Expression, epitope prediction and IgE‑binding of the Tyrophagus putrescentiae group 13 allergen
Affiliations: Department of Basic Medicine, Jiangsu Vocational College of Medicine, Yancheng, Jiangsu 224005, P.R. China, Department of Pediatrics Laboratory, Wuxi Children's Hospital, Wuxi, Jiangsu 214023, P.R. China, Department of Clinical Laboratory, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, P.R. China
- Published online on: August 27, 2020 https://doi.org/10.3892/etm.2020.9149
- Article Number: 21
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
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The non-predatory storage mite Tyrophagus putrescentiae is distributed worldwide (1) and can be found in a variety of stored goods, particularly in high-fat and protein-rich stores (2). In addition to causing damage to food stores, T. putrescentiae can cause human allergic diseases (3,4). Allergic diseases caused by T. putrescentiae are more common in Asia (5) and Europe (6).
At present, the diagnosis and allergen immunotherapy (AIT) of mite allergic diseases depend on the use of natural allergen extracts. However, natural allergen extracts are associated with a number of issues, including unpredictable amounts of allergens and non-allergenic components in natural extracts, which can lead to serious immediate and late side effects (7,8). The majority of these issues in allergy diagnosis and AIT can be circumvented with the use of recombinant allergens and peptides (9). In order to improve the safety of AIT, researchers have turned their attention to recombinant allergens with modified B-cell epitopes, which can prevent IgE binding, but with intact T-cell epitopes to produce an immunotherapeutic benefit (10). With regards to allergy diagnosis, studies on food allergens have revealed the potential role of B-cell epitopes (IgE-binding epitopes) as biomarkers for characterizing various phenotypes (11). Thus, peptide-ELISA or epitope-ELISA has the potential to lead to advances in the clinical diagnosis of allergic diseases.
A better understanding of T. putrescentiae-produced allergens may aid the diagnosis and treatment of allergies to this mite. A series of studies on the T. putrescentiae allergens from China have thus been conducted. Using molecular cloning techniques, the sequencing and characterization of group 4, 28, 35 and 36 T. putrescentiae allergens from China were reported in our previous studies (12,13). The T. putrescentiae group 13 allergen (Tyr p 13) was another allergic component in our series of studies. The identification of B-cell epitopes of Tyr p 13 may be helpful in designing sequences for more accurate and safer peptide-based allergen diagnosis and immunotherapeutic agents. However, the tertiary structure and epitope of Tyr p 13 remain unclear.
The aim of the present study was to clone and express the full-length sequence encoding Tyr p 13 from China. Combined with the prediction analysis of Tyr p 13 structure and B-cell epitopes, these results were intended for use in the diagnosis and treatment of anaphylaxis caused by T. putrescentiae and may lay the foundation for the design of recombinant Tyr p 13 (rTyr p 13) with modified B-cell epitopes.
Materials and methods
Peripheral blood samples were collected from patients with allergic asthma at the Respiratory Department, Wuxi People's Hospital (Wuxi, China). The peripheral blood samples were placed at room temperature for 1 h, and then centrifuged at 1,000 x g for 10 min at 4˚C to obtain the serum. Patients had not received any treatment and asthma diagnosis was performed following the World Health Organization criteria (14). The diagnosis of allergy to T. putrescentiae allergens was established on the basis of a suggestive clinical history and positive serum IgE (sIgE) to T. putrescentiae extracts, as determined by the Allergy Screening test panel for atopy (Mediwiss Analytic GmbH). The inclusion criteria were as follows: i) Allergy to T. putrescentiae extracts with >1 year of follow-up, and ii) levels of sIgE to T. putrescentiae extract >3 kU/l at the time of inclusion in the study. A total of 38 patients (male:female ratio, 18:20), with a mean age of 35.263±8.374 years, were enrolled as serum-positive for T. putrescentiae and their serum was used to detect specific IgE-binding to recombinant allergens. In addition, serum from five non-atopic healthy people (malefemale ratio=2:3), with a mean age of 35.8±8.727 years, was used as the negative control. All participants provided written informed consent. The study protocol was approved by the Ethics Committee for Clinical Investigation of Wuxi People's Hospital Affiliated to Nanjing Medical University (Wuxi, China).
T. putrescentiae culture and isolation of adult mites
To isolate T. putrescentiae, flour and dust were obtained from the floors of a flour storage warehouse in Yancheng, China. The collected sample was placed in a 9-cm glass petri dish and the sample was observed under a stereomicroscope. The suspected pregnant T. putrescentiae were picked under a stereomicroscope and placed separately in different culture flasks containing culture medium for cultivation. Under the stereomicroscope, pregnant mites moved slowly, crept on the bottom of the petri dish, formed egg cells in the body and their body shape was larger. After ~2 months, the whole culture in the flask was examined under the stereomicroscope. According to the movement speed, body type and body surface of the T. putrescentiae, the species of mites in the culture medium could be simply judged (15). Subsequently, mites considered to be T. putrescentiae were cultivated in small culture flasks for pure culture. After a further 2 months, the mites previously isolated and cultured were identified again. The identification of T. putrescentiae was performed in strict accordance with the morphological characteristics of T. putrescentiae (15). If the mite was confirmed to be T. putrescentiae, all mites in that flask were considered to be T. putrescentiae. During the cultivation process, these T. putrescentiae were raised in an atmosphere of 25˚C and 85% relative humidity. In the culture process of T. putrescentiae, the culture medium consisted of rice bran medium, flour and yeast powder (11). The isolation method of pure T. putrescentiae mites from the culture medium was the same as that of pure Dermatophagoides farinae mites (16).
Preparation of Tyr p 13 cDNA
Total RNA was obtained from T. putrescentiae using the Takara MiniBEST Universal RNA Extraction Kit (cat. no. 9767; Takara Biotechnology Co., Ltd.). Based on the published sequence of Tyr p 13 (GenBank accession no. DQ983316; https://www.ncbi.nlm.nih.gov/nuccore/DQ983316.1/), a pair of primers were designed: Forward, 5'-GGAATTCCATATGTCGGTCGAAGAAC-3' and reverse, 5'-CCGCTCGAGTTAATCACCAGTCATCATCTCC-3', with an NdeI and an XhoI site at their 5' ends (underlined), respectively. Reverse transcription (RT)-PCR was performed using total mite RNA and the High-Fidelity PrimeScript RT Reagent Kit with gDNA Eraser (cat. no. DRR047A; Takara Biotechnology Co., Ltd.) on a PCR Thermal Cycler Dice (cat. no. TP600; Takara Biotechnology Co., Ltd.). gDNA was removed from the total RNA samples; briefly, the reaction mixture (10 µl) contained total RNA (1 µl), 5X gDNA Eraser Buffer (2 µl), gDNA Eraser (1 µl) and RNase-free H2O (6 µl). The mixture was incubated at 42˚C for 2 min, and RT was performed according to the manufacturer's instructions. PrimeScript RT Enzyme Mix I (1 µl), RT Primer Mix (1 µl), 5X PrimeScript Buffer 2 (4 µl) and RNase-free dH2O (4 µl) were added to the aforementioned reaction mixture. The reaction mixture (20 µl) was incubated at 37˚C for 15 min and 85˚C for 5 sec. The RT product was used as the template for PCR using PrimeSTAR® HS DNA Polymerase (cat. no. R010A; Takara Biotechnology Co., Ltd.). The PCR mix comprised RT products (1 µl), 5X PrimeSTAR PCR buffer (10 µl), 2.5 mmol/l dNTP mixture (4 µl), 20 µmol/l forward primer (1 µl), 20 µmol/l reverse primer (1 µl), 2.5 U/µl PrimeSTAR HS DNA polymerase (0.5 µl) and dH2O (32.5 µl). For PCR, samples were incubated for 2 min at 94˚C, followed by 30 cycles at 98˚C for 10 sec, 65˚C for 30 sec and 72˚C for 40 sec. Finally, samples were incubated for 10 min at 72˚C and 5 min at 10˚C, and amplicons (5 µl) were analyzed by agarose gel electrophoresis (1.0%) containing ethidium bromide (cat. no. E7637; Sigma-Aldrich, Merck KGaA) and visualized with ImageMaster® VDS (Pharmacia Biotech).
Cloning and DNA sequencing
The PCR-amplified DNA was recovered from the gel using the Agarose Gel DNA Purification kit v2.0 (cat. no. DV805; Takara Biotechnology Co., Ltd.) and a poly-A tail was added using the DNA A-Tailing Kit (cat. no. 6109; Takara Biotechnology Co., Ltd.). The poly-A tailed product was cloned into the simple vector pMD20-T (cat. no. D107A; Takara Biotechnology Co., Ltd. according to the manufacturer's instructions. Competent Escherichia coli (E. coli) JM109 cells (cat. no. 9052; Takara Biotechnology Co., Ltd.) were transformed with the recombinant plasmid pMD20T-Tyr p 13 using the heat-shock method. Positive clones were selected by blue/white screening on Luria-Bertani (LB) plates containing 100 µg/ml ampicillin and samples were sequenced using Bca BEST sequencing primer RV-M/M13-47 (Takara Biotechnology Co., Ltd.) on an ABI PRISM 377XL DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
DNA sequence retrieval and analysis
The sequences were edited to remove the vector sequence and extra restriction sites using SeqMan on DNASTAR Lasergene software suite v7.1 (DNASTAR, Inc.). The open reading frame (ORF) was identified using the ORF finder (http://www.bioinformatics.org/sms2/orf_find.html) from the National Center for Biotechnology Information.
Physicochemical analysis and analysis of patterns
The sequence of Tyr p 13 was obtained by sequencing after pMD20T-Tyr p 13 was transformed into E. coli JM109 cells. The amino acid sequence of Tyr p 13 was predicted using Translate Tools in ExPaSy (https://web.expasy.org/protparam/). The family classification of rTyr p 13 was analyzed using InterPro v56.0 (www.ebi.ac.uk/interpro/) (17). The signal peptide sequence of rTyr p 13 was predicted by SignalP 4.1 software (http://www.cbs.dtu.dk/services/SignalP/), as previously described (17). Physiochemical analyses, including theoretical pI, aliphatic index, grand average of hydropathicity (GRAVY) and instability index of rTyr p 13, were predicted using ProtParam (web.expasy.org/protparam/) (17) and ProtScale tools (https://web.expasy.org/protscale/) (18). The phosphorylation sites of rTyr p 13 were determined using the NetPhos3.1 server (http://www.cbs.dtu.dk/services/NetPhos/), as previously described (17). The secondary structure was predicted using GOR4.0 (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html), as previously described (19). The TMHMM server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane protein helices (20).
Homology modeling was used to construct a tertiary structure of Tyr p 13. A BLASTP (blast.ncbi.nlm.nih.gov/Blast.cgi) search with default parameters was performed against the Protein Data Bank (PDB; www.rcsb.org/pdb/) to identify a suitable template for Tyr p 13. Based on this template, the tertiary structure of Tyr p 13 was predicted using SWISS-MODEL (https://www.swissmodel.expasy.org/). The generated model was evaluated using QMEAN, Global Model Quality Estimation (GMQE), ERRAT, VERIFY_3D, PROCHECK and ProSA-Web. QMEAN was used to to provide the global (for the entire structure) and local (per residue) error estimates on the basis of a single model, while GMQE was used to reflect the expected accuracy of a model built with that alignment and template and the coverage of the target, ERRAT (services.mbi.ucla.edu/SAVES) was used to analyze the statistics of non-bond interactions between different atom types, VERIFY_3D (services.mbi.ucla.edu/SAVES) was used to determine the compatibility of the atomic model (3D) with its amino acid sequence (1D) and compare the results to an advantageous structure, PROCHECK (services.mbi.ucla.edu/SAVES) was used to verify the stereochemical quality of the Tyr p 13 structure, and ProSA-Web (https://prosa.services.came.sbg.ac.at/prosa.php) was used to analyze whether the interaction between each residue of the model was within a reasonable range (21). Superimposition of the query and template structure and visualization of the generated models was performed using UCSF Chimera 1.10.2 (www.cgl.ucsf.edu/chimera/).
Structure-based prediction of Bcell epitopes
Although the reliable prediction of epitopes is challenging, the prediction of allergen epitopes is beneficial to the immunological diagnosis of allergic diseases. Linear and discontinuous B-cell epitopes were predicted using ElliPro: Antibody Epitope Prediction; IEDB analysis resource (http://tools.immuneepitope.org/tools/ElliPro/tutorial.jsp) (22). The rTyr p 13 protein sequence was analyzed using different analytical parameters, such as secondary structure, hydropathy, antigenicity, amphilicity, surface probability and flexibility in the Protean software of the DNASTAR Lasergene software suite v7.1(23).
Construction of expression plasmids pET28a(+)-Tyr p 13
The recombinant pMD20-T-Tyr p 13 plasmid was digested with NdeI and XhoI to release the Tyr p 13 cDNA. The cDNA was separated by agarose gel electrophoresis and purified from the gel using the Agarose Gel DNA Purification kit v2.0. The DNA Ligation kit (cat. no. D6023; Takara Biotechnology Co., Ltd.) was used to sub-clone the cDNA into the expression vector pET28a(+) (cat. no. N72770; Novagen; Merck KGaA) to create pET28a(+)-Tyr p 13. E. coli DH5α cells (cat. no. D9057; Takara Biotechnology Co., Ltd.) were transformed with pET28a(+)-Tyr p 13 plasmids. Positive clones were selected by blue/white screening on LB plates containing 50 µg/ml kanamycin and verified by restriction enzyme analysis with NdeI and XhoI.
Production and purification of recombinant allergen
The pET-28a (+)-Tyr p 13 plasmid (0.5 µl) was purified using the MiniBEST Plasmid Purification Kit Ver. 4.0 (cat. no. 9760; Takara Biotechnology Co., Ltd.) and used to transform 100 µl E. coli BL21 (DE3) cells (cat. no. 200131; Agilent Technologies, Inc.). The pET28a(+)-Tyr p13-transformed E. coli BL21 cells were cultured in 3 ml LB liquid medium containing 50 µg/ml kanamycin for 8 h at 37˚C. After 8 h of cell culture, 2 ml cell culture was diluted into 100 ml LB + kanamycin in a glass tube and cultured at 37˚C until an approximate absorbance of 0.5 at 600 nm was reached. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mmol/l. The sample was incubated for 2 h at 30˚C. E. coli cells were then harvested by centrifugation (8,000 x g) at 4˚C for 10 min, resuspended in 15 ml Tris-HCl (pH 7.5) buffer and sonicated (20 kHz; ultrasound duration 3 sec, interval 10 sec) in an ice bath until the suspension became transparent. The cell debris and supernatant were separated by centrifugation (5,000 x g) at 4˚C for 40 min. The presence of the recombinant protein was verified by 10% SDS-PAGE and Coomassie Brilliant Blue R-250 (CBB-R250) staining.
The supernatant was applied to a Ni-NTA affinity chromatography column (cat. no. 70971; Novagen; Merck KGaA) pre-equilibrated with buffer [50 mmol/l Tris-HCl (pH 7.5), 0.3 mol/l NaCl]. The column was washed with 10 ml washing buffer 1 [50 mmol/l Tris-HCl (pH 7.5), 10 mmol/l imidazole and 0.3 mol/l NaCl] to remove unbound proteins. Recombinant protein was eluted by a gradient of increasing concentrations of imidazole elution buffer. The elution flow rate was 1 ml/min. Eluents at different imidazole concentrations were collected and verified by 10% SDS-PAGE. The eluents containing high purity recombinant protein samples were mixed and desalinated by 12-h dialysis with several rounds of buffer replacement. The purified product was separated by 10% SDS-PAGE and observed by Coomassie Brilliant Blue R-250 (CBB-R250) staining.
Specific IgE-binding to recombinant allergen
The ability of rTyr p 13 to bind to IgE was assessed by ELISA. The rTyr p 13 used for ELISA was purified by Ni NTA affinity chromatography. For antigen coating, 100 µl rTyr p 13 (1 µg/ml) diluted in coating buffer (0.1 mol/l PBS)was added to a 96-well microtiter plate and incubated at 8˚C overnight. Wells were washed with 300 µl PBS-0.05%Tween-20 (PBS-T) buffer and blocked with 3% bovine serum albumin (cat. no. A1933, Sigma-Aldrich; Merck KGaA) in PBS-T for 1 h at 37˚C. Subsequently, 100 µl patient serum and 100 µl healthy donor serum (diluted at 1:4 with PBS-T) were added, followed by overnight incubation at 4˚C. Secondary antibody (50 µl; HRP-mouse anti-human IgE; 1:5,000; Sigma-Aldrich; Merck KGaA) was added and samples were incubated at 37˚C for 1 h. To detect binding, 100 µl TMB substrate solution (cat. no. P0209; Beyotime Institute of Biotechnology) was added and samples were incubated for 15 min at room temperature. A total of 2 mol/l H2SO4 (50 µl) was added to stop the reactions. Optical density (OD) was measured for triplicate samples at a wavelength of 450 nm using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Inc.). The reactivity was considered positive if the OD values of the detected serum were higher than the cutoff ELISA value (mean ELISA value of healthy donors + 3 SD).
Cloning Tyr p 13
Total RNA was isolated from adult mites, and Tyr p 13 cDNA was amplified by RT-PCR. A product of the expected size (486 bp) was produced (Fig. 1A). The product was cloned into the pMD20T vector and sequenced. Using the ORF Finder, a complete ORF was identified in the Tyr p 13 cDNA. The length from the start codon ATG to the stop codon TAA was 486 bp (Fig. 1B).
Cloning and sequencing of cDNA encoding Tyr p 13. (A) Amplification of Tyr p 13 cDNA by RT-PCR. Total RNA was isolated from mites and subjected to RT-PCR. The PCR products were separated on a 1% agarose gel containing ethidium bromide. Lane M, DNA Marker DL2, 000; Lane 1, PCR product (486 bp). (B) Nucleotide sequence and deduced amino acid sequence of Tyr p 13. Tyr p 13, Tyrophagus putrescentiae group 13 allergen; RT-PCR, reverse transcription-PCR.
Inferred amino acid sequence, and its structural and functional prediction
Family classification revealed that Tyr p 13 belongs to the intracellular lipid-binding protein family. An amino acid sequence of 161 residues was predicted for Tyr p 13 using the Translate Tools on the ExPaSy web server. A signal peptide sequence from amino acids 1 to 22 was predicted using SignalP 4.1 software. The removal of the signal peptide sequence predicted by the SignalP 4.0 software (Fig. S1) yielded a predicted mature Tyr p 13 protein of 139 amino acid residues with a theoretical pI of 8.07. The instability index was 26.7, indicating a stable amino acid sequence. The grand average of GRAVY was -0.49, indicating that Tyr p 13 was hydrophilic. The NetPhos3.1 server was used to search the Tyr p 13 amino acid sequence for phosphorylation sites. Nineteen sites were predicted (Fig. S2), including seven serine sites positioned at residues 6, 17, 39, 63, 71, 87 and 103, nine threonine sites at residues 12, 53, 58, 61, 68, 84, 97, 107 and 133, and three tyrosine sites at residues 19, 43 and 120. The secondary structure of Tyr p 13 was predicted to comprise an α-helix (15 peptides, 10.79%), an extension chain (47 peptides, 33.81%) and a random coil (77 peptides, 55.40%; Fig. 2). The Tyr p 13 protein sequences were entered into the TMHMM Server 2.0, which predicted no transmembrane helices, and inferred that all protein sequences were located outside the membrane.
Secondary structure analysis of the mature Tyrophagus putrescentiae group 13 allergen by GOR4.0 software. In the sequence, h, e, and c represent α-helix, extended strand and random coil, respectively.
Homology modeling and model evaluation
The fatty acid-binding protein in brain tissue of Drosophila melanogaster (PDB accession no. 5GKB) belongs to the intracellular lipid-binding protein family (InterPro no. IPR031259), has a high sequence identity with Tyr p 13 (36.43%), and was used as the template for homology modeling (Fig. 3A). In the Ramachandran plot, 96.6% of the residues were in favored regions, 2.6% were in additional allowed parts, 0.8% were in generously allowed regions and 0% percent were in outlier regions (Fig. 3B). Based on the ERRAT results, the overall quality factor was 96.64%, indicating that the tertiary structure of Tyr p 13 had a high resolution. The Verify3D test requires an average 3D-1D score of ≥80% of the amino acids to be ≥0.2. The VERIFY 3D results showed that 84.44% of residues had an average 3D1D score of ≥0.2, which suggested that the structures were favorable. ProSA analysis revealed that the predicted model was comparable to other acceptable proteins with a z score of -4.78 (Fig. 3C). The QMEAN server results revealed that the QMEAN Zscore was 1.61, thus suggesting that the protein model variation rate was low, overall folding and local structures had high accuracy rates, and stereochemistry was reasonable. Furthermore, the Q value was 0.7, which indicated that the predicted model of Tyr p 13 was reliable. These findings revealed that the tertiary structure model of Tyr p 13 was reliable and was suitable for use in the current study.
Three-dimensional structure and validation of Tyr p 13 homology model. (A) Structure of Tyr p 13 homology model. (B) Ramachandran plot of the Tyr p 13. (C) ProSA protein structure analysis results. Tyr p 13, Tyrophagus putrescentiae group 13 allergen.
Prediction of B-cell epitopes by an integrated strategy
α-Helices and β-sheets have been reported to have high chemical bond energies and to be unlikely to form epitope sequences (24). By contrast, β-turns and random coils are located in surface-exposed regions of a protein, which often contain epitope sequences (21). The secondary structure, hydrophilicity, antigenicity, amphilicity, surface probability and flexibility of Tyr p 13 were analyzed using the Protean Software (for Protein Structure Analysis and Prediction) of the DNASTAR Lasergene software suite Ver. 7.1 (Fig. 4). Based on these results, linear and discontinuous B-cell epitopes were predicted by ElliPro software (Table I). Five amino acid peptide sequences were identified as promising linear epitopes (Fig. 5A) and three clusters were predicted to form discontinuous epitopes (Fig. 5B).
Analysis of Tyrophagus putrescentiae group 13 allergen secondary structure, hydropathy, antigenicity, surface probability and flexibility by Protean software.
Possible B-cell epitopes within the 3D structure of Tyr p 13. (A) Linear Bcell epitopes within the 3D structure of Tyr p 13. (B) Discontinuous B cell epitopes within the 3D structure of Tyr p 13. Each color represents different linear/discontinuous epitopes, and the numbers correspond to the epitope numbers displayed in Table I. Tyr p 13, Tyrophagus putrescentiae group 13 allergen.
Expression and purification of recombinant allergen (rTyr p 13)
Tyr p 13 cDNA was sub-cloned into the pET-28a (+) vector to produce a recombinant His-tagged protein. The recombinant His-tagged protein (rTyr p 13) was successfully purified by Ni-NTA affinity chromatography (Fig. 6).
Expression of rTyr p 13 in E. coli BL21 cells. (A) SDS-PAGE of rTyr p 13 expressed in E. coli BL21 cells. Lane M, Takara Protein Marker (Broad); Lane 1, whole cell lysate of E. coli BL21 cells containing pET-28a as a negative control; Lane 2, whole-cell lysate of E.coli BL21 cells containing pET-28a (+)-Tyr p 13; Lane 3, supernatant of cells containing pET-28a (+)-Tyr p 13; Lane 4, pellet of cells containing pET-28a (+)-Tyr p 13. (B) SDS-PAGE of purified rTyr p 13. Lane M, Takara Protein Marker (Broad); Lane 1, protein flow-through from the column; Lanes 2, 3, 4 and 5, eluted fractions with 0, 10, 50 and 250 mmol/l imidazole elution buffer, respectively. rTyr p 13, recombinant Tyrophagus putrescentiae group 13 allergen.
IgE reactivity to rTyr p 13
ELISA was used to determine whether E. coli-produced rTyr p 13 had an IgE-binding ability with purified rTyr p 13 as a coat antigen. The mean OD value of the five healthy donors was 0.12 and the standard deviation (SD) was 0.0726. The postulated cutoff value was 0.34 (the mean OD value of healthy donors + three SD) (Fig. 7). Those with OD values >0.34 were judged to be IgE-positive. Positive IgE reactions to rTyr p 13 were detected in the serum of 13.2% (5/38) of the T. putrescentiae-allergic patients. The results indicated that rTyr p 13 had the ability to bind to IgE.
IgE reactivity of serum to recombinant Tyrophagus putrescentiae group 13 allergen. Each dot on the grapH indicates an individual serum sample. The cutoff value (straight line) is the mean OD value of healthy donors + three SD. OD, optical density.
Allergic diseases caused by T. putrescentiae, a type of storage mite, are common in several countries worldwide, including China. The positive skin prick test prevalence for T. putrescentiae in 2012 was 63% in Guangzhou, China (25). A better understanding of T. putrescentiae allergens may aid the diagnosis and treatment of T. putrescentiae allergies. For example, polymorphisms have been described for several storage mite allergens from different regions (25,26). Polymorphisms can have an important effect on the epitopes recognized by T lymphocytes, monoclonal antibodies and IgE of allergic patients (27). Therefore, in the present study, cDNA encoding Tyr p 13 was amplified from mites collected from a flour storage warehouse in China; subsequently, rTyr p 13 was expressed in an E. coli expression system and was purified by affinity chromatography.
Bioinformatics tools are crucial to allergy research, particularly the characterization of allergens by identification of structural motifs and epitopes and are complementary to experimental studies of allergens. Bioinformatics analysis of the sequence of Tyr p 13 revealed it to be a hydrophilic and stable protein, with no transmembrane helices and all protein sequences located outside the membrane. In addition, homology modeling based on a template was used to predict the tertiary structure of rTyr p 13. The key to homology modeling is to identify the right template; notably, quality of the homology model is dependent on high quality sequence alignment and template structure. The fatty acid-binding protein in brain tissue of D. melanogaster has a high sequence identity with Tyr p 13 (36.43%), and both proteins belong to the intracellular lipid-binding protein family (IPR031259). Therefore, the fatty acid-binding protein in brain tissue of D. melanogaster can be used as a modeling template. Following homology modeling using SWISS-MODEL, various additional parameters/programs were incorporated to establish a reliable model of Tyr p 13. Future work on these features can improve our understanding of Tyr p 13.
The specific interaction of allergens with IgE antibodies is a key event in allergic diseases. The present study assessed the allergenicity of rTyr p 13 and revealed that rTyr p 13 bound with serum from 13.2% (5/38) of patients allergic to T. putrescentiae, according to the results of ELISA. Furthermore, studies have determined a correlation between the severity or persistence of allergic diseases and the diversity of B-cell epitopes (IgE-binding epitopes) (27). The identification of B-cell epitopes of allergens is valuable for accurate and safe peptide-based allergen diagnosis, such as peptide-ELISA or epitope-ELISA, and immunotherapy. The in-silico prediction of B-cell epitopes is considered a useful tool for selecting B-cell epitopes from immunologically relevant proteins and has been reported to be well correlated with the experimental approach (23). In most cases, B-cell epitopes are located on the surface of antigen molecules. Secondary and tertiary protein structures also contain important information regarding B-cell epitope prediction. For example, β-turns and random coils are detected in surface-exposed protein regions of, which often contain epitope sequences (21). Numerous algorithms have been generated to predict B-cell epitopes on protein sequences; these algorithms are based on the propensity values of the amino-acid properties of hydrophilicity, antigenicity, flexibility and accessibility. By integrating DNAStar Protean and ElliPro analysis results, and combining information from secondary and tertiary structures, the present study identified potential B-cell linear and discontinuous epitopes. However, further experimental verification is required for these predicted epitopes. Therefore, the findings of the present study may be useful not only for further work on Tyr p 13 but may also lay the foundation for the study of peptide-based allergen diagnosis and immunotherapy.
In conclusion, the present study demonstrated that the cloning, expression, characterization and B cell epitopes of recombinant Tyr p 13 protein provided initial evaluation of its potency as an allergen in mite-allergic individuals. These findings provided a foundation for which to explore the structural biology and biochemistry of Tyr p 13 protein, thereby enabling future work with ASIT.
Signal peptide prediction (eukaryotic networks) of Tyrophagus putrescentiae group 13 allergen by SignalP 4.0 software.
Phosphorylation site analysis of Tyrophagus putrescentiae group 13 allergen using NetPhos 3.1 Server.
This work was supported by the National Sciences Foundation of China (grant nos. NSFC30060166, NSFC81001330 and NSFC31272369).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
NW and YC conceived and designed the study. NW and YZ performed the experiments and wrote the manuscript. YC revised the manuscript critically. MW and HZ collected the patients' samples and analyzed the data. All authors contributed to the preparation of the final manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All participants provided written informed consent. The present study was approved by the Ethics Committee for Clinical Investigation of Wuxi People's Hospital Affiliated to Nanjing Medical University.
Patient consent for publication
The authors declare that they have no competing interests.
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