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Goat milk (GM), as compared to cow milk (CM), is easier for humans to digest. It also has antioxidant and anti-inflammatory effects and can improve minor digestive disorders and prevent allergic diseases in infants. It is unclear whether GM consumed in pregnant mothers has any protective effects on allergic diseases in infants. In this experimental study with mice, we found GM feeding enhanced immunoglobulin production, antigen-specific (ovalbumin, OVA) immune responses, and phagocytosis activity. The GM-fed mice had an increasing proportion of CD3 + T lymphocytes in the spleen. Splenocytes isolated from these animals also showed significantly increased production of cytokines IFN-γ and IL-10. More importantly, GM feeding during pregnancy and lactation periods can confer protective activity onto offspring by alleviating the airway inflammation of allergic asthma induced by mite allergens. There was a remarkably different composition of gut microbiota between offspring of pregnant mice fed with water or with milk (GM or CM). There was a greater proportion of beneficial bacterial species, such as Akkermansia muciniphila, Bacteroides eggerthii , and Parabacteroides goldsteinii in the gut microbiota of offspring from GM- or CM-fed pregnant mice compared to the offspring of water-fed pregnant mice. These results suggested that improving the nutrition of pregnant mice can promote immunological maturation and colonization of gut microbiota in offspring. This mother-to-child biological action may provide a protective effect on atopy development and alleviate allergen-induced airway inflammation in offspring., (Copyright © 2020 Kao, Wang, Tseng, Wu, Tsai, Hsieh, Chen, Kuo, Liu, Liu and Wang.)

Interleukin (IL)-10-expressing B cells play a critical role in the immune homeostasis in the body; its regulation has not been fully understood. Micro-RNA (miR)-17-92 cluster has strong regulation in the immunity. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells. In this study, peripheral B cells were collected from patients with allergic rhinitis (AR). The B cells were treated with specific allergens, dust mite extracts, in the culture. The expressions of miR-17-92 cluster and IL-10 in the culture were assessed by real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that the levels of miR-19a, but not the rest of the 5 members (miR-17, miR-18a, miR-19b, miR-20a, and miR-92a), were significantly higher in peripheral B cells from AR patients as than in B cells from healthy participants. Exposure of B cells from AR patients to specific allergen, dust mite extracts, significantly increased the levels if miR-19a and suppressed the expression of IL-10 in B cells. The levels of histone deacetylase 11 and acetylated H3K9 were higher, and the RNA polymerase II and c-Maf (the IL-10 transcription factor) were lower, at the IL-10 promoter locus. In conclusion, miR-19a mediates the allergen-specific immune response-decreased IL-10 expression in B cells., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies., Objective: We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens., Methods: A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests., Results: The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome., Conclusion: An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: To clone, express, and identify a fragment of Cor h 1 from Corylus heterophylla., Methods: Through bioinformatics predication, the antigenic epitope of Cor h 1 was selected. A fragment gene of Cor h 1 was amplified by PCR and cloned into pMD18-T vector for sequencing. Then the fragment gene was sub cloned into pET-32a expression vector for expression, and then purified by metal (Ni(2+);) chelating affinity chromatography. The immunogenicity was tested by Western blot., Results: The length of the fragment gene was 243 bp, coding 81 amino acids; the relative molecular mass of recombinant protein was 9 000. And the fragment of Cor h 1 was mainly expressed as soluble protein, purified protein has good immunogenicity., Conclusion: The fragment gene of Cor h 1 was successfully cloned and expressed in this study, and the recombinant protein possessed good IgE-binding capacity.

Recombinant proteins expressed by prokaryotic expression system are normally in the form of inclusion. In the present paper, refolding process of recombinant pan-allergen profilin protein induced by urea has been investigated by using circular dichroism spectra, fluorescence spectra, synchronous fluorescence spectra systematically. And the spectral characteristics of the renaturation were obtained. In addition, bioinformatics methods including predications of secondary and tertiary structures have also been used to explain the spectral characteristics and analyze the conformational changes of the protein during renaturation in vitro. Results from this study should be useful to the establishment of a spectral method examining the extent of protein renaturation, and be helpful to the understanding of the mechanism of renaturation of recombinant protein.

Objective: Allergic disease caused by airborne pollen is a major health problem in China. Intensive study on pollen allergens can be of great help for preventing and treating pollinosis. Four aspects of the study on pollen allergens in China including major allergic pollen in our country, analysis and purification of pollen allergen composition, recombinant pollen allergens and clinical application of pollen allergens are described in this paper., (Copyright © 2010 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.)

In the present paper, the authors aimed to detect the binding of monoclonal antibody and polyclonal antibodies in sera of patients with recombinant house dust mite allergen (rDer p2) by surface plasmon resonance (SPR) technique. This technique is superior to other methods, like enzyme-linked immunosorbent assays (ELISAs) in studying the interaction between biomolecules because no labeling and sample separation are needed. The allergen rDer p2 was immobilized on carboxymethyldextran-modified sensor chip surface by amine coupling. Surface plasmon resonance measurements of monoclonal antibody and polyclonal antibodies in patients' sera revealed that their bindings diverge widely; the binding of patients' sera was remarkably lower than that of monoclonal antibody. At the same time, the binding of patients' sera with rDer p2 varied among patients allergic to dust mite. This study could provide an easy, fast and real-time way for clinical allergic diseases diagnosis.

Objective: To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity., Method: The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindIII. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA)., Result: The cloned cDNA ORF sequence (Accession no. EU429466) contained 1068 bp and encoded 365 amino acids. Its sequence homology with the published one (Accession no. AY563004) was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 (DE3) as a soluble protein mainly with the molecular weight of about Mr 45000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA., Conclusion: The recombinant cockroach arginine kinase has been obtained with proper allergenicity.

Objective: To identify and purify the allergens of Tyrophagus putrescentiae., Methods: Tyrophagus putrescentiae extract was analyzed by SDS-PAGE and Western blotting, and was partially purified by DE52 anion-exchange chromatography and HiLoad 16, 60 Superdex 200 prep grade Size-exclusion chromatography., Results: 23 bands were found after SDS-PAGE with comparative molecular weight (Mr) of 177,000, 118,000, 107,000, 70,000, 67,000, 60,000, 52,000, 45,000, 41,000, 40,000, 38,000, 37,000, 35,000, 27,000, 23,000, 22,000, 18,000, 17,000, 16,000, 15,000, 14,000, 13,000 and 12,000. Five allergens were detected by Western blotting with Mr 128,000, 67,000-70,000, 36,000-37,000, 18,000 and 16,000, respectively. The positive reaction rate of 3 allergens, with Mr 128,000, 67,000-70,000 and 36,000-37,000, were 100%, while that of other 2 allergens with Mr 18,000 and 16,000 was 77.8% and 44.4% respectively. The allergen with Mr 18,000 was purified by anion-exchange chromatography and Size-exclusion chromatography., Conclusion: Four major allergens and one minor allergen from Tyrophagus putrescentiae have been identified.

Objective: To detect mite allergens in the dust of air conditioner filter-net and floating air in room., Methods: Samples were collected from rooms of asthma patient and normal families with or without air conditioner. Der p1, Der f1 and Der 2 were determined by two monoclonal antibody-based ELISA., Results: In asthma patient families, the concentration of airborne Der p1, Der f1 and Der 2 was (0.23 +/- 0.13), (2.62 +/- 1.08), (0.93 +/- 0.41) ng/m3, and (0.56 +/- 0.25), (4.74 +/- 1.22), (2.33 +/- 0.64) ng/m3 respectively before and after the air conditioner switched on, all showing a significant difference (P < 0.05). In families without asthma patient, the concentration of Der p1, Der f1 and Der 2 was (0.33 +/- 0.11), (11.50 +/- 3.08) and (2.10 +/- 0.80) ng/m3, and (0.63 +/- 0.23), (19.80 +/- 4.30) and (3.60 +/- 1.00) ng/m3 respectively before and after the air conditioner switched on, also showing a significant difference (P < 0.05). From the filter-net dust of air conditioner in family with allergic asthma patient, the concentration of Der p1, Der f1 and Der 2 was (0.52 +/- 0.19), (3.34 +/- 0.63), (2.53 +/- 0.65) microg/g dust, while that of normal families was (1.30 +/- 0.35), (5.16 +/- 0.92), (3.47 +/- 1.13) microg/g dust respectively. The concentration of Der f1 and Der 2 at both asthma families and normal families was higher than 2 microg/g, an allergen concentration threshold., Conclusion: Mite allergens exist in the filter-net dust of air conditioner, which may be an important source of indoor allergens and a cause of the increasing prevalence of allergic asthma.

Objective: To express the major allergen of Blattella germanica (Bla g 2) in Pichia pastoris and obtain the soluble protein., Methods: The known Bla g 2 gene was used to design the primers which had the restriction enzyme sites. PCR method was applied to obtain the Bla g 2 gene. The gene fragment was then cut and ligated with the Pichia expression vector pGAPZaA, resulting in a recombinant plasmid pGAPZaA-Bla g 2. The linearized pGAPZaA-Bla g 2 was transformed into Pichia pastoris GS115 through electroporation, then screened to positive transformants, and the protein was expressed in YPD medium. Purification of the recombinant protein was achieved by metal (Ni2+) chelating affinity chromatography and Western-blotting assay indicated its IgE binding capacity., Results: With the expressed recombinant protein, SDS-PAGE showed the presence of the product in the supernatant of the culture with Mr 45 000. After 3 days culture, the recombinant protein occupied 50% of the total proteins in the supernatant. The recombinant protein was purified and Western-blot demonstrated an adequate IgE binding capacity of the product., Conclusion: A recombinant protein of Bla g 2 has been obtained, which is soluble in the supernatant and therefore can avoid a process of denaturalization and renaturation of the recombinant.

Objective: To localize Der f 2 in the body of Dermatophagoides farinae., Methods: Live mites were embedded. Serial mite paraffin sections were made and checked under light microscope. The sections were incubated with anti-recombinant Der f 2 monoclonal antibody as the first antibody. They were then reacted with the fluorescent isothiocyanate conjugated goat anti-mouse IgG as the secondary antibody. The sections were examined with fluorescent light microscopy., Results: Der f 2 reacting with immunofluorescent antibodies was found localized in the tissue and contents of the mid-gut of the mite., Conclusion: The major allergen Der f 2 distributes in the gut and fecal pellets of Dermatophagoides farinae.

An efficient preparation of Periplaneta americana nymphae allergen, Cr PI (54 kDa) is described. It was expressed as a GST-tag fusion protein in Escherichia coli, strain BL21 (DE3). Expression of recombinant Cr PI (rCr PI), denaturation/renaturation of the inclusion bodies and the effects of protein and L-arginine concentration on inclusion body aggregation were optimized. The fusion protein was purified by affinity chromatography and size exclusion chromatography, and Cr PI fusion protein was purified to >95%. rCr PI bound strongly to IgE in the sera of individuals with cockroach allergies as shown by western blot and ELISA. Highly refolded and purified recombinant protein was obtained, providing a basis for the large-scale preparation of Cr PI allergen.

Objective: To construct, purify and characterize a recombinant expression plasmid containing Der f6 gene of Dermatophagoides farinae., Methods: A pair of primers was designed according to the known sequence of Der f6 gene. The live mites identified and cultured locally were picked and the total RNA was extracted. The Der f6 gene fragment was amplified by RT-PCR, and cloned into pMD18-T vector, and then transferred into E. coli Top10. The target gene obtained from the recombinant plasmid by digestion with EcoR I and Xho I was connected to the prokaryotic expression vector pET-24a. The expressed recombinant plasmid containing Der f6 gene was constructed by cloning target gene into pET-24a and first transferred into E. coli Top10, then into E. coli B121 (DE3). The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting, and was purified by immobilized metal ion affinity chromatography (IMAC)., Results: The two recombinant plasmids, pMD18-T-Der f6 and pET24a-Der f6, were constructed. SDS-PAGE showed a correct molecular weight of the recombinant Der f6 protein. After purification by affinity chromatography, the protein showed only one strip on SDS-PAGE gel and appropriate combination ability with IgE in sera of allergic patients., Conclusion: The Der f6 gene has been cloned into plasmid pMD18-T vector and sub-cloned into the expression vector pET-24a, the recombinant plasmid pET24a-Der f6 has been expressed in E. coli BL21 (DE3), purified by IMAC, and showed appropriate IgE-combined ability.

The recombinant allergen, Bla g 2, was expressed by prokaryotic vector E. coli and eukaryotic vector P. Pastoris. The different structures and configurations of the Bla g 2 from E. coli and P. Pastoris were studied by fluorescence and circular dichroism. The secondary structures of Bla g 2 in solution, and the composition besides the type of its tertiary structure were proposed. These studies help understand the differences between prokaryotic and eukaryotic expression systems, reveal the relationship between the structure and the function of Bla g 2, and improve the production of this significant allergen.

Objective: To study the localization of specific allergen of Dermatophagoides pteronyssinus., Methods: Through optical microscope, the specific allergens of D. pteronyssinus were observed in paraffin sections using D. pteronyssinus-specific IgE antibodies from the patient sera., Results and Conclusion: The digestive system was found occupying large parts of body cavity of D. pteronyssinus by HE staining, while the specific allergens of D. pteronyssinus were mostly occurred in the midgut tissue, gut contents, cuticle and reproductive system in the immunostained sections. The results also showed that many parts of D. pteronyssinus were recognized by the specific IgE antibodies obtained from allergic individuals to D. pteronyssinus, which provided a theoretic base for further study of isolation and purification of the specific allergen.

Lychee fruits can cause severe anaphylactic reactions and has cross-reactivity with other allergens. Panallergen profilin is partly responsible for the cross-reactivity between allergens. In the present study, the cDNA of lychee profilin was performed by RT-PCR and 3'RACE from total RNA. The lychee profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant lychee profilin was carried out in Escherichia coli BL21 (DE3) using vector PET-28a, and the purification of the recombinant protein was performed via affinity chromatography with Ni+ coupled to sepharose. IgE reactivity of recombinant lychee profilin was investigated by immunoblot, and five of 15 lychee-allergic patients tested presented specific IgE antibodies to recombinant lychee profilin. IgE inhibition and ELISA inhibition experiments were performed to analyze lychee profilin cross-reactivity with profilins from birch pollen, and a high cross-reactivity between lychee profilin and birch profilin has been found in sera from lychee allergic patients. [ABSTRACT FROM AUTHOR]

Abstract: Food allergen labeling has not yet been implemented in China. Therefore, a gold immunochromatography assay (GICA) was developed using two monoclonal antibodies (mAb) against the peanut allergen Ara h1. The GICA was specific for standard peanut samples with a sensitivity of 10ng/ml. Peanut protein traces extracted from 124 food products imported and exported by China Customs were easily and rapidly detected by GICA. 68 food samples originally labeled as containing peanuts were positive for Ara h1 and 54 food samples labeled as not containing peanuts were negative for Ara h1, indicating that the labels from the manufacturers were accurate. However, 2 food samples labeled as not containing peanuts tested positive for Ara h1. The present GICA provides a fast, simple, semi-quantitative method for the determination of peanut allergens in foods. This detection system can be used to ensure the safety of food imported and exported by China Customs. [Copyright &y& Elsevier]