Tyrophagus putrescentiae group 4 allergen allergenicity and epitope prediction

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Leecyous, F. Bakhtiar, M.M. Tang, Z.H.M. Yadzir, N. Abdullah" "autores" => array:5 [ 0 => array:2 [ "nombre" => "B." "apellidos" => "Leecyous" ] 1 => array:2 [ "nombre" => "F." "apellidos" => "Bakhtiar" ] 2 => array:2 [ "nombre" => "M.M." "apellidos" => "Tang" ] 3 => array:2 [ "nombre" => "Z.H.M." "apellidos" => "Yadzir" ] 4 => array:2 [ "nombre" => "N." 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A: representation of the specific IgG in AU/ml. B: representation of the specific IgG1 in AU/ml. C: representation of the specific IgG2a in AU/ml. D: representation of the specific IgE in AU/ml. E: representation of the specific IgG against Fel d 1 antigen in AU/ml." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "J.P. Sola González, E. Bravo Hernández, A. Cerezo Abellán, M. Peñalver-Mellado" "autores" => array:4 [ 0 => array:2 [ "nombre" => "J.P. Sola" "apellidos" => "González" ] 1 => array:2 [ "nombre" => "E. Bravo" "apellidos" => "Hernández" ] 2 => array:2 [ "nombre" => "A. Cerezo" "apellidos" => "Abellán" ] 3 => array:2 [ "nombre" => "M." "apellidos" => "Peñalver-Mellado" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0301054620300744?idApp=UINPBA00004N" "url" => "/03010546/0000004800000006/v1_202011271043/S0301054620300744/v1_202011271043/en/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "Original Article" "titulo" => "Tyrophagus putrescentiae group 4 allergen allergenicity and epitope prediction" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "619" "paginaFinal" => "625" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "F.-X. Teng, H.-F. Huang, D.-Z. Ge, L.-L. Yu, C. Xu, Y.-B. Cui" "autores" => array:6 [ 0 => array:3 [ "nombre" => "F.-X." "apellidos" => "Teng" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "H.-F." "apellidos" => "Huang" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "D.-Z." "apellidos" => "Ge" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 3 => array:3 [ "nombre" => "L.-L." "apellidos" => "Yu" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 4 => array:3 [ "nombre" => "C." "apellidos" => "Xu" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 5 => array:4 [ "nombre" => "Y.-B." "apellidos" => "Cui" "email" => array:1 [ 0 => "ybcui1975@hotmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "d" "identificador" => "aff0020" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "Department of Basic Medicine, Jiangsu Vocational College of Medicine, Yancheng, Jiangsu 224005, China" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Department of Dermatology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, China" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Department of Internal Medicine, The University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52246, United States" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "Department of Clinical Laboratory, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, China" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 499 "Ancho" => 2500 "Tamanyo" => 156371 ] ] "descripcion" => array:1 [ "en" => "Phylogenetic tree of Tyr p 4 protein." ] ] ] "textoCompleto" => "IntroductionMites allergens are a common trigger of allergies in children and adults worldwide, and produce asthma, atopic dermatitis, and perennial rhinitis, among other symptoms.<a class="elsevierStyleCrossRef" href="#bib0005">1</a> Allergens from house dust mites (HDMs), in particular those from the most common HDMs, Dermatophagoides farinae (Der f) and Dermatophagoides pteronyssinus (Der p), are major environmental factors for allergic diseases.<a class="elsevierStyleCrossRefs" href="#bib0010">2,3</a> However, many allergens from other mite species, such as Tyrophagus putrescentiae in storage mites, also contribute to allergic disease.<a class="elsevierStyleCrossRefs" href="#bib0020">4,5</a> Since there are currently few studies on allergens of T. putrescentiae, only eight groups of mite allergens from T. putrescentiae have been designated in the WHO/IUIS Allergen Nomenclature Database (<a href="http://www.allergen.org/">http://www.allergen.org/</a>). In addition to these eight groups of allergens, there are other allergens in T. putrescentiae that are not listed in the nomenclature database, like Tyr p 4, because their characterization and allergenicity are still unclear.At present, treatment options for mite allergic diseases include allergen avoidance, pharmacotherapy, and allergen-specific immunotherapy (ASIT).<a class="elsevierStyleCrossRef" href="#bib0030">6</a> Allergen avoidance and pharmacotherapy are currently the mainstay treatments of these diseases, but they are not easily able to change the course of disease.<a class="elsevierStyleCrossRef" href="#bib0030">6</a> ASIT is the only allergic disease-modifying therapy available.<a class="elsevierStyleCrossRef" href="#bib0035">7</a> However, ASIT with crude mite extracts, a mixture of allergens, nonallergens, and other proteins cannot be fully standardized and can cause severe side effects such as anaphylaxis.Recombinant allergens are an interesting approach to improve allergy diagnosis and ASIT, because they can be modified to reduce allergenicity and retain immunogenicity, which may increase safety and efficacy of allergy treatments.<a class="elsevierStyleCrossRef" href="#bib0040">8</a> Thus, researchers are working toward the development of recombinant allergens that mimic the native proteins and can be used for ASIT.<a class="elsevierStyleCrossRef" href="#bib0040">8</a>Our laboratory has previously cloned and expressed several recombinant allergens from mites,<a class="elsevierStyleCrossRefs" href="#bib0045">9,10</a> which are expected to be used in ASIT in the future. Here, in order to better understand the allergens in T. putrescentiae and provide more recombinant allergens for ASIT, we sought to clone and express recombinant Tyr p 4 allergen protein, and then describe characterization, allergenicity and possible antigenic epitopes of Tyr p 4. These results provide a foundation for further study of this allergen in the diagnosis and ASIT of storage mite allergy.Materials and methodsTyrophagus putrescentiae extractsTyrophagus putrescentiae extracts were supplied by Greer Laboratories (Lenoir, IL, USA) as a lyophilized powder from cleaned mites. It was dissolved in phosphate-buffered saline (PBS, pH 7.4), dialyzed against PBS, and diluted to a final concentration of 1g/L for skin prick test of serum provider.Serum samplesSerum samples were obtained from 120 workers at five wheat milling factories in Xinghua City, Jiangsu Province, China. The 120 workers (aged 20–60 years, mean 36.57 years; 95 males and 25 females; mean working age, 3.98 years) were in close contact with wheat flour during work and all had allergic symptoms. Based on personal information and doctor's diagnosis, 89 of them had allergic rhinitis, 35 had mild asthma, and 28 had atopic dermatitis. All of them had positive skin reactions by the skin prick test (the ratio of the wheal area ≥++, the detailed steps are below) to T. putrescentiae extracts. None of these participants had undergone allergic treatment. Sera from 10 healthy adult volunteers with no history of any allergic symptom and a negative skin prick test response to common mite allergens were obtained as negative controls. All participants provided written informed consent. The study was approved by the Clinical and New Technology Ethics Committee of Wuxi People's Hospital (approval number is KYLLH2018034), Wuxi City, Jiangsu Province, China. Confidentiality is maintained.Sequencing Tyr p 4Tyrophagus putrescentiae was cultured and total RNA was isolated as previously described.<a class="elsevierStyleCrossRef" href="#bib0025">5</a> DNA primers were designed and synthesized based on the sequence of Tyr p 4 (GenBank accession no. DQ983318.1). The sequence of the forward primer was 5′-CATGCCATGGCTTTCAACAGCTACATTCTTC-3′ and the reverse primer was 5′-CCGCTCGATGCTTGGCATGCACGTGAATTG-3′. The reaction was performed as previously reported<a class="elsevierStyleCrossRef" href="#bib0045">9</a>: 2min at 98°C, 30 cycles of 30s at 98°C, 30s at 60°C, 10min at 72°C, and 5min at 10°C. 5μL of PCR product was analyzed by agarose gel electrophoresis (2.0%) and visualized with ImageMaster® VDS.TA cloningA deoxyadenosine nucleotide (A) was added to the 3′ end of the PCR product using a Takara DNA A-tailing kit (Code No. 6109). The reaction mix of 50μL consisted of t10× A-Tailing Buffer (5μL), 2.5mM of dNTP Mixture (4μL), 5U/μL of A-tailing enzyme (0.5μL), smooth-ended DNA fragments (5μg), and ddH2O (35.5μL) and was incubated at 72°C for 20min before being placed on ice for 2min.The A-tailed fragment was subjected to an enzymatic reaction with the pMD20T plasmid using NEB T4 DNA ligase (Code No. M0202). The reaction mixture of 10μL consisted of 10× T4 DNA ligase buffer (1μL), pMD20T vector DNA (4kb, 1μL), A-tail DNA fragments (6μL), T4 DNA ligase (0.5μL), and nuclease-free water (1.5μL) and was incubated at 4°C for 8h. E. coli JM109 (Takara Bio Inc., Code No. D9052) was then transformed with the recombinant plasmid pMD20T-Tyr p 4 and positive clones were selected by blue/white screening on Luria Bertani (LB) plates containing 100mg/mL ampicillin. The plasmid was then extracted for verification.Construction of expression plasmid pET-28a(+)-Tyr p 4pMD20T-Tyr p 4 and pET-28a(+) were double digested. The 105-μL reaction mixture consisted of pMD20T-Tyr p 4 (50μL), 10× K buffer (5μL), 0.1% BSA (5μL), pET28a(+) plasmid (35μL), NcoI (2.5μL), XhoI (2.5μL), and DDH2O (5μL) and was incubated at 37°C for 6h. The product was then recovered by cutting fragments from the agarose gel. Then, 10× T4 DNA ligase buffer (1μL), pET-28a(+) plasmid recovered by double digestion (4kb, 2μL), Tyr p 4 recovered by double digestion (2μL), T4 DNA ligase (0.5μL), and nuclease-free water (4.5μL) were incubated at 4°C for 8h. E. coli DH5α competent cells (Takara Bio Inc., Code No. 9057) were transformed with the recombinant plasmid pET-28a(+)-Tyr p 4 and positive clones were selected by blue/white screening on Luria Bertani (LB) plates containing 50mg/mL kanamycin. The plasmid was then extracted for sequencing.Production and purification of recombinant Tyr p 4 proteinThe pET-28a(+)-Tyr p 4 plasmids were transformed and expressed in E. coli BL21 (DE3, Stratagene, La Jolla, CA, USA), purified by nickel affinity chromatography, verified by SDS-PAGE and Western blotting, and analyzed by MALDI-TOF/TOF mass spectrometer (model 4800, Applied Biosystems) as previously described.<a class="elsevierStyleCrossRef" href="#bib0050">10</a>Skin prick testParticipants were free of skin scratches and had no skin damage or infection at the test site. All stopped taking anti-allergy drugs and hormones for at least 3 days before the experiment. The skin of the inner forearm was cleaned with saline and the negative control solution (0.9% NaCl solution), the allergen solution (1g/L T. putrescentiae extracts or 1g/L recombinant Tyr p 4 protein), and the positive control solution (10g/L histamine phosphate solution) were sequentially applied to the skin of each participant. The distance between application sites was 3cm. Avoiding blood vessels, a needle was passed through the droplets to slightly puncture the skin without bleeding. The needle was removed after 1s, and the skin was observed after 15min. The ratio of the wheal area caused by T. putrescentiae extracts or recombinant Tyr p 4 protein liquid (S1) to the wheal area caused by positive control solution (S2) was determined. If S1/S2 was between 0 and 25% it was scored as negative (−), between 26% and 50% it was slightly positive (+), between 51% and 100% was positive (++), between 101 and 200% was (+++), and more than 200% was (++++). The result was judged to be positive at (++) or above.<a class="elsevierStyleCrossRef" href="#bib0055">11</a>IgE-binding reactivity of recombinant Tyr p 4 proteinThe IgE-binding reactivity of Tyr p 4 allergen was detected by ELISA as previously described.<a class="elsevierStyleCrossRef" href="#bib0045">9</a> Recombinant Tyr p 4 protein was coated onto a microplate, blocked in bovine serum albumin (BSA), and patient serum was added to the microtiter plate. Secondary antibody and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution were added to the microtiter plate to carry out the reaction. Finally, H2SO4 was added to terminate the reaction. The optical density was measured using an iMark microplate absorbance reader (Bio-Rad). Reactivity was considered positive if the optical density (OD) of the detected sera was higher than the cut-off ELISA value (within three standard deviations of the mean ELISA value of healthy donors). The specific IgE classes are defined using six calibrators: 0, 0.35, 0.7, 3.5, 17.5 and 100kUA/L (Class 0: from 0 to <0.35kUA/L; Class 1: from 0.35 to <0.7kUA/L; Class 2: from 0.70 to <3.5kUA/L; Class 3: from 3.50 to <17.5kUA/L; Class 4: from 17.5 to <50kUA/L; Class 5: from 50 to <100kUA/L; and Class 6: from ≥100kUA/L).<a class="elsevierStyleCrossRef" href="#bib0060">12</a>Characterization of Tyr p 4 proteinThe amino acid sequence of Tyr p 4 protein (GenBank accession no. ABM53754.1) was obtained from the protein database of the National Center for Biotechnology Information (<a href="http://www.allergen.org/">www.allergen.org</a>). The open reading frame (ORF) was identified using ORF server.<a class="elsevierStyleCrossRef" href="#bib0065">13</a> Transmembrane protein helices were predicted by TMHMM server 2.0.<a class="elsevierStyleCrossRef" href="#bib0070">14</a> The physicochemical properties and hydrophilicity of the Tyr p 4 amino acid sequence was predicted using ProtParam,<a class="elsevierStyleCrossRef" href="#bib0075">15</a> and phosphorylation sites were predicted using NetPhos 3.1 server.<a class="elsevierStyleCrossRef" href="#bib0080">16</a> Phylogenetic tree construction and identification of Tyr p 4 amino acid sequence alignment were performed by MEGA 5.02<a class="elsevierStyleCrossRef" href="#bib0085">17</a> and VECTOR NTI 11.0,<a class="elsevierStyleCrossRef" href="#bib0090">18</a> respectively. Secondary structure was predicted using the JPred4 server.<a class="elsevierStyleCrossRef" href="#bib0095">19</a>Structure prediction and homology modelingTemplates of Tyr p 4 protein were analyzed for homology modeling in the Protein Data Bank (PDB)<a class="elsevierStyleCrossRef" href="#bib0100">20</a> using Protein BLAST. Based on high score, low e-value, and maximum sequence identity, the appropriate templates were selected. The tertiary structure of Tyr p 4 protein was predicted by homology modeling using MODELLER 9.22.<a class="elsevierStyleCrossRef" href="#bib0105">21</a> PROCHECK determined the stereochemical quality of Tyr p 4 protein structure by analyzing residue-by-residue geometry and overall structural geometry.<a class="elsevierStyleCrossRef" href="#bib0110">22</a> VERIFY 3D was used to detect the compatibility of an atomic model (3D) of Tyr p 4 protein with its own amino acid sequence (1D).<a class="elsevierStyleCrossRef" href="#bib0115">23</a> ERRAT analyzed the statistics of non-bonded interactions between different atom types.<a class="elsevierStyleCrossRef" href="#bib0120">24</a> ProSA was used to analyze the Z-score to determine the degree of match between the template and Tyr p 4 protein.<a class="elsevierStyleCrossRef" href="#bib0125">25</a> QMEAN derived both global (the entire structure) and local (per residue) absolute quality estimates on the basis of one single model.<a class="elsevierStyleCrossRef" href="#bib0130">26</a> Interactive visualization and analysis of molecular structures were completed using UCSF Chimera 1.13.1.<a class="elsevierStyleCrossRef" href="#bib0135">27</a>Prediction of epitopesBcePred,<a class="elsevierStyleCrossRef" href="#bib0140">28</a> ABCpred,<a class="elsevierStyleCrossRef" href="#bib0145">29</a> and BCPreds<a class="elsevierStyleCrossRef" href="#bib0150">30</a> were used to predict the B-cell antigenic epitopes of Tyr p 4. NetMHCpan 4.0,<a class="elsevierStyleCrossRef" href="#bib0155">31</a> NetMHCII 2.3,<a class="elsevierStyleCrossRef" href="#bib0160">32</a> and NetMHCIIpan 3.2<a class="elsevierStyleCrossRef" href="#bib0165">33</a> were used to predict the T-cell antigenic epitopes of Tyr p 4.ResultsGene cloning, expression, and purification of recombinant Tyr p 4 proteinPlasmids from sequenced kanamycin-resistant clones were used to successfully transform pET-28a(+)-Tyr p 4 into E. coli DH5α cells and expressed under IPTG (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A). A single and specific band matching the predicted molecular weight of Tyr p 4 using ProtParam was observed (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>B).<elsevierMultimedia ident="fig0005"></elsevierMultimedia>Characterization of Tyr p 4 proteinThe Tyr p 4 gene contains a complete open reading frame, with 1557bp between the initiation codon ATG and the stop codon TAA, encoding a protein of 518 amino acids. Family classification shows that Tyr p 4 protein belongs to the alpha amylase family (InterPro no. IPR006046). Tyr p 4 protein has no transmembrane helices, and all amino acids of the Tyr p 4 protein are outside the membrane. ProtParam indicates that Tyr p 4 protein has a relative molecular weight of 58.4kDa and a theoretical isoelectric point (pI) of 6.76. The instability index (II) was computed to be 36.11, classifying the protein as stable. The grand average of hydropathicity is −0.431, indicating that Tyr p 4 protein is a hydrophilic protein. Tyr p 4 protein contains 42 phosphorylation sites, includes 27 serine sites (amino acids 28, 69, 95, 97, 166, 185, 193, 194, 221, 266, 361, 384, 424, 503, 504, and 505), five tyrosine sites (amino acids 196, 269, 362, 436, and 471), and one threonine site (amino acid 457). Secondary structure prediction shows that Tyr p 4 protein contains α-helices, β-sheets, and random coils. The analysis of the phylogenetic tree and sequence identity showed the closest genetic relationship between Tyr p 4 and Aca s 4 (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a> and <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>).<elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="tbl0005"></elsevierMultimedia>Skin prick test and IgE-binding reactivity of recombinant Tyr p 4 proteinThe skin prick test of recombinant Tyr p 4 protein revealed seven + cases, twenty-two ++ cases, and one +++ case. The positive response rate was 19.2% (23 participants out of 120). The serum specific IgE test of recombinant Tyr p 4 protein revealed ten cases of Class 1, eight cases of Class 2, and two cases of Class 3. The total positive response rate was 16.7% (20/120). Sixteen participants were positive in both skin prick test and sera specific IgE-binding test, with a positive rate of 13.3% (16/120).Homology modeling of Tyr p 4 protein tertiary structureThe sequence identity of alpha-amylase (PDB accession no. 1PPI) and Tyr p 4 protein is 52.32%. The parameters of the Tyr p 4 protein tertiary structure are similar to 1PPI (<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>). PROCHECK shows high stereochemical quality of Tyr p 4 protein structure and the ERRAT value suggests high resolution of the tertiary structure of Tyr p 4 protein. VERIFY 3D suggests that the Tyr p 4 protein structure is favorable. The Z-scores of the template and Tyr p 4 protein show a high level of matching between the tertiary structures of the protein. The Q value suggests that the predicted model of Tyr p 4 protein is reliable. Based on this evaluation, we determined the tertiary structure of Tyr p 4 protein obtained by homologous modeling to be reliable. The tertiary structure of Tyr p 4 protein also contains α-helices, β-sheets, and random coils, and similar proportions of these elements are found in the secondary and tertiary structures. The proportions of α-helices, β-sheets and random coils in the secondary structure are 28.19% (15 domains), 18.34% (20 domains) and 53.47%, and are 25.87% (18 domains), 12.55% (13 domains) and 61.58% in the tertiary structure.<elsevierMultimedia ident="tbl0010"></elsevierMultimedia>Tyr p 4 protein epitope predictionEight B-cell epitopes (amino acid positions 26–32, 90–99, 137–145, 160–175, 256–265, 369–381, 431–436, and 502–508) and three T-cell epitopes (amino acid positions 204–208, 280–288, and 347–355) of Tyr p 4 were predicted (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a> and <a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>).<elsevierMultimedia ident="fig0015"></elsevierMultimedia><elsevierMultimedia ident="tbl0015"></elsevierMultimedia>DiscussionAlthough it was a potential allergen, there is little information regarding the physical and chemical properties of Tyr p 4, its allergenicity, or antigenic epitopes. It has not been included in the official website of the systematic allergen nomenclature approved by the WHO/IUIS Allergen Nomenclature Database (<a href="http://www.allergen.org/">http://www.allergen.org/</a>). We determined the allergenicity of recombinant Tyr p 4 protein by skin prick test and ELISA. The skin prick positive rate was 19.2%, the rate of serum specific IgE positives was 16.7%, and the total positive response to both methods was 13.3%. The total positive rate of the skin prick test was higher than the serum specific IgE test, this is likely due to a false positive result on the skin prick test; thus, combining the two methods allows for a more accurate determination. Phylogenetic analysis shows that Tyr p 4 has the highest homology with Aca s 4 (Acarus siro) and Blo t 4 (Blomia tropicalis). Interestingly, all three are storage mites, and Blo t 4 induced positive skin prick tests in 10% of 200.<a class="elsevierStyleCrossRef" href="#bib0170">34</a> Based on these results, the allergenicity of groups 4 allergens is weak compared to groups 1 and 2 allergens. Cross-reactivity sometimes occurs between the same group allergens of different mite species, but no reports on cross-reactivity between different mite species group 4 allergens have been found to date. This will be the focus of our further research.Although Tyr p 4 protein contains α-helices, β-sheets, and random coils, the number of amino acids in these elements in the tertiary structure was found to vary marginally from the secondary structure. This discrepancy may be due to different structural prediction methods.After determining the allergenicity of Tyr p 4, the B-cell and T-cell epitopes of Tyr p 4 were predicted. The properties of the antigen epitope, their number, and spatial configuration determine antigen specificity.<a class="elsevierStyleCrossRefs" href="#bib0175">35,36</a> α-helices and β-sheets have higher chemical bond energy and have difficulty forming epitope sequences. By contrast, β-turns and random coils are located in surface-exposed regions of a protein, which often contain epitope sequences.<a class="elsevierStyleCrossRef" href="#bib0185">37</a> Epitopes usually contain 6–8 amino acids residues and in general contain <20 amino acid residues. In addition, allergen epitopes usually contain high proportions of hydrophobic amino acid residues, including Ala, Ser, Asn, Gly, and Lys.<a class="elsevierStyleCrossRef" href="#bib0190">38</a> Based on the results of the epitope prediction software and the tertiary structure of the Tyr p 4 protein, the presence of eight B-cell epitopes and three T-cell epitopes is likely. However, these predicted epitopes require further experimental verification.In conclusion, we demonstrate that the cloning, expression, characterization, allergenicity, and epitopes of recombinant Tyr p 4 protein has enabled an initial evaluation of its potency as an allergen in mite-allergic individuals. These findings provide a foundation from which to explore the structural biology and biochemistry of Tyr p 4 protein, thereby enabling future work with ASIT.Conflict of interestThe authors have no conflict of interest to declare." "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres1422135" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction and objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Materials and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1300241" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xpalclavsec1300240" "titulo" => "Abbreviations" ] 3 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 4 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:11 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Tyrophagus putrescentiae extracts" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Serum samples" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Sequencing Tyr p 4" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "TA cloning" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Construction of expression plasmid pET-28a(+)-Tyr p 4" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Production and purification of recombinant Tyr p 4 protein" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Skin prick test" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "IgE-binding reactivity of recombinant Tyr p 4 protein" ] 8 => array:2 [ "identificador" => "sec0055" "titulo" => "Characterization of Tyr p 4 protein" ] 9 => array:2 [ "identificador" => "sec0060" "titulo" => "Structure prediction and homology modeling" ] 10 => array:2 [ "identificador" => "sec0065" "titulo" => "Prediction of epitopes" ] ] ] 5 => array:3 [ "identificador" => "sec0070" "titulo" => "Results" "secciones" => array:5 [ 0 => array:2 [ "identificador" => "sec0075" "titulo" => "Gene cloning, expression, and purification of recombinant Tyr p 4 protein" ] 1 => array:2 [ "identificador" => "sec0080" "titulo" => "Characterization of Tyr p 4 protein" ] 2 => array:2 [ "identificador" => "sec0085" "titulo" => "Skin prick test and IgE-binding reactivity of recombinant Tyr p 4 protein" ] 3 => array:2 [ "identificador" => "sec0090" "titulo" => "Homology modeling of Tyr p 4 protein tertiary structure" ] 4 => array:2 [ "identificador" => "sec0095" "titulo" => "Tyr p 4 protein epitope prediction" ] ] ] 6 => array:2 [ "identificador" => "sec0100" "titulo" => "Discussion" ] 7 => array:2 [ "identificador" => "sec0105" "titulo" => "Conflict of interest" ] 8 => array:2 [ "identificador" => "xack496383" "titulo" => "Acknowledgments" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2019-12-01" "fechaAceptado" => "2020-02-17" "PalabrasClave" => array:1 [ "en" => array:2 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1300241" "palabras" => array:5 [ 0 => "Storage mites" 1 => "Immunoglobulin E (IgE)" 2 => "Molecular cloning" 3 => "Recombinant mite allergen" 4 => "Homology modeling" ] ] 1 => array:4 [ "clase" => "abr" "titulo" => "Abbreviations" "identificador" => "xpalclavsec1300240" "palabras" => array:12 [ 0 => "Aca s" 1 => "ASIT" 2 => "Blo the" 3 => "BSA" 4 => "Der f" 5 => "Der p" 6 => "HDMs" 7 => "OD" 8 => "ORF" 9 => "PDB" 10 => "TMB" 11 => "Tyr p" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "Introduction and objectivesAllergen-specific immunotherapy (ASIT) is the only allergic disease-modifying therapy available for children and adults, and recombinant allergens are an interesting approach to improve allergy diagnosis and ASIT. Tyrophagus putrescentiae is a common storage mite that produces potent allergens. The aim of this study was to express and characterize recombinant group 4 allergen protein of T. putrescentiae (Tyr p 4), and to further investigate allergenicity and potential epitopes of Tyr p 4. Materials and methodsThe cDNA encoding Tyr p 4 was generated by RT-PCR and subcloned into pET-28a(+) plasmid. The plasmid was then transformed into E. coli cells for expression. After purification by nickel affinity chromatography and identification by SDS-PAGE, recombinant Tyr p 4 protein was used for a skin prick test and an ELISA to determine the allergic response. ResultsStudy participants’ allergic response rate to Tyr p 4 protein was 13.3% (16/120). Eight B-cell epitopes and three T-cell epitopes of Tyr p 4 were predicted. ConclusionsSimilar to group 4 allergens of other species of mite, allergenicity of Tyr p 4 is weak. The expression, characterization and epitope prediction of recombinant Tyr p 4 protein provide a foundation for further study of this allergen in the diagnosis and ASIT of storage mite allergy." "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction and objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Materials and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] ] "multimedia" => array:6 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1362 "Ancho" => 2083 "Tamanyo" => 133342 ] ] "descripcion" => array:1 [ "en" => "Expression and purification of rTyr p 4 protein. (A) SDS-PAGE demonstrating expression of rTyr p 4 protein in BL21 cells. Lane M, protein marker (TransGen Biotech); lane 1, the pellet of cells containing pET-28a(+)-Tyr p 4 3h after induction; lane 2, supernatant from cells containing pET-28a(+)-Tyr p 4 3h after induction; lane 3, whole BL21 cells containing pET-28b (+)-Tyr p 4 before IPTG induction; lane 4, whole BL21 cells containing pET-28a(+) before IPTG induction. (B) Tyr p 4 protein purified from BL21 cells. Lane M, protein marker (TransGen Biotech); lane 1, purified Tyr p 4 protein." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 499 "Ancho" => 2500 "Tamanyo" => 156371 ] ] "descripcion" => array:1 [ "en" => "Phylogenetic tree of Tyr p 4 protein." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1143 "Ancho" => 2500 "Tamanyo" => 398426 ] ] "descripcion" => array:1 [ "en" => "3D structure and epitopes of Tyr p 4 protein. (A-1 and A-2) 3D structure of Tyr p 4 protein. (B-1 and B-2) B-cell epitopes on the 3D structure of Tyr p 4 protein. (C-1 and C-2) T-cell epitopes on the predicted 3D structure of Tyr p 4 protein." ] ] 3 => array:8 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at1" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Tyr p 4 \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Aca s 4 \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Blo t 4 \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Der p 4 \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Der f 4 \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Eur m 4 \t\t\t\t\t\t\n \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Tyr p 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">72 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">72 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">69 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">64 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">66 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Aca s 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">68 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">66 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">62 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">62 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Blo t 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">70 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">65 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">66 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Der p 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">89 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">90 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Der f 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">87 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Eur m 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab2439214.png" ] ] ] ] "descripcion" => array:1 [ "en" => "Percent identity between Tyr p 4 and related allergens." ] ] 4 => array:8 [ "identificador" => "tbl0010" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at2" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Protein \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Structural assessment method \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">PROCHECK (%) \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">ERRAT value \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">VERIFY 3D (%) \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Z-score \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Q-value \t\t\t\t\t\t\n \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Tyr p 4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">SAVES \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">87.9 core \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">95.9916 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">97.95% \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">10.6 allowed \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.4 generously allowed \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.0 disallowed \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">ProSa \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">−9.25 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">QMEAN \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.78 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1PPI \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">SAVES \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">89.8 core \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">96.0168 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">100.00% \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">9.8 allowed \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.5 generously allowed \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.0 disallowed \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">ProSa \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">−9.29 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">QMEAN \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.65 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab2439213.png" ] ] ] ] "descripcion" => array:1 [ "en" => "Evaluation of the tertiary structure of Tyr p 4 protein." ] ] 5 => array:8 [ "identificador" => "tbl0015" "etiqueta" => "Table 3" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at3" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:2 [ "leyenda" => "Bold letters represent the hydrophobic amino acid residues." "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Type of epitope \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Prediction server \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Epitope prediction (peptide) \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Confirmed epitopes (peptide) \t\t\t\t\t\t\n \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " rowspan="3" align="left" valign="middle">B-cell epitopes</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">BcePred \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">26–32, 87–99, 127–145, 157–175, 188–194, 243–250, 255–265, 270–277, 290–293, 317–325, 369–387, 400–405, 431–436, 477–487, 502–508 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " rowspan="3" align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">B1(26–32, PYSHPHF)B2(90–99, SYRIISRSGN)B3(137–145, GSHYNGNSM)B4(160–175, HESCPTSDLEIHNYDN)B5(256–265, VIYYGGNGIK)B6(369–381, QNGHDVNDWMGPP)B7(431–436, WDNGDY)B8(502–508, NSSSDDD)</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">ABCpred \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">23–38, 90–105, 133–148, 160–175, 185–200, 242–257, 253–268, 272–287, 289–304, 318–333, 366–381, 398–413, 426–441, 481–496, 500–515 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">BCPred \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">21–40, 56–75, 83–102, 137–156, 160–179, 256–275, 290–309, 315–334, 367–386, 425–444, 453–472, 491–510 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " colspan="4" align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t"></td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " rowspan="3" align="left" valign="middle">T-cell epitopes</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">NetMHCpan \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">200–208, 204–213, 223–232, 231–239, 347–355 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " rowspan="3" align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">T1(204–208, FLNKL)T2(280–288, YYRELANVL)T3(347–355, FMLAWPYGV)</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">NetMHCII \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">280–288, 347–355, 447–455 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">NetMHCIIpan \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">280–288, 341–349, 412–420 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab2439215.png" ] ] ] ] "descripcion" => array:1 [ "en" => "Predicted B and T cell epitopes of Tyr p 4." ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0015" "bibliografiaReferencia" => array:38 [ 0 => array:3 [ "identificador" => "bib0005" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Mites and allergy" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:3 [ 0 => "E. 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Original Article

F.-X. Tenga, H.-F. Huangb, D.-Z. Gec, L.-L. Yua, C. Xuc, Y.-B. Cuid,

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ybcui1975@hotmail.com

Corresponding author.

a Department of Basic Medicine, Jiangsu Vocational College of Medicine, Yancheng, Jiangsu 224005, China

b Department of Dermatology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, China

c Department of Internal Medicine, The University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52246, United States

d Department of Clinical Laboratory, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, China

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such as <span class="elsevierStyleItalic">Tyrophagus putrescentiae</span> in storage mites&#44; also contribute to allergic disease&#46;<a class="elsevierStyleCrossRefs" href="#bib0020"><span class="elsevierStyleSup">4&#44;5</span></a> Since there are currently few studies on allergens of <span class="elsevierStyleItalic">T&#46; putrescentiae</span>&#44; only eight groups of mite allergens from <span class="elsevierStyleItalic">T&#46; putrescentiae</span> have been designated in the WHO&#47;IUIS Allergen Nomenclature Database &#40;<a href="http://www.allergen.org/">http&#58;&#47;&#47;www&#46;allergen&#46;org&#47;</a>&#41;&#46; In addition to these eight groups of allergens&#44; there are other allergens in <span class="elsevierStyleItalic">T&#46; putrescentiae</span> that are not listed in the nomenclature database&#44; like Tyr p 4&#44; because their characterization and allergenicity are still unclear&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">At present&#44; treatment options for mite allergic diseases include allergen avoidance&#44; pharmacotherapy&#44; and allergen-specific immunotherapy &#40;ASIT&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a> Allergen avoidance and pharmacotherapy are currently the mainstay treatments of these diseases&#44; but they are not easily able to change the course of disease&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a> ASIT is the only allergic disease-modifying therapy available&#46;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a> However&#44; ASIT with crude mite extracts&#44; a mixture of allergens&#44; nonallergens&#44; and other proteins cannot be fully standardized and can cause severe side effects such as anaphylaxis&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Recombinant allergens are an interesting approach to improve allergy diagnosis and ASIT&#44; because they can be modified to reduce allergenicity and retain immunogenicity&#44; which may increase safety and efficacy of allergy treatments&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a> Thus&#44; researchers are working toward the development of recombinant allergens that mimic the native proteins and can be used for ASIT&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">Our laboratory has previously cloned and expressed several recombinant allergens from mites&#44;<a class="elsevierStyleCrossRefs" href="#bib0045"><span class="elsevierStyleSup">9&#44;10</span></a> which are expected to be used in ASIT in the future&#46; Here&#44; in order to better understand the allergens in <span class="elsevierStyleItalic">T&#46; putrescentiae</span> and provide more recombinant allergens for ASIT&#44; we sought to clone and express recombinant Tyr p 4 allergen protein&#44; and then describe characterization&#44; allergenicity and possible antigenic epitopes of Tyr p 4&#46; These results provide a foundation for further study of this allergen in the diagnosis and ASIT of storage mite allergy&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050"><span class="elsevierStyleItalic">Tyrophagus putrescentiae</span> extracts</span><p id="par0025" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Tyrophagus putrescentiae</span> extracts were supplied by Greer Laboratories &#40;Lenoir&#44; IL&#44; USA&#41; as a lyophilized powder from cleaned mites&#46; It was dissolved in phosphate-buffered saline &#40;PBS&#44; pH 7&#46;4&#41;&#44; dialyzed against PBS&#44; and diluted to a final concentration of 1<span class="elsevierStyleHsp" style=""></span>g&#47;L for skin prick test of serum provider&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Serum samples</span><p id="par0030" class="elsevierStylePara elsevierViewall">Serum samples were obtained from 120 workers at five wheat milling factories in Xinghua City&#44; Jiangsu Province&#44; China&#46; The 120 workers &#40;aged 20&#8211;60 years&#44; mean 36&#46;57 years&#59; 95 males and 25 females&#59; mean working age&#44; 3&#46;98 years&#41; were in close contact with wheat flour during work and all had allergic symptoms&#46; Based on personal information and doctor&#39;s diagnosis&#44; 89 of them had allergic rhinitis&#44; 35 had mild asthma&#44; and 28 had atopic dermatitis&#46; All of them had positive skin reactions by the skin prick test &#40;the ratio of the wheal area &#8805;&#43;&#43;&#44; the detailed steps are below&#41; to <span class="elsevierStyleItalic">T&#46; putrescentiae</span> extracts&#46; None of these participants had undergone allergic treatment&#46; Sera from 10 healthy adult volunteers with no history of any allergic symptom and a negative skin prick test response to common mite allergens were obtained as negative controls&#46; All participants provided written informed consent&#46; The study was approved by the Clinical and New Technology Ethics Committee of Wuxi People&#39;s Hospital &#40;approval number is KYLLH2018034&#41;&#44; Wuxi City&#44; Jiangsu Province&#44; China&#46; Confidentiality is maintained&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Sequencing Tyr p 4</span><p id="par0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Tyrophagus putrescentiae</span> was cultured and total RNA was isolated as previously described&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> DNA primers were designed and synthesized based on the sequence of Tyr p 4 &#40;GenBank accession no&#46; DQ983318&#46;1&#41;&#46; The sequence of the forward primer was 5&#8242;-CATGCCATGGCTTTCAACAGCTACATTCTTC-3&#8242; and the reverse primer was 5&#8242;-CCGCTCGATGCTTGGCATGCACGTGAATTG-3&#8242;&#46; The reaction was performed as previously reported<a class="elsevierStyleCrossRef" href="#bib0045"><span class="elsevierStyleSup">9</span></a>&#58; 2<span class="elsevierStyleHsp" style=""></span>min at 98<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 30 cycles of 30<span class="elsevierStyleHsp" style=""></span>s at 98<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 30<span class="elsevierStyleHsp" style=""></span>s at 60<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; 10<span class="elsevierStyleHsp" style=""></span>min at 72<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and 5<span class="elsevierStyleHsp" style=""></span>min at 10<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; 5<span class="elsevierStyleHsp" style=""></span>&#956;L of PCR product was analyzed by agarose gel electrophoresis &#40;2&#46;0&#37;&#41; and visualized with ImageMaster&#174; VDS&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">TA cloning</span><p id="par0040" class="elsevierStylePara elsevierViewall">A deoxyadenosine nucleotide &#40;A&#41; was added to the 3&#8242; end of the PCR product using a Takara DNA A-tailing kit &#40;Code No&#46; 6109&#41;&#46; The reaction mix of 50<span class="elsevierStyleHsp" style=""></span>&#956;L consisted of t10&#215; A-Tailing Buffer &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; 2&#46;5<span class="elsevierStyleHsp" style=""></span>mM of dNTP Mixture &#40;4<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; 5<span class="elsevierStyleHsp" style=""></span>U&#47;&#956;L of A-tailing enzyme &#40;0&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; smooth-ended DNA fragments &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;g&#41;&#44; and ddH<span class="elsevierStyleInf">2</span>O &#40;35&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; and was incubated at 72<span class="elsevierStyleHsp" style=""></span>&#176;C for 20<span class="elsevierStyleHsp" style=""></span>min before being placed on ice for 2<span class="elsevierStyleHsp" style=""></span>min&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall">The A-tailed fragment was subjected to an enzymatic reaction with the pMD20T plasmid using NEB T4 DNA ligase &#40;Code No&#46; M0202&#41;&#46; The reaction mixture of 10<span class="elsevierStyleHsp" style=""></span>&#956;L consisted of 10&#215; T4 DNA ligase buffer &#40;1<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; pMD20T vector DNA &#40;4<span class="elsevierStyleHsp" style=""></span>kb&#44; 1<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; A-tail DNA fragments &#40;6<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; T4 DNA ligase &#40;0&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; and nuclease-free water &#40;1&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; and was incubated at 4<span class="elsevierStyleHsp" style=""></span>&#176;C for 8<span class="elsevierStyleHsp" style=""></span>h&#46; <span class="elsevierStyleItalic">E&#46; coli</span> JM109 &#40;Takara Bio Inc&#46;&#44; Code No&#46; D9052&#41; was then transformed with the recombinant plasmid pMD20T-Tyr p 4 and positive clones were selected by blue&#47;white screening on Luria Bertani &#40;LB&#41; plates containing 100<span class="elsevierStyleHsp" style=""></span>mg&#47;mL ampicillin&#46; The plasmid was then extracted for verification&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Construction of expression plasmid pET-28a&#40;&#43;&#41;-Tyr p 4</span><p id="par0050" class="elsevierStylePara elsevierViewall">pMD20T-Tyr p 4 and pET-28a&#40;&#43;&#41; were double digested&#46; The 105-&#956;L reaction mixture consisted of pMD20T-Tyr p 4 &#40;50<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; 10&#215; K buffer &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; 0&#46;1&#37; BSA &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; pET28a&#40;&#43;&#41; plasmid &#40;35<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; NcoI &#40;2&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; XhoI &#40;2&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; and DDH<span class="elsevierStyleInf">2</span>O &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; and was incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 6<span class="elsevierStyleHsp" style=""></span>h&#46; The product was then recovered by cutting fragments from the agarose gel&#46; Then&#44; 10&#215; T4 DNA ligase buffer &#40;1<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; pET-28a&#40;&#43;&#41; plasmid recovered by double digestion &#40;4<span class="elsevierStyleHsp" style=""></span>kb&#44; 2<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; Tyr p 4 recovered by double digestion &#40;2<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; T4 DNA ligase &#40;0&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; and nuclease-free water &#40;4&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; were incubated at 4<span class="elsevierStyleHsp" style=""></span>&#176;C for 8<span class="elsevierStyleHsp" style=""></span>h&#46; <span class="elsevierStyleItalic">E&#46; coli</span> DH5&#945; competent cells &#40;Takara Bio Inc&#46;&#44; Code No&#46; 9057&#41; were transformed with the recombinant plasmid pET-28a&#40;&#43;&#41;-Tyr p 4 and positive clones were selected by blue&#47;white screening on Luria Bertani &#40;LB&#41; plates containing 50<span class="elsevierStyleHsp" style=""></span>mg&#47;mL kanamycin&#46; The plasmid was then extracted for sequencing&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Production and purification of recombinant Tyr p 4 protein</span><p id="par0055" class="elsevierStylePara elsevierViewall">The pET-28a&#40;&#43;&#41;-Tyr p 4 plasmids were transformed and expressed in <span class="elsevierStyleItalic">E&#46; coli</span> BL21 &#40;DE3&#44; Stratagene&#44; La Jolla&#44; CA&#44; USA&#41;&#44; purified by nickel affinity chromatography&#44; verified by SDS-PAGE and Western blotting&#44; and analyzed by MALDI-TOF&#47;TOF mass spectrometer &#40;model 4800&#44; Applied Biosystems&#41; as previously described&#46;<a class="elsevierStyleCrossRef" href="#bib0050"><span class="elsevierStyleSup">10</span></a></p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Skin prick test</span><p id="par0060" class="elsevierStylePara elsevierViewall">Participants were free of skin scratches and had no skin damage or infection at the test site&#46; All stopped taking anti-allergy drugs and hormones for at least 3 days before the experiment&#46; The skin of the inner forearm was cleaned with saline and the negative control solution &#40;0&#46;9&#37; NaCl solution&#41;&#44; the allergen solution &#40;1<span class="elsevierStyleHsp" style=""></span>g&#47;L <span class="elsevierStyleItalic">T&#46; putrescentiae</span> extracts or 1<span class="elsevierStyleHsp" style=""></span>g&#47;L recombinant Tyr p 4 protein&#41;&#44; and the positive control solution &#40;10<span class="elsevierStyleHsp" style=""></span>g&#47;L histamine phosphate solution&#41; were sequentially applied to the skin of each participant&#46; The distance between application sites was 3<span class="elsevierStyleHsp" style=""></span>cm&#46; Avoiding blood vessels&#44; a needle was passed through the droplets to slightly puncture the skin without bleeding&#46; The needle was removed after 1<span class="elsevierStyleHsp" style=""></span>s&#44; and the skin was observed after 15<span class="elsevierStyleHsp" style=""></span>min&#46; The ratio of the wheal area caused by <span class="elsevierStyleItalic">T&#46; putrescentiae</span> extracts or recombinant Tyr p 4 protein liquid &#40;S1&#41; to the wheal area caused by positive control solution &#40;S2&#41; was determined&#46; If S1&#47;S2 was between 0 and 25&#37; it was scored as negative &#40;&#8722;&#41;&#44; between 26&#37; and 50&#37; it was slightly positive &#40;&#43;&#41;&#44; between 51&#37; and 100&#37; was positive &#40;&#43;&#43;&#41;&#44; between 101 and 200&#37; was &#40;&#43;&#43;&#43;&#41;&#44; and more than 200&#37; was &#40;&#43;&#43;&#43;&#43;&#41;&#46; The result was judged to be positive at &#40;&#43;&#43;&#41; or above&#46;<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a></p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">IgE-binding reactivity of recombinant Tyr p 4 protein</span><p id="par0065" class="elsevierStylePara elsevierViewall">The IgE-binding reactivity of Tyr p 4 allergen was detected by ELISA as previously described&#46;<a class="elsevierStyleCrossRef" href="#bib0045"><span class="elsevierStyleSup">9</span></a> Recombinant Tyr p 4 protein was coated onto a microplate&#44; blocked in bovine serum albumin &#40;BSA&#41;&#44; and patient serum was added to the microtiter plate&#46; Secondary antibody and 3&#44;3&#8242;&#44;5&#44;5&#8242;-tetramethylbenzidine &#40;TMB&#41; substrate solution were added to the microtiter plate to carry out the reaction&#46; Finally&#44; H<span class="elsevierStyleInf">2</span>SO<span class="elsevierStyleInf">4</span> was added to terminate the reaction&#46; The optical density was measured using an iMark microplate absorbance reader &#40;Bio-Rad&#41;&#46; Reactivity was considered positive if the optical density &#40;OD&#41; of the detected sera was higher than the cut-off ELISA value &#40;within three standard deviations of the mean ELISA value of healthy donors&#41;&#46; The specific IgE classes are defined using six calibrators&#58; 0&#44; 0&#46;35&#44; 0&#46;7&#44; 3&#46;5&#44; 17&#46;5 and 100<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L &#40;Class 0&#58; from 0 to &#60;0&#46;35<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#59; Class 1&#58; from 0&#46;35 to &#60;0&#46;7<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#59; Class 2&#58; from 0&#46;70 to &#60;3&#46;5<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#59; Class 3&#58; from 3&#46;50 to &#60;17&#46;5<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#59; Class 4&#58; from 17&#46;5 to &#60;50<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#59; Class 5&#58; from 50 to &#60;100<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#59; and Class 6&#58; from &#8805;100<span class="elsevierStyleHsp" style=""></span>kU<span class="elsevierStyleInf">A</span>&#47;L&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">12</span></a></p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Characterization of Tyr p 4 protein</span><p id="par0070" class="elsevierStylePara elsevierViewall">The amino acid sequence of Tyr p 4 protein &#40;GenBank accession no&#46; ABM53754&#46;1&#41; was obtained from the protein database of the National Center for Biotechnology Information &#40;<a href="http://www.allergen.org/">www&#46;allergen&#46;org</a>&#41;&#46; The open reading frame &#40;ORF&#41; was identified using ORF server&#46;<a class="elsevierStyleCrossRef" href="#bib0065"><span class="elsevierStyleSup">13</span></a> Transmembrane protein helices were predicted by TMHMM server 2&#46;0&#46;<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> The physicochemical properties and hydrophilicity of the Tyr p 4 amino acid sequence was predicted using ProtParam&#44;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a> and phosphorylation sites were predicted using NetPhos 3&#46;1 server&#46;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">16</span></a> Phylogenetic tree construction and identification of Tyr p 4 amino acid sequence alignment were performed by MEGA 5&#46;02<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">17</span></a> and VECTOR NTI 11&#46;0&#44;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">18</span></a> respectively&#46; Secondary structure was predicted using the JPred4 server&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">19</span></a></p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Structure prediction and homology modeling</span><p id="par0075" class="elsevierStylePara elsevierViewall">Templates of Tyr p 4 protein were analyzed for homology modeling in the Protein Data Bank &#40;PDB&#41;<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">20</span></a> using Protein BLAST&#46; Based on high score&#44; low e-value&#44; and maximum sequence identity&#44; the appropriate templates were selected&#46; The tertiary structure of Tyr p 4 protein was predicted by homology modeling using MODELLER 9&#46;22&#46;<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">21</span></a> PROCHECK determined the stereochemical quality of Tyr p 4 protein structure by analyzing residue-by-residue geometry and overall structural geometry&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> VERIFY 3D was used to detect the compatibility of an atomic model &#40;3D&#41; of Tyr p 4 protein with its own amino acid sequence &#40;1D&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">23</span></a> ERRAT analyzed the statistics of non-bonded interactions between different atom types&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a> ProSA was used to analyze the <span class="elsevierStyleItalic">Z</span>-score to determine the degree of match between the template and Tyr p 4 protein&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">25</span></a> QMEAN derived both global &#40;the entire structure&#41; and local &#40;per residue&#41; absolute quality estimates on the basis of one single model&#46;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> Interactive visualization and analysis of molecular structures were completed using UCSF Chimera 1&#46;13&#46;1&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">27</span></a></p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Prediction of epitopes</span><p id="par0080" class="elsevierStylePara elsevierViewall">BcePred&#44;<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">28</span></a> ABCpred&#44;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> and BCPreds<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">30</span></a> were used to predict the B-cell antigenic epitopes of Tyr p 4&#46; NetMHCpan 4&#46;0&#44;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">31</span></a> NetMHCII 2&#46;3&#44;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">32</span></a> and NetMHCIIpan 3&#46;2<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">33</span></a> were used to predict the T-cell antigenic epitopes of Tyr p 4&#46;</p></span></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Results</span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Gene cloning&#44; expression&#44; and purification of recombinant Tyr p 4 protein</span><p id="par0085" class="elsevierStylePara elsevierViewall">Plasmids from sequenced kanamycin-resistant clones were used to successfully transform pET-28a&#40;&#43;&#41;-Tyr p 4 into <span class="elsevierStyleItalic">E&#46; coli</span> DH5&#945; cells and expressed under IPTG &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41;&#46; A single and specific band matching the predicted molecular weight of Tyr p 4 using ProtParam was observed &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>B&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Characterization of Tyr p 4 protein</span><p id="par0090" class="elsevierStylePara elsevierViewall">The Tyr p 4 gene contains a complete open reading frame&#44; with 1557<span class="elsevierStyleHsp" style=""></span>bp between the initiation codon ATG and the stop codon TAA&#44; encoding a protein of 518 amino acids&#46; Family classification shows that Tyr p 4 protein belongs to the alpha amylase family &#40;InterPro no&#46; IPR006046&#41;&#46; Tyr p 4 protein has no transmembrane helices&#44; and all amino acids of the Tyr p 4 protein are outside the membrane&#46; ProtParam indicates that Tyr p 4 protein has a relative molecular weight of 58&#46;4<span class="elsevierStyleHsp" style=""></span>kDa and a theoretical isoelectric point &#40;pI&#41; of 6&#46;76&#46; The instability index &#40;II&#41; was computed to be 36&#46;11&#44; classifying the protein as stable&#46; The grand average of hydropathicity is &#8722;0&#46;431&#44; indicating that Tyr p 4 protein is a hydrophilic protein&#46; Tyr p 4 protein contains 42 phosphorylation sites&#44; includes 27 serine sites &#40;amino acids 28&#44; 69&#44; 95&#44; 97&#44; 166&#44; 185&#44; 193&#44; 194&#44; 221&#44; 266&#44; 361&#44; 384&#44; 424&#44; 503&#44; 504&#44; and 505&#41;&#44; five tyrosine sites &#40;amino acids 196&#44; 269&#44; 362&#44; 436&#44; and 471&#41;&#44; and one threonine site &#40;amino acid 457&#41;&#46; Secondary structure prediction shows that Tyr p 4 protein contains &#945;-helices&#44; &#946;-sheets&#44; and random coils&#46; The analysis of the phylogenetic tree and sequence identity showed the closest genetic relationship between Tyr p 4 and Aca s 4 &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a> and <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="tbl0005"></elsevierMultimedia></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Skin prick test and IgE-binding reactivity of recombinant Tyr p 4 protein</span><p id="par0095" class="elsevierStylePara elsevierViewall">The skin prick test of recombinant Tyr p 4 protein revealed seven &#43; cases&#44; twenty-two &#43;&#43; cases&#44; and one &#43;&#43;&#43; case&#46; The positive response rate was 19&#46;2&#37; &#40;23 participants out of 120&#41;&#46; The serum specific IgE test of recombinant Tyr p 4 protein revealed ten cases of Class 1&#44; eight cases of Class 2&#44; and two cases of Class 3&#46; The total positive response rate was 16&#46;7&#37; &#40;20&#47;120&#41;&#46; Sixteen participants were positive in both skin prick test and sera specific IgE-binding test&#44; with a positive rate of 13&#46;3&#37; &#40;16&#47;120&#41;&#46;</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Homology modeling of Tyr p 4 protein tertiary structure</span><p id="par0100" class="elsevierStylePara elsevierViewall">The sequence identity of alpha-amylase &#40;PDB accession no&#46; 1PPI&#41; and Tyr p 4 protein is 52&#46;32&#37;&#46; The parameters of the Tyr p 4 protein tertiary structure are similar to 1PPI &#40;<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>&#41;&#46; PROCHECK shows high stereochemical quality of Tyr p 4 protein structure and the ERRAT value suggests high resolution of the tertiary structure of Tyr p 4 protein&#46; VERIFY 3D suggests that the Tyr p 4 protein structure is favorable&#46; The <span class="elsevierStyleItalic">Z</span>-scores of the template and Tyr p 4 protein show a high level of matching between the tertiary structures of the protein&#46; The <span class="elsevierStyleItalic">Q</span> value suggests that the predicted model of Tyr p 4 protein is reliable&#46; Based on this evaluation&#44; we determined the tertiary structure of Tyr p 4 protein obtained by homologous modeling to be reliable&#46; The tertiary structure of Tyr p 4 protein also contains &#945;-helices&#44; &#946;-sheets&#44; and random coils&#44; and similar proportions of these elements are found in the secondary and tertiary structures&#46; The proportions of &#945;-helices&#44; &#946;-sheets and random coils in the secondary structure are 28&#46;19&#37; &#40;15 domains&#41;&#44; 18&#46;34&#37; &#40;20 domains&#41; and 53&#46;47&#37;&#44; and are 25&#46;87&#37; &#40;18 domains&#41;&#44; 12&#46;55&#37; &#40;13 domains&#41; and 61&#46;58&#37; in the tertiary structure&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Tyr p 4 protein epitope prediction</span><p id="par0105" class="elsevierStylePara elsevierViewall">Eight B-cell epitopes &#40;amino acid positions 26&#8211;32&#44; 90&#8211;99&#44; 137&#8211;145&#44; 160&#8211;175&#44; 256&#8211;265&#44; 369&#8211;381&#44; 431&#8211;436&#44; and 502&#8211;508&#41; and three T-cell epitopes &#40;amino acid positions 204&#8211;208&#44; 280&#8211;288&#44; and 347&#8211;355&#41; of Tyr p 4 were predicted &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a> and <a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><elsevierMultimedia ident="tbl0015"></elsevierMultimedia></span></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Discussion</span><p id="par0110" class="elsevierStylePara elsevierViewall">Although it was a potential allergen&#44; there is little information regarding the physical and chemical properties of Tyr p 4&#44; its allergenicity&#44; or antigenic epitopes&#46; It has not been included in the official website of the systematic allergen nomenclature approved by the WHO&#47;IUIS Allergen Nomenclature Database &#40;<a href="http://www.allergen.org/">http&#58;&#47;&#47;www&#46;allergen&#46;org&#47;</a>&#41;&#46; We determined the allergenicity of recombinant Tyr p 4 protein by skin prick test and ELISA&#46; The skin prick positive rate was 19&#46;2&#37;&#44; the rate of serum specific IgE positives was 16&#46;7&#37;&#44; and the total positive response to both methods was 13&#46;3&#37;&#46; The total positive rate of the skin prick test was higher than the serum specific IgE test&#44; this is likely due to a false positive result on the skin prick test&#59; thus&#44; combining the two methods allows for a more accurate determination&#46; Phylogenetic analysis shows that Tyr p 4 has the highest homology with Aca s 4 &#40;<span class="elsevierStyleItalic">Acarus siro</span>&#41; and Blo t 4 &#40;<span class="elsevierStyleItalic">Blomia tropicalis</span>&#41;&#46; Interestingly&#44; all three are storage mites&#44; and Blo t 4 induced positive skin prick tests in 10&#37; of 200&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">34</span></a> Based on these results&#44; the allergenicity of groups 4 allergens is weak compared to groups 1 and 2 allergens&#46; Cross-reactivity sometimes occurs between the same group allergens of different mite species&#44; but no reports on cross-reactivity between different mite species group 4 allergens have been found to date&#46; This will be the focus of our further research&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">Although Tyr p 4 protein contains &#945;-helices&#44; &#946;-sheets&#44; and random coils&#44; the number of amino acids in these elements in the tertiary structure was found to vary marginally from the secondary structure&#46; This discrepancy may be due to different structural prediction methods&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">After determining the allergenicity of Tyr p 4&#44; the B-cell and T-cell epitopes of Tyr p 4 were predicted&#46; The properties of the antigen epitope&#44; their number&#44; and spatial configuration determine antigen specificity&#46;<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">35&#44;36</span></a> &#945;-helices and &#946;-sheets have higher chemical bond energy and have difficulty forming epitope sequences&#46; By contrast&#44; &#946;-turns and random coils are located in surface-exposed regions of a protein&#44; which often contain epitope sequences&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">37</span></a> Epitopes usually contain 6&#8211;8 amino acids residues and in general contain &#60;20 amino acid residues&#46; In addition&#44; allergen epitopes usually contain high proportions of hydrophobic amino acid residues&#44; including Ala&#44; Ser&#44; Asn&#44; Gly&#44; and Lys&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">38</span></a> Based on the results of the epitope prediction software and the tertiary structure of the Tyr p 4 protein&#44; the presence of eight B-cell epitopes and three T-cell epitopes is likely&#46; However&#44; these predicted epitopes require further experimental verification&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">In conclusion&#44; we demonstrate that the cloning&#44; expression&#44; characterization&#44; allergenicity&#44; and epitopes of recombinant Tyr p 4 protein has enabled an initial evaluation of its potency as an allergen in mite-allergic individuals&#46; These findings provide a foundation from which to explore the structural biology and biochemistry of Tyr p 4 protein&#44; thereby enabling future work with ASIT&#46;</p></span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Conflict of interest</span><p id="par0130" class="elsevierStylePara elsevierViewall">The authors have no conflict of interest to declare&#46;</p></span></span>"
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          "titulo" => "Abstract"
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              "titulo" => "Introduction and objectives"
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              "titulo" => "Materials and methods"
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          "titulo" => "Keywords"
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        2 => array:2 [
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          "titulo" => "Abbreviations"
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        3 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
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        4 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:11 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Tyrophagus putrescentiae extracts"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Serum samples"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Sequencing Tyr p 4"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "TA cloning"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Construction of expression plasmid pET-28a&#40;&#43;&#41;-Tyr p 4"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Production and purification of recombinant Tyr p 4 protein"
            ]
            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Skin prick test"
            ]
            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "IgE-binding reactivity of recombinant Tyr p 4 protein"
            ]
            8 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "Characterization of Tyr p 4 protein"
            ]
            9 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Structure prediction and homology modeling"
            ]
            10 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Prediction of epitopes"
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          ]
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        5 => array:3 [
          "identificador" => "sec0070"
          "titulo" => "Results"
          "secciones" => array:5 [
            0 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Gene cloning&#44; expression&#44; and purification of recombinant Tyr p 4 protein"
            ]
            1 => array:2 [
              "identificador" => "sec0080"
              "titulo" => "Characterization of Tyr p 4 protein"
            ]
            2 => array:2 [
              "identificador" => "sec0085"
              "titulo" => "Skin prick test and IgE-binding reactivity of recombinant Tyr p 4 protein"
            ]
            3 => array:2 [
              "identificador" => "sec0090"
              "titulo" => "Homology modeling of Tyr p 4 protein tertiary structure"
            ]
            4 => array:2 [
              "identificador" => "sec0095"
              "titulo" => "Tyr p 4 protein epitope prediction"
            ]
          ]
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        6 => array:2 [
          "identificador" => "sec0100"
          "titulo" => "Discussion"
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        7 => array:2 [
          "identificador" => "sec0105"
          "titulo" => "Conflict of interest"
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        8 => array:2 [
          "identificador" => "xack496383"
          "titulo" => "Acknowledgments"
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          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2019-12-01"
    "fechaAceptado" => "2020-02-17"
    "PalabrasClave" => array:1 [
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        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1300241"
          "palabras" => array:5 [
            0 => "Storage mites"
            1 => "Immunoglobulin E &#40;IgE&#41;"
            2 => "Molecular cloning"
            3 => "Recombinant mite allergen"
            4 => "Homology modeling"
          ]
        ]
        1 => array:4 [
          "clase" => "abr"
          "titulo" => "Abbreviations"
          "identificador" => "xpalclavsec1300240"
          "palabras" => array:12 [
            0 => "Aca s"
            1 => "ASIT"
            2 => "Blo the"
            3 => "BSA"
            4 => "Der f"
            5 => "Der p"
            6 => "HDMs"
            7 => "OD"
            8 => "ORF"
            9 => "PDB"
            10 => "TMB"
            11 => "Tyr p"
          ]
        ]
      ]
    ]
    "tieneResumen" => true
    "resumen" => array:1 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction and objectives</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Allergen-specific immunotherapy &#40;ASIT&#41; is the only allergic disease-modifying therapy available for children and adults&#44; and recombinant allergens are an interesting approach to improve allergy diagnosis and ASIT&#46; <span class="elsevierStyleItalic">Tyrophagus putrescentiae</span> is a common storage mite that produces potent allergens&#46; The aim of this study was to express and characterize recombinant group 4 allergen protein of <span class="elsevierStyleItalic">T&#46; putrescentiae</span> &#40;Tyr p 4&#41;&#44; and to further investigate allergenicity and potential epitopes of Tyr p 4&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Materials and methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">The cDNA encoding Tyr p 4 was generated by RT-PCR and subcloned into pET-28a&#40;&#43;&#41; plasmid&#46; The plasmid was then transformed into <span class="elsevierStyleItalic">E&#46; coli</span> cells for expression&#46; After purification by nickel affinity chromatography and identification by SDS-PAGE&#44; recombinant Tyr p 4 protein was used for a skin prick test and an ELISA to determine the allergic response&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Study participants&#8217; allergic response rate to Tyr p 4 protein was 13&#46;3&#37; &#40;16&#47;120&#41;&#46; Eight B-cell epitopes and three T-cell epitopes of Tyr p 4 were predicted&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Similar to group 4 allergens of other species of mite&#44; allergenicity of Tyr p 4 is weak&#46; The expression&#44; characterization and epitope prediction of recombinant Tyr p 4 protein provide a foundation for further study of this allergen in the diagnosis and ASIT of storage mite allergy&#46;</p></span>"
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            "titulo" => "Results"
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          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Expression and purification of rTyr p 4 protein&#46; &#40;A&#41; SDS-PAGE demonstrating expression of rTyr p 4 protein in BL21 cells&#46; Lane M&#44; protein marker &#40;TransGen Biotech&#41;&#59; lane 1&#44; the pellet of cells containing pET-28a&#40;&#43;&#41;-Tyr p 4 3<span class="elsevierStyleHsp" style=""></span>h after induction&#59; lane 2&#44; supernatant from cells containing pET-28a&#40;&#43;&#41;-Tyr p 4 3<span class="elsevierStyleHsp" style=""></span>h after induction&#59; lane 3&#44; whole BL21 cells containing pET-28b &#40;&#43;&#41;-Tyr p 4 before IPTG induction&#59; lane 4&#44; whole BL21 cells containing pET-28a&#40;&#43;&#41; before IPTG induction&#46; &#40;B&#41; Tyr p 4 protein purified from BL21 cells&#46; Lane M&#44; protein marker &#40;TransGen Biotech&#41;&#59; lane 1&#44; purified Tyr p 4 protein&#46;</p>"
        ]
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          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Phylogenetic tree of Tyr p 4 protein&#46;</p>"
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        "etiqueta" => "Fig&#46; 3"
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          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">3D structure and epitopes of Tyr p 4 protein&#46; &#40;A-1 and A-2&#41; 3D structure of Tyr p 4 protein&#46; &#40;B-1 and B-2&#41; B-cell epitopes on the 3D structure of Tyr p 4 protein&#46; &#40;C-1 and C-2&#41; T-cell epitopes on the predicted 3D structure of Tyr p 4 protein&#46;</p>"
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Der f 4&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Eur m 4&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">Tyr p 4&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t">64&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">68&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">66&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">62&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">62&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Blo t 4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">70&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">65&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">66&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Der p 4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">89&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">90&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Der f 4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
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                  \t\t\t\t">87&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Eur m 4&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
                  """
              ]
              "imagenFichero" => array:1 [
                0 => "xTab2439214.png"
              ]
            ]
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Percent identity between Tyr p 4 and related allergens&#46;</p>"
        ]
      ]
      4 => array:8 [
        "identificador" => "tbl0010"
        "etiqueta" => "Table 2"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "at2"
            "detalle" => "Table "
            "rol" => "short"
          ]
        ]
        "tabla" => array:1 [
          "tablatextoimagen" => array:1 [
            0 => array:2 [
              "tabla" => array:1 [
                0 => """
                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Protein&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Structural assessment method&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">PROCHECK &#40;&#37;&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">ERRAT value&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">VERIFY 3D &#40;&#37;&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">Z</span>-score&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">Q</span>-value&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Tyr p 4&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">87&#46;9 core&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="char" valign="\n
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                  \t\t\t\t  " align="char" valign="\n
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                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">10&#46;6 allowed&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&#46;4 generously allowed&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;0 disallowed&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">ProSa&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;9&#46;25&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">QMEAN&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;78&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1PPI&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">SAVES&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">89&#46;8 core&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t">96&#46;0168&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">100&#46;00&#37;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">9&#46;8 allowed&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;5 generously allowed&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;0 disallowed&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">ProSa&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;9&#46;29&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">QMEAN&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;65&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
                  """
              ]
              "imagenFichero" => array:1 [
                0 => "xTab2439213.png"
              ]
            ]
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Evaluation of the tertiary structure of Tyr p 4 protein&#46;</p>"
        ]
      ]
      5 => array:8 [
        "identificador" => "tbl0015"
        "etiqueta" => "Table 3"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "at3"
            "detalle" => "Table "
            "rol" => "short"
          ]
        ]
        "tabla" => array:2 [
          "leyenda" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Bold letters represent the hydrophobic amino acid residues&#46;</p>"
          "tablatextoimagen" => array:1 [
            0 => array:2 [
              "tabla" => array:1 [
                0 => """
                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Type of epitope&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Prediction server&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Epitope prediction &#40;peptide&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Confirmed epitopes &#40;peptide&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " rowspan="3" align="left" valign="middle">B-cell epitopes</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">BcePred&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">26&#8211;32&#44; 87&#8211;99&#44; 127&#8211;145&#44; 157&#8211;175&#44; 188&#8211;194&#44; 243&#8211;250&#44; 255&#8211;265&#44; 270&#8211;277&#44; 290&#8211;293&#44; 317&#8211;325&#44; 369&#8211;387&#44; 400&#8211;405&#44; 431&#8211;436&#44; 477&#8211;487&#44; 502&#8211;508&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " rowspan="3" align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">B1&#40;26&#8211;32&#44; PY<span class="elsevierStyleBold">S</span>HPHF&#41;B2&#40;90&#8211;99&#44; <span class="elsevierStyleBold">S</span>YRII<span class="elsevierStyleBold">S</span>R<span class="elsevierStyleBold">SGN</span>&#41;B3&#40;137&#8211;145&#44; <span class="elsevierStyleBold">GS</span>HY<span class="elsevierStyleBold">NGNS</span>M&#41;B4&#40;160&#8211;175&#44; HE<span class="elsevierStyleBold">S</span>CPT<span class="elsevierStyleBold">S</span>DLEIH<span class="elsevierStyleBold">N</span>YD<span class="elsevierStyleBold">N</span>&#41;B5&#40;256&#8211;265&#44; VIYY<span class="elsevierStyleBold">GGNG</span>I<span class="elsevierStyleBold">K</span>&#41;B6&#40;369&#8211;381&#44; Q<span class="elsevierStyleBold">NG</span>HDV<span class="elsevierStyleBold">N</span>DWM<span class="elsevierStyleBold">G</span>PP&#41;B7&#40;431&#8211;436&#44; WD<span class="elsevierStyleBold">NG</span>DY&#41;B8&#40;502&#8211;508&#44; <span class="elsevierStyleBold">NSSS</span>DDD&#41;</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">ABCpred&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">23&#8211;38&#44; 90&#8211;105&#44; 133&#8211;148&#44; 160&#8211;175&#44; 185&#8211;200&#44; 242&#8211;257&#44; 253&#8211;268&#44; 272&#8211;287&#44; 289&#8211;304&#44; 318&#8211;333&#44; 366&#8211;381&#44; 398&#8211;413&#44; 426&#8211;441&#44; 481&#8211;496&#44; 500&#8211;515&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">BCPred&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">21&#8211;40&#44; 56&#8211;75&#44; 83&#8211;102&#44; 137&#8211;156&#44; 160&#8211;179&#44; 256&#8211;275&#44; 290&#8211;309&#44; 315&#8211;334&#44; 367&#8211;386&#44; 425&#8211;444&#44; 453&#8211;472&#44; 491&#8211;510&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " colspan="4" align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleVsp" style="height:0.5px"></span></td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " rowspan="3" align="left" valign="middle">T-cell epitopes</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">NetMHCpan&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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Article information

ISSN: 03010546
Original language: English

DOI:10.1016/j.aller.2020.02.006

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	4	0	4
2024 October	116	9	125
2024 September	166	12	178
2024 August	99	25	124
2024 July	95	16	111
2024 June	102	9	111
2024 May	128	19	147
2024 April	111	21	132
2024 March	112	11	123
2024 February	107	16	123
2024 January	126	11	137
2023 December	105	10	115
2023 November	123	8	131
2023 October	209	10	219
2023 September	123	2	125
2023 August	113	16	129
2023 July	112	3	115
2023 June	105	11	116
2023 May	151	15	166
2023 April	81	12	93
2023 March	107	9	116
2023 February	66	7	73
2023 January	91	7	98
2022 December	87	14	101
2022 November	78	19	97
2022 October	70	21	91
2022 September	97	17	114
2022 August	80	16	96
2022 July	57	9	66
2022 June	42	19	61
2022 May	35	18	53
2022 April	37	23	60
2022 March	58	16	74
2022 February	37	14	51
2022 January	39	6	45
2021 December	49	15	64
2021 November	42	30	72
2021 October	65	36	101
2021 September	60	22	82
2021 August	19	28	47
2021 July	18	22	40
2021 June	28	15	43
2021 May	32	18	50
2021 April	72	35	107
2021 March	80	18	98
2021 February	25	27	52
2021 January	21	20	41
2020 December	6	0	6
2020 June	0	2	2

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Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

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