| Artemisia pollen is one of the most important allergenic sources in Northern China,which has wide cross-reactivity among various species,and Artemisia Pollen-food syndrome is the most common cause of severe anaphylaxis in Chinese allergic patients.In recent years,with the development of molecular biology technology and a new era of precision medicine,allergen component-resolved diagnosis(CRD)has become an important approach in the diagnosis and treatment of allergic diseases.To date,seven allergen components of mugwort(Artemisia vulgaris)were discovered,Art v 1-Art v 6,and Art v 60 kDa has no official name due to the lack of molecular information,which is an important reference for our study on Chinese Artemisia pollen allergens.The lack of research on the molecular biology and component-resolved diagnosis of Artemisia pollen allergy in China hindered the development of the precise diagnosis and treatment.In this study,a 62 kDa protein of Artemisia annua pollen is identified as putative galactose oxidase and designated Art an 7,using combined transcriptome,proteome and mass spectrometry approaches.Its physicochemical properties and immunological activity were studied.Seven allergen gene sequences of six Artemisia species were analyzed based on expression profile and transcriptome sequencing,showed Art v 3 gene was most variable among species.Concentrations of Art v 1,Art v 2 and Art v 3 homologues in A.annua,-argyi,-capillaris,-gmelinii,-lavandulifolia,-sieversiana and-vulgaris pollen extracts are determined using sandwich ELISA,allergenic activity among different pollen extracts are also compared,implying the mixture of A.annua,A.argyi,and A.sieversiana extracts would be representative and will improve the accuracy of allergy diagnosis and treatment.Recognition of sIgE antibody from Chinese Artemisia-allergic patients to four Artemisia allergen components(Art v 1,Art ar 2,Art v 3,Art an 7)are determined,to analyze correlation between sensitization pattern to geographical and symptoms differences.The main findings are as follows:1,Three major allergen bands were observed in immunoblots from Chinese Artemisia-allergic patients against A.annua,-argyi,-capillaris,-gmelinii,-lavandulifolia,-sieversiana and-vulgaris pollen extracts,located at 12,25-35 and 60-70 kDa,respectively,Art v 1 and Art v 3 were major allergens in Chinese Artemisia-allergic patients.Binding of specific IgE(sIgE)antibody of 79 patients confirmed sensitized to Europe mugwort and six Chinese Artemisia pollen extracts were tested by ELISA assay,which showed highest sensitization to A.annua but lowest to A.sieversiana.All pollen extracts but A.sieversiana could completely inhibited sIgE antibody bind to major allergen Art v 1 and Art v 3.However,A.sieversiana showed a better inhibition in sIgE antibody of Art v 1 and Art v 3-negative patients' sera.2,5.8 Gb sequence data and 69,007 Unigene were obtained from transcriptome sequencing of A.annua pollen,317,291 peptide spectrum and 2035 protein were identitied from proteomic sequencing.A 60-70 kDa allergen protein was successfully purified from A.annua pollen,identified as putative galactose oxidase,with a high sensitization of 94%in Chinese Artemisia-allergic patients,designated Art an 7.0101 by International Nomenclature Committee for Allergens(IUIS).The full-length ORF of acidic glycoprotein Art an 7 gene was 1785 bp,with a molecular weight of 61954 Da,the highest similarity(74%)was found between Art an 7 and a putative galactose oxidase gene of sunflower from Compositae,and a N-linked glycan binding site(NTL)at 412-414aa was observed.Recombinant allergens Art an 1.01 and Art an 3.02 were expressed in E.coli,both showed high allergic reactivity to sIgE antibody from Artemisia-allergic patients,could be further developed and applied in allergy diagnosis.3,7.3-7.7 Gb data were obtained from transcriptome sequencing of A.argyi,-capillaris,-gmelinii,-anethifolia and sieversiana,higher proportion of similarity sequences were found in A.argyi,-anethifolia and-capillaris.Expression prfiles showed most relevant expression levels of gene was A.vulgaris vs A.argyi(>0.97).The of transcriptome,sequences of seven Artemisia allergens were aligned(Art v 1-Art v 7 and homologous),in which the Art v 3 homologues were the most variable(amino acid sequence similarity 85%-100%),sequences of Art v 1,Art v 2,Art v 3 and Art an 7 have been submitted to the NCBI database,cluster analysis based on seven allergen sequences could divided Artemisia species into two groups:one is A.annua,-capillaris and-sieversiana;the other is A.vulgaris,-argyi,-lavandulifolia and-gmelinii.The gene expression profiles of A.annua,-argyi,-capillaris,-gmelinii,-sieversiana and-vulgaris showed that Art v 1.01,Art v 4.02,Art v 4.01 and Art v 5.01 were the most highly expressed in A.annua,-argyi,-gmelinii and-sieversiana,respectively,Art 2.01,Art v 6.01 and Art v 7.01 were most highly expressed in A.vulgaris,the accuracy of Art v 3 expression determination decreased due to more variable gene sequences,Art v 3 had the highest expression in A.annua.4,Monoclonal antibodies and polyclonal antibodies were produced by immunizing mice and rabbit with A.annua and A.argyi pollen extracts,the natural Artemisia allergens were successfully purified using monoclonal antibodies against Art v 1 and Art v 2,which can be applied for the diagnosis.Three mAb-pAb sandwich ELISA methods were established to quantify allergens Art v 1,Art v 2 and Art v 3,and applied to test seven Artemisia pollen extracts.Concentration of Art v 1 and Art v 2 homologous in pollen extracts were little difference among species,the highest were A.annua(7.1%)and A.lavandulifolia(3.3%),in contrast,concentration of Art v 3 homologues were significant different,with the highest content of A.argyi(13.9%)and the lowest was A.sieversiana(0.07%).The total contents of the three allergens were as follows:A.argyi(22.3%),A.vulgaris(12.6%),A.lavandulifolia(12.0%),A.annua(11.2%),A.capillaris(8.5%),A.gmelinii(8.1%)and A.sieversiana(7.3%).5,sIgE antibody levels of mugwort,Art v 1 and Art v 3 in 331 Chinese Artemisia-allergic patients,slgE antibody levels of Art ar 2 and Art an 7 in 209 patients were determined using ImmunoCAP and analyzed,The prevalence of Art v 1,Art ar 2,Art v 3 and Art an 7 were 77%,48%,60%and 85%,respectively,and their slgE antibody levels were 6.4 kUA/L,0.3 kUA/L,6.6 kUA/L and 2.8 kUA/L,respectively,significant differences were found in the sIgE antibody levels against mugwort,Art ar 2 and Art an 7 among children,teenagers and adults,patients from Northern China had a higher proportion of asthma than that from Southern China(42%vs 6%),multi-component sensitized allergic patients had a higher risk of asthma. |
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