The written informed consent was obtained from each participant for the use of peripheral blood samples and SPT. The serum and SPT of 37 allergic patients (22 female, 15 male; 8–86 years) were from The First Affiliated Hospital of Guangzhou Medical University. The sera of 3 healthy subjects (3 male, 8–15 years) were recruited from Shenzhen Children's Hospital. The 11 children among the subjects were approved by legal guardians. The samples were collected between January 2014 and December 2015. the present study was approved by the ethic Committee of the Institutional Review Board of the School of Medicine, Shenzhen University.

In a previous study, the draft genome of Dermatophagoides farinae were assembled using high-throughput sequencing (20). The gene sequence of Der f TEF 2 was obtained by high-throughput sequencing from Dermatophagoides farinae (Hong Kong Bioinformatics Centre, Hong Kong) and then the gene of Der f TEF 2 was synthesizing by Sangon Biotech in Shanghai. The 18 µl cDNA mixed with 2 µl (1/10 volume) of 10X DNA loading buffer (Beijing Solarbio Science & Technology Co., Ltd.) were added to the sample tank and the electrophoresis started. After the electrophoresis, the gel was stained by ethidium bromide, and images were captured under a UV lamp with a wavelength of 302 nm.

The cDNA of Der f TEF 2 was inserted into a pMD-18T vector (Takara Bio, Inc.), cloned into the BamHI and HindIII sites and the plasmids were heat-transformed into E. coli Top10 (Invitrogen; Thermo Fisher Scientific, Inc. The bacteria were incubated in LB medium (Beijing Solarbio Science & Technology Co., Ltd.) with 100 mg/l ampicillin and cultured overnight at 37°C. The recombinant colonies were transferred into LB medium with ampicillin to expand the culture, followed by plasmid extraction using plasmid DNA extraction kit (Omega Bio-Tek, Inc). The recombinant plasmid was characterized using BamHI and sequenced by BGI Group. After sequencing, the correct clone was ligated into the pET-32a expression vector (Novagen; Merck KGaA) at 37°C for 4 h. Initial cloning was performed with the E. coli strain Top10 cultured overnight at 37°C; a single colony was picked, and the plasmid was extracted for identification using BamHI and HindIII restriction enzymes. pET-32a-TEF 2 was transformed into E. coli BL21 (Invitrogen; Thermo Fisher Scientific, Inc.) cells for expression. The cells were grown in LB medium supplemented with 50 µg/ml ampicillin until the logarithmic phase was reached (A600 nm =0.6–0.9). Expression of TEF 2 was induced by adding 20 µl of isopropyl-D-thiogalactopyranoside (IPTG; 1 mol/l) to the LB medium. After 4 h of continued incubation at 37°C, the bacteria were harvested via centrifugation at 11,180 × g at room temperature for 5 min and 1 ml of the bacterial culture resuspended in 100 µl deionized water and mixed with 20–25 µl 10X protein SDS-PAGE loading buffer (Beijing Solarbio Science & Technology Co., Ltd.). The expression of the recombinant proteins was analyzed using 12% SDS-PAGE electrophoresis.

Following the induced expression of the recombinant protein, the samples were lysed using ultrasonic treatment at 4°C and an amplitude of 38% for 5 min (1 sec pulse on and 0.1 sec pulse off). The supernatant was purified using a balanced Ni-NTA column (ShangHai, Sangon Biotech, cat. no. C600792, 5 ml, 90 µm) at a speed of 2 ml/min and 4°C. After washing with a washing buffer (50 mM Tris, 40 mM imidazole and 0.5 M NaCl, pH 8), the protein was eluted slowly using an elution buffer (50 mM Tris, 0.3 M imidazole and 0.2 M NaCl, pH 8). The elution peak was collected by elution buffer (50 mM Tris, 0.3 M imidazole and 0.2 M NaCl, pH 8).

The HDMs allergic sera IgE antibodies specific to TEF 2 were measured indirectly using ELISAs. Briefly, 96-well microtiter plates were coated with 100 ng/well of TEF 2 at a concentration of 1 µg/ml in carbonate buffered solution (15 mM Na₂CO₃ and 35 mM NaHCO₃, pH 9.5) at 4°C overnight and then blocked with 200 µl 3% BSA in PBS at 37°C for 120 min. Each well was prepared with the serum (diluted 1:5; 100 µl/well) as the primary antibody or 3% BSA (as a negative control, Beijing Solarbio Science & Technology Co., Ltd.) and incubated at 37°C for 60 min. The plates were incubated with peroxidase-conjugated goat anti-human IgE (1:2,000, A18793, Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 60 min. Each incubation step was followed by three washes with PBS-0.05% Tween 20 (PBST). Color was developed by adding 100 µl/well TMB at 37°C for 10 mins and terminated by the addition of 2 M H2SO4 (50 µl/well). Absorbance was determined using a microplate reader at 450 nm.

The serum of patients allergic to TEF 2 was used for inhibition experiments to determine the cross reactivity among TEF 2 and HDMs. Patients' sera (supplied pre-diluted in 2% BSA in PBST) were pre-incubated with purified Der f TEF 2 or dust mite extract (DME) obtained from mites that were cryopreserved in liquid nitrogen and then ground to a powder (final concentration: 0.00001, 0.0001, 0.001, 0.01 or 1 g/ml) at 4°C overnight. Pre-incubated Der f TEF 2 or DME as the primary antibody were added to the microtiter plates pre-coated with TEF 2 or DME (0.1 µg/well) and the ELISA was performed as in the previous section. According to ELISA test procedures, data from three experiments were collected. The inhibition rates were calculated according to the following formula: Inhibition (%)=(OD₀-OD_inhibitor)/(OD₀-OD_BSA), where: OD₀=the optical density of antigen without any inhibitor, OD_inhibitor=the optical density after adding an inhibitor (0.00001, 0.0001, 0.001, 0.01 or 1 g/ml TEF2 or DME), and OD_BSA=the optical density with only BSA in the plate.

SPTs were performed with purified and endotoxin-removed TEF 2 (0.01 mg/ml). dissolved in phosphate buffer (pH 7.4, 50 mM phosphate buffer, 100 mM NaCl). Glycerin was added to a final concentration of 50%. The sample contained histamine phosphate (5 mg/ml) as the positive control or saline as a negative control. The results were checked 20 min after the SPT. The results were classified as follows: The prick spot became a wheal and fleck surrounding the wheal, it was positive (+); 3+, the response was the same as or stronger that of the histamine control; 2+, the response was weaker than that of the histamine control but stronger than that of the negative control; 1+, the response was significantly weaker than that of the histamine control but slightly stronger than that of the negative control; negative, no response (21).

SPTs and sera from non-allergic children were used to assess immunogenicity. TEF 2 was subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane for immunoblot analysis. The membrane was blocked with 3% BSA for 1 h at 37°C and incubated with SPTs and sera from non-allergic children (diluted 1:5 with 5% BSA-PBST, PBST: PBS containing 0.05% Tween 20; 100 µl/well) for 1 h at room temperature. After washing five times with TBST, the membrane was incubated with a secondary antibody (peroxidase-conjugated goat anti-human IgE, 1:2,000, A18793, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at 37°C. Finally, after washing three times with TBS-T, the membrane was developed with a DAB kit (Invitrogen; Thermo Fisher Scientific, Inc.).

The open reading frame (ORF) of TEF 2 was analyzed using the NCBI database (http://www.ncbi.nlm.nih.gov/), its physicochemical properties were predicted using the ProtParam Tool (http://web.expasy.org/protparam/), and the amino acid sequence was predicted using the Translate Tool (http://web.expasy.org/translate/) (22,23). NetPhos3.1 (http://www.cbs.dtu.dk/services/NetPhos/) was used to predict phosphorylation sites, and Subcellular localization of cathepsin D was predicted by CELLO 2.5 (24). ProtScale (http://web.expasy.org/protscale/) was used to assess the hydrophilicity, average flexibility and the relative mutability (25,26). Secondary structural elements were obtained using DNAStar. InterPro5.0 (http://www.ebi.ac.uk/interpro/), ScanProsite (http://prosite.expasy.org/scanprosite/) and MotifScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan) was used to assess functional sites. The phylogenetic tree was constructed using BLAST, MEGA 5.0 and ClustalX 2.1 software (27). The Bioinformatics Predicted Antigenic Peptides (BPAP) system (http://imed.med.ucm.es/Tools/antigenic.pl), DNAStar Protean system (DNAStar Inc, Madison, WI, USA) and the BepiPred 1.0 server (http://www.cbs.dtu.dk/services/BepiPred/) were used to predict the B cell epitopes of TEF 2 (28–30). Furthermore, the Immune Epitope Database, Propre, SYFPEITHI, Net-MHCII 2.2 server and NetMHCIIpan-3.0 server were used to study the T cell epitopes of Der f TEF 2 (28–30).

All data are presented as the mean ± SD and analyzed SPSS 18.0 statistical software (SPSS, Inc.). Student's t-test was used for the mean differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

The cDNA of Der f TEF 2 showed bright bands at ~2,000 bp after double enzymatic digestion (Fig. 1A). The sequencing results showed that one ORF of Der f TEF 2 is 2,535 bp long and encodes 844 amino acids from the ATG start codon to the TAA stop codon (Fig. 1B). Alignment between Der f TEF 2 and the homologous amino acid sequences from different species of Insecta, Merostomata and Arachnida was conducted using BLAST. Der f TEF 2 is 94, 93 and 87% similar to Sarcoptes scabiei (GenBank: KPM11996.1), Rhagoletis zephyria (GenBank: XP_017490130.1) and Galendromus occidentalis (GenBank: XP_003744110.1), respectively, indicating high homology (Fig. 2A). The homologous sequence was output in FASTA format after using MEGA 5.0 and ClustalX 2.1 software to construct the molecular evolutionary tree. The results showed that the TEF 2 gene from Der f has a relatively close relationship with the TEF 2 gene from Lasius niger, D. farina, Camponotus floridanus, Harpegnathos saltator, Melipona quadrifasciata and Sarcoptes scabiei (Fig. 2B).

The SDS-PAGE analysis showed that TEF 2 was successfully expressed had a molecular weight of ~100 kDa as predicted (Fig. 3A). To determine the allergenicity of recombinant TEF 2, immunoblot analysis was used to determine if the recombinant protein could react with serum from patients that are allergic to HDMs; the IgE-binding band had a molecular weight of ~100 kDa as predicted (Fig. 3B). The serum IgE from patients allergic to HDMs that was bound by recombinant TEF 2 was >6-fold higher compared with the serum IgE from healthy controls when quantified via ELISA (Fig. 3C). For ELISA inhibition assays, dilutions of recombinant TEF 2 or DME were incubated with the serum. The incubation inhibited the IgE binding of the serum obtained from patients to the coated DME or recombinant TEF 2 in a dose-dependent manner. The inhibition rate of recombinant TEF 2 against DME was ~60% at 0.1 mg/ml (Fig. 3D). These results suggested that TEF 2 may be a novel allergen from the HDM allergen family that leads to type I hypersensitivity.

SPTs were performed with Der f TEF2 in 37 patients sensitized to HDMs; 6 patients (16.2%) showed a positive reaction (Table I).

As shown, there was no cleavage site in the amino acid sequence as predicted using SignalP 4.1 (Fig. 4A). Prediction of the physicochemical properties of Der f TEF 2 using the ProtParam Tool estimated that the molecular formula was C_4,216H_6,703N_1,141O_1,240S₄₇, with a molecular weight of 94,722.32 Da and a theoretical isoelectric point of 6.25. The grand average of hydrophobicity (GRAVY) was predicted to be −0.222, the aliphatic index was 87.86 and the instability index was predicted to be 34.13. According to the definition provided by ProtParam, when the instability coefficient is <40, the protein is predicted to be stable; therefore, the TEF 2 protein was classified as stable. Combined with the analysis from the ProtScale software scale ‘Hphob./Kyte & Doolittle’, these results indicated the hydrophilicity of the protein. It showed that the most hydrophilic site of Der f TEF 2 appeared at amino acid position 62 with a score of −3.3456, and the most hydrophobic site appeared at amino acid position 132 with a score of 2.333 (Fig. 4B). The maximum score for the average flexibility was calculated to be 0.501 at amino acid position 706; the minimum score at amino acid position 481 was 0.376 (Fig. 4C). The relative mutability was calculated to have a maximum score of 103.000 at amino acid position 191; the minimum score, at amino acid position 716, was 47.889 (Fig. 4D). Using the Polarity/Zimmerman scale, the individual values were scored at a maximum of 39.766 at residue 62 and at a minimum of 0.237 at residue 745 (Fig. 4E). The accessible residues (%) were calculated as a minimum score of 3.478 at residues 279 and 280, and a maximum score of 7.900 at residues 314–316 (Fig. 4F).

NetPhos3.1 identified 31 phosphorylation sites. In total, 17 Ser residues (23, 41, 76, 97, 109, 116, 211, 214, 264, 391, 404, 488, 564, 570, 573, 664 and 833), 8 Thr residues (59, 69, 298, 390, 473, 558, 568, and 824) and 6 Tyr residues (179, 271, 359, 420, 443 and 840) were identified as potential phosphorylation sites (Fig. 5). The secondary structure prediction of Der f TEF 2 with DNAStar predicted 27 α-helices and 6 β-sheets in the protein (Fig. 5). The subcellular localization of Der f TEF 2 was predicted to be in the plasma membrane and extracellular space (Table II). Moreover, Der f TEF 2 was predicted to be a P-loop-containing nucleoside triphosphate hydrolase, Translation protein family member with G_TR_2 and G_TR_1 peptidase domains (Table III).

BPAP, DNAStar Protean and BepiPred 1.0 were used to predict the B cell epitopes of Der f TEF 2. The hydrophilicity, surface accessibility and flexibility of proteins play an important role in the formation of antigens. When the hydrophilic score is >0, the antigen index is >0 and surface accessibility is >l, an epitope is likely to be formed. Consolidating the predictions of these three tools, 17 potential B cell epitopes were identified on Der f TEF 2 (amino acids 29–35, 55–64, 92–99, 173–200, 259–272, 311–318, 360–365, 388–395, 422–428, 496–502, 512–518, 567–572, 580–586, 602–617, 785–790, 811–817 and 827–836; Fig. 5). The SYFPEITHI, NetMHCII 2.2 server, NetMHCIIpan-3.0 server, Immune Epitope Database and Propred were applied to predict the T cell epitopes of Der f TEF 2. Based on the results of these five immunoinformatics tools, the potential epitopes of Der f TEF 2 were predicted to comprise 14 peptide sequences (amino acids 1–15, 65–79, 120–134, 144–159, 236–250, 275–289, 404–418, 426–440, 463–477, 510–524, 644–658, 684–698, 716–730 and 816–830; Table IV and Fig. 5).