important for antigen-binding (Table III; Fig. S3). As the peptide 1-32 aa region (red domain in Fig. 5) of Der f 24 was located in a protruding region of the structure, the N-terminal 32-residue region may be more easily accessible for binding by specific IgEs.