RESUMEN
Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.
Asunto(s)
Alérgenos , Antígenos de Plantas , Aptámeros de Nucleótidos , Arachis , Ensayo de Inmunoadsorción Enzimática , Proteínas de Plantas , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/inmunología , Arachis/química , Arachis/inmunología , Antígenos de Plantas/inmunología , Antígenos de Plantas/análisis , Antígenos de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/genética , Alérgenos/inmunología , Alérgenos/análisis , Hipersensibilidad al Cacahuete/inmunología , Glicoproteínas/inmunología , Glicoproteínas/química , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/genética , Humanos , Técnica SELEX de Producción de Aptámeros/métodosRESUMEN
Pollen from Salsola kali, i.e., saltwort, Russian thistle, is a major allergen source in the coastal regions of southern Europe, in Turkey, Central Asia, and Iran. S. kali-allergic patients mainly suffer from hay-fever (i.e., rhinitis and conjunctivitis), asthma, and allergic skin symptoms. The aim of this study was to investigate the importance of individual S. kali allergen molecules. Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5, and Sal k 6 were expressed in Escherichia coli as recombinant proteins containing a C-terminal hexahistidine tag and purified by nickel affinity chromatography. The purity of the recombinant allergens was analyzed by SDS-PAGE. Their molecular weight was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and their fold and secondary structure were studied by circular dichroism (CD) spectroscopy. Sera from clinically well-characterized S. kali-allergic patients were used for IgE reactivity and basophil activation experiments. S. kali allergen-specific IgE levels and IgE levels specific for the highly IgE cross-reactive profilin and the calcium-binding allergen from timothy grass pollen, Phl p 12 and Phl p 7, respectively, were measured by ImmunoCAP. The allergenic activity of natural S. kali pollen allergens was studied in basophil activation experiments. Recombinant S. kali allergens were folded when studied by CD analysis. The sum of recombinant allergen-specific IgE levels and allergen-extract-specific IgE levels was highly correlated. Sal k 1 and profilin, reactive with IgE from 64% and 49% of patients, respectively, were the most important allergens, whereas the other S. kali allergens were less frequently recognized. Specific IgE levels were highest for profilin. Of note, 37% of patients who were negative for Sal k 1 showed IgE reactivity to Phl p 12, emphasizing the importance of the ubiquitous cytoskeletal actin-binding protein, profilin, for the diagnosis of IgE sensitization in S. kali-allergic patients. rPhl p 12 and rSal k 4 showed equivalent IgE reactivity, and the clinical importance of profilin was underlined by the fact that profilin-monosensitized patients suffered from symptoms of respiratory allergy to saltwort. Accordingly, profilin should be included in the panel of allergen molecules for diagnosis and in molecular allergy vaccines for the treatment and prevention of S. kali allergy.
Asunto(s)
Alérgenos , Reacciones Cruzadas , Inmunoglobulina E , Polen , Profilinas , Salsola , Humanos , Profilinas/inmunología , Profilinas/química , Inmunoglobulina E/inmunología , Alérgenos/inmunología , Alérgenos/genética , Salsola/inmunología , Femenino , Polen/inmunología , Masculino , Reacciones Cruzadas/inmunología , Adulto , Proteínas Recombinantes/inmunología , Rinitis Alérgica Estacional/inmunología , Persona de Mediana Edad , Basófilos/inmunología , Basófilos/metabolismo , Antígenos de Plantas/inmunología , Antígenos de Plantas/genética , Adulto Joven , Adolescente , Proteínas de Plantas/inmunología , Proteínas de Plantas/genéticaRESUMEN
Soybean ß-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in ß-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean ß-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the ß-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and ß-subunits of soybean ß-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the ß-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and ß-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein ß-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the ß-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.
Asunto(s)
Antígenos de Plantas , Sistemas CRISPR-Cas , Edición Génica , Globulinas , Glycine max , ARN Largo no Codificante , Proteínas de Almacenamiento de Semillas , Proteínas de Soja , Proteínas de Almacenamiento de Semillas/genética , Proteínas de Almacenamiento de Semillas/química , Globulinas/genética , Globulinas/metabolismo , Globulinas/química , Glycine max/genética , Glycine max/metabolismo , Antígenos de Plantas/genética , Antígenos de Plantas/química , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Proteínas de Soja/química , ARN Largo no Codificante/genética , Regulación de la Expresión Génica de las Plantas , Semillas/genética , Semillas/metabolismo , Semillas/químicaRESUMEN
Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.
Asunto(s)
Actinidia , Alérgenos , Reacciones Cruzadas , Hipersensibilidad a los Alimentos , Inmunoglobulina E , Látex , Musa , Humanos , Reacciones Cruzadas/inmunología , Hipersensibilidad a los Alimentos/inmunología , Alérgenos/inmunología , Alérgenos/genética , Musa/inmunología , Musa/genética , Inmunoglobulina E/inmunología , Actinidia/inmunología , Femenino , Látex/inmunología , Masculino , Proteínas de Plantas/inmunología , Proteínas de Plantas/genética , Adulto , Antígenos de Plantas/inmunología , Antígenos de Plantas/genética , Secuencia de Aminoácidos , Epítopos de Linfocito T/inmunología , Persona de Mediana Edad , Adolescente , Niño , Adulto JovenRESUMEN
Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.
Asunto(s)
Alérgenos , Ambrosia , Antígenos de Plantas , Inmunoglobulina E , Proteínas Recombinantes , Humanos , Antígenos de Plantas/inmunología , Antígenos de Plantas/genética , Antígenos de Plantas/química , Inmunoglobulina E/inmunología , Animales , Alérgenos/inmunología , Alérgenos/genética , Ambrosia/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Femenino , Adulto , Proteínas de Plantas/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/química , Rinitis Alérgica Estacional/inmunología , Masculino , Persona de Mediana Edad , Extractos Vegetales/químicaRESUMEN
Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I (151SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG181) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 (157DQMPRRFYLANGNQE170) showed allergenicity, and after replacing it with alanine, it was found that amino acids D157, Q158, M159, and Y164 were the key amino acids that affected its antigenicity, while Q158, M159, R162, and N168 affected allergenicity.
Asunto(s)
Alérgenos , Globulinas , Calor , Proteínas de Soja , Alérgenos/inmunología , Alérgenos/química , Humanos , Globulinas/química , Globulinas/inmunología , Proteínas de Soja/química , Proteínas de Soja/inmunología , Secuencia de Aminoácidos , Hipersensibilidad a los Alimentos/inmunología , Epítopos/química , Epítopos/inmunología , Dominios Proteicos , Antígenos de Plantas/inmunología , Antígenos de Plantas/química , Antígenos de Plantas/genética , Glycine max/química , Glycine max/inmunología , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501-1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee.
Asunto(s)
Corylus , Hipersensibilidad , Humanos , Anciano , Alérgenos , Proteínas de Plantas/metabolismo , Polen/metabolismo , Betulaceae/metabolismo , Betula/metabolismo , ARN Mensajero , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismoRESUMEN
Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.
Asunto(s)
Betula , Hipersensibilidad , Humanos , Betula/genética , Células CACO-2 , Interleucina-4/genética , Polen , Interleucina-10/genética , Técnicas de Cocultivo , Regulación hacia Arriba , Escherichia coli/genética , Proteínas de Plantas/genética , Antígenos de Plantas/genética , Alérgenos/genética , Expresión Génica , Proteínas RecombinantesRESUMEN
Ambrosia trifida (giant ragweed) is an invasive plant that can cause serious damage to natural ecosystems and severe respiratory allergies. However, the genomic basis of invasive adaptation and pollen allergens in Ambrosia species remain largely unknown. Here, we present a 1.66 Gb chromosome-scale reference genome for giant ragweed and identified multiple types of genome duplications, which are responsible for its rapid environmental adaptation and pollen development. The largest copies number and species-specific expansions of resistance-related gene families compared to Heliantheae alliance might contribute to resist stresses, pathogens and rapid adaptation. To extend the knowledge of evolutionary process of allergic pollen proteins, we predicted 26 and 168 potential pollen allergen candidates for giant ragweed and other Asteraceae plant species by combining machine learning and identity screening. Interestingly, we observed a specific tandemly repeated array for potential allergenic pectate lyases among Ambrosia species. Rapid evolutionary rates on putative pectate lyase allergens may imply a crucial role of nonsynonymous mutations on amino acid residues for plant biological function and allergenicity. Altogether, this study provides insight into the molecular ecological adaptation and putative pollen allergens prediction that will be helpful in promoting invasion genomic research and evolution of putative pollen allergy in giant ragweed.
Asunto(s)
Ambrosia , Hipersensibilidad , Ambrosia/genética , Antígenos de Plantas/genética , Ecosistema , Alérgenos/genética , Alérgenos/química , Polen/genética , CromosomasRESUMEN
The term allergy was coined in 1906 by the Austrian scientist and pediatrician Clemens Freiherr von Pirquet. In 1976, Dietrich Kraft became the head of the Allergy and Immunology Research Group at the Department of General and Experimental Pathology of the University of Vienna. In 1983, Kraft proposed to replace natural extracts used in allergy diagnostic tests and vaccines with recombinant allergen molecules and persuaded Michael Breitenbach to contribute his expertise in molecular cloning as one of the mentors of this project. Thus, the foundation for the Vienna School of Molecular Allergology was laid. With the recruitment of Heimo Breiteneder as a young molecular biology researcher, the work began in earnest, resulting in the publication of the cloning of the first plant allergen Bet v 1 in 1989. Bet v 1 has become the subject of a very large number of basic scientific as well as clinical studies. Bet v 1 is also the founding member of the large Bet v 1-like superfamily of proteins with members-based on the ancient conserved Bet v 1 fold-being present in all three domains of life, i.e., archaea, bacteria and eukaryotes. This suggests that the Bet v 1 fold most likely already existed in the last universal common ancestor. The biological function of this protein was probably related to lipid binding. However, during evolution, a functional diversity within the Bet v 1-like superfamily was established. The superfamily comprises 25 families, one of which is the Bet v 1 family, which in turn is composed of 11 subfamilies. One of these, the PR-10-like subfamily of proteins, contains almost all of the Bet v 1 homologous allergens from pollen and plant foods. Structural and functional comparisons of Bet v 1 and its non-allergenic homologs of the superfamily will pave the way for a deeper understanding of the allergic sensitization process.
Asunto(s)
Alérgenos , Hipersensibilidad , Humanos , Betula , Proteínas de Plantas/química , Antígenos de Plantas/genética , Polen/genéticaRESUMEN
Raw strawberries contain allergens that cause oral allergic syndrome. Fra a 1 is one of the major allergens in strawberries and might decrease their allergenicity by heating, likely due to structural changes in the allergen leading to decreased recognition of the allergens in the oral cavity. In the present study, to understand the relationship between allergen structure and allergenicity, the expression and purification of 15N-labeled Fra a 1 were examined and the sample was used for NMR analysis. Two isoforms, Fra a 1.01 and Fra a 1.02, were used and expressed in E. coli BL21(DE3) in M9 minimal medium. Fra a 1.02 was purified as a single protein by using the GST tag approach, whereas histidine × 6-tag (his6-tag) Fra a 1.02 was obtained both as the full-length (â¼20 kDa) and a truncated (â¼18 kDa) form. On the other hand, his6-tag Fra a 1.01 was purified as a homogeneous protein. 15N-labeled HSQC NMR spectra suggested that Fra a 1.02 was thermally denatured at lower temperatures than Fra a 1.01, despite the high amino acid sequence homology (79.4%) of these isoforms. Furthermore, the samples in the present study allowed us to analyze ligand binding that probably affects structural stability. In conclusion, GST tag was effective for obtaining a homogeneous protein when his6-tag failed to give a single form, and the present study provided a sample that could be used for NMR studies of the details of the allergenicity and structure of Fra a 1.
Asunto(s)
Alérgenos , Fragaria , Alérgenos/genética , Alérgenos/química , Proteínas de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Fragaria/genética , Fragaria/química , Escherichia coli/genética , Escherichia coli/metabolismo , Isoformas de ProteínasRESUMEN
Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis.
Asunto(s)
Hipersensibilidad a los Alimentos , Proteínas de Plantas , Humanos , Mapeo Epitopo , Proteínas de Plantas/metabolismo , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Secuencia de Aminoácidos , Glicoproteínas , Epítopos , Péptidos , Alérgenos , Arachis , Inmunoglobulina E/metabolismoRESUMEN
Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.
Asunto(s)
Alérgenos , Antígenos de Plantas , Hipersensibilidad a la Nuez , Humanos , Alérgenos/química , Antígenos de Plantas/química , Antígenos de Plantas/genética , Escherichia coli/genética , Inmunoglobulina E/química , Macadamia/genética , Hipersensibilidad a la Nuez/diagnóstico , Hipersensibilidad a la Nuez/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Isoformas de Proteínas , LeguminasRESUMEN
Birch belongs to the order Fagales and the family Betulaceae. Birch pollen is one of the most important airborne inhaled allergens in the north temperate zone, leading to allergic rhinitis, asthma and pollen-related food allergy. The sensitization rate to birch pollen is about 8-16% in the general population and 7-57% in patients seen at various allergy centers. Seven birch pollen allergens have been recognized by the International Allergen Nomenclature Sub-committee, with Bet v 1 as the sole major allergen. Component-resolved diagnostics can help to discriminate broad cross-reactivity and false-positive diagnoses of pollen allergy caused by specific IgE to pan-allergens such as Bet v 2, 4 or Bet v 7 from true birch allergy represented by the major allergen Bet v 1-specific IgE. Patients with allergic symptoms to birch pollen showed significantly higher serum anti-Bet v 1 IgE concentrations than asymptomatic individuals with birch sensitization. A higher level of IgE to Bet v 1 also predicted oral allergy syndrome after the ingestion of Rosaceae fruits, nuts, or Apiaceae vegetables, which have cross-reactive homologous allergens with birch allergens. Bet v 1 is one of the first allergens developed using recombinant technology. Many forms of genetically modified Bet v 1 hypo-allergens have been developed and have shown benefit in animal models or even clinical trials of allergen immunotherapy.
Asunto(s)
Alérgenos , Hipersensibilidad a los Alimentos , Animales , Humanos , Betula , Antígenos de Plantas/genética , Polen , Inmunoglobulina E , Reacciones Cruzadas , Proteínas de Plantas/genéticaRESUMEN
In this work, three allergen-encoding genes (Ana o 1, Ana o 2, Ana o 3) were investigated for the detection of cashew nut as an allergenic food. Normalised and single-tube nested real-time PCR approaches targeting the Ana o 2 or Ana o 3 genes are proposed and compared. Normalised real-time PCR detected 10 pg, while single-tube nested real-time PCR achieved 1 pg of cashew nut DNA. Single-tube nested real-time PCR targeting Ana o 3 allowed the best relative sensitivities (10 mg/kg cashew nut in dough/biscuit), being successfully validated regarding precision/accuracy. The normalised real-time PCR did not show acceptable accuracy for both targets. Sensitivity of single-tube nested real-time PCR was affected by the matrix (pasta), but not by thermal processing (dough/biscuit). Herein, two highly sensitive and specific single-tube nested real-time PCR targeting allergen-encoding genes are proposed for the first time as quantitative/validated tools for cashew nut analysis as an allergenic food.
Asunto(s)
Anacardium , Hipersensibilidad a los Alimentos , Hipersensibilidad a la Nuez , Alérgenos/genética , Antígenos de Plantas/genética , Inmunoglobulina E , Hipersensibilidad a la Nuez/genética , Nueces , Proteínas de Plantas/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Gibberellin-regulated protein (GRP) is a fruit severe allergen. The amounts of GRP expression normalized against actin in peach were determined by reverse transcription-quantitative PCR (RT-qPCR). The results were consistent with those determined by enzyme-linked immunosorbent assay (ELISA). The GRP expression was more evident in flesh than peel and increased rapidly in the maturing period. This approach is applicable to estimate the amount of GRP in other plants.
Asunto(s)
Prunus persica , Actinas/metabolismo , Alérgenos/metabolismo , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Frutas/metabolismo , Giberelinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prunus persica/genética , Prunus persica/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción ReversaRESUMEN
Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteine-protease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen-specific immunotherapy.
Asunto(s)
Proteasas de Cisteína , Hipersensibilidad , Rinitis Alérgica Estacional , Alérgenos/química , Alérgenos/genética , Ambrosia/genética , Ambrosia/metabolismo , Antígenos de Plantas/genética , Proteasas de Cisteína/genética , Humanos , Inmunoglobulina E/metabolismo , Extractos Vegetales , Proteínas de Plantas/química , Proteínas de Plantas/genética , PolenRESUMEN
The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.
Asunto(s)
Albuminas 2S de Plantas/genética , Antígenos de Plantas/genética , Arachis/genética , Edición Génica , Protoplastos , Arachis/inmunología , Sistemas CRISPR-Cas , Marcación de Gen , Vectores Genéticos/genética , Proyectos Piloto , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida , Plantones , Temperatura , Transfección/métodosRESUMEN
Reducing allergenicity and increasing oleic content are important goals in groundnut breeding studies. Ara h 1 is a major allergen gene and Delta(12)-fatty-acid desaturase (FAD2) is responsible for converting oleic into linoleic acid. These genes have homoeologues with one copy in each subgenome, identified as Ara h 1.01, Ara h 1.02, ahFAD2A and ahFAD2B in tetraploid groundnut. To alter functional properties of these genes we have generated an Ethyl Methane Sulfonate (EMS) induced mutant population to be used in Targeting Induced Local Lesions in Genomes (TILLING) approach. Seeds were exposed to two EMS concentrations and the germination rates were calculated as 90.1% (1353 plants) for 0.4% and 60.4% (906 plants) for 1.2% EMS concentrations in the M1 generation. Among the 1541 M2 mutants, 768 were analyzed by TILLING using four homoeologous genes. Two heterozygous mutations were identified in the ahFAD2B and ahFAD2A gene regions from 1.2% and 0.4% EMS-treated populations, respectively. The mutation in ahFAD2B resulted in an amino acid change, which was serine to threonine predicted to be tolerated according to SIFT analysis. The other mutation causing amino acid change, glycine to aspartic acid was predicted to affect protein function in ahFAD2A. No mutations were detected in Ara h 1.01 and Ara h 1.02 for both EMS-treatments after sequencing. We estimated the overall mutation rate to be 1 mutation every 2139 kb. The mutation frequencies were also 1/317 kb for ahFAD2A in 0.4% EMS and 1/466 kb for ahFAD2B in 1.2% EMS treatments. The results demonstrated that TILLING is a powerful tool to interfere with gene function in crops and the mutagenized population developed in this study can be used as an efficient reverse genetics tool for groundnut improvement and functional genomics.
Asunto(s)
Antígenos de Plantas/genética , Arachis/genética , Arachis/metabolismo , Ácido Graso Desaturasas/genética , Genoma de Planta/genética , Proteínas de la Membrana/genética , Proteínas de Plantas/genética , Mutación Puntual , Genética Inversa/métodos , Alérgenos/metabolismo , Arachis/inmunología , Ácido Linoleico/metabolismo , Ácido Oléico/metabolismo , Hipersensibilidad al Cacahuete/prevención & controlRESUMEN
Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.