Recombinant Derp5 allergen with αS1-casein signal peptide secreted in murine milk protects against dust mite allergen–induced airway inflammation

Recent advances in recombinant technology make transgenic animals that produce pharmaceutical proteins in their milk more feasible. The group 5 allergen isolated from Dermatophagoides pteronyssinus (Derp5) is one of the most important dust mite allergens in humans. The aims of this study were to develop transgenic mice that could secrete recombinant Derp5-containing milk and to demonstrate that ingesting recombinant milk protects against allergic airway inflammation. Two transgenes were constructed separately. The α-LA-Derp5f transgene consisted of the bovine α-lactalbumin (α-LA) promoter and full-length Derp5 cDNA. The α-LA-CN-Derp5t transgene included the α-LA promoter, a leader sequence of α_S1-casein (CN), and signal peptide-truncated Derp5 cDNA. Both species of transgenic mice were confirmed to have successful transgene integration and stable germline transmission. Western blot analysis of the milk obtained from the offspring of transgenic mice demonstrated that recombinant Derp5 was secreted successfully in the milk of αLA-CN-Derp5t transgenic mice but not in that of αLA-Derp5f transgenic mice. This study provides new evidence that transgenic mice can secrete recombinant Derp5 efficiently in milk by adding a signal peptide of α_S1-casein. The antigenic activity of recombinant Derp5 milk was demonstrated to have a protective effect against allergic airway inflammation in a murine model in which the ingestion of recombinant Derp5-containing milk was used as pretreatment.     

The panel of egg allergens, Gal d 1-Gal d 5: Their improved purification and characterization

Egg proteins represent one of the most important sources evoking food allergic reactions. In order to improve allergy diagnosis, purified and well-characterized proteins are needed. Although the egg white allergens Gal d 1, 2, 3 and 4 (ovomucoid, ovalbumin, ovotransferrin, and lysozyme) are commercially available, these preparations contain impurities, which affect exact in vitro diagnosis. The aim of the present study was to set up further purification protocols and to extend the characterization of the physicochemical and immunological properties of the final batches. The egg white allergens Gal d 1-4 were purified from commercial preparations, whereas Gal d 5 (alpha-livetin) was purified from egg yolk. The final batches of Gal d 1-5 consisted of a range of isoforms with defined tertiary structure. In addition, the IgE binding capacity of the purified egg allergens was tested using allergic patients' sera. The allergen batches will be further used to set up allergen specific diagnostic assays and to screen a larger collection of patients' sera.     

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.     

Clinical effects of Lactobacillus acidophilus strain L-92 on perennial allergic rhinitis: a double-blind, placebo-controlled study

Studies in animals have suggested that lactic acid bacteria alleviate allergic diseases, however, little information is available on their clinical effect on allergy in humans. Thus, we examined the efficacy of orally administered Lactobacillus acidophilus strain L-92 (L-92) on perennial allergic rhinitis. In a randomized, double-blind, placebo-controlled clinical trial, 49 patients with perennial allergic rhinitis were randomized to receive either 100 mL of heat-treated fermented milk containing L-92 (n = 25) or acidified milk without lactic acid bacteria (placebo; n = 24) for 8 wk. The severity of symptoms was evaluated based on the changes in the scores of clinical symptoms. Oral administration of milk fermented with L-92 resulted in a statistically significant improvement of nasal symptom-medication scores. Ocular symptom-medication scores of patients in the L-92 intervention group tended to improve compared with those in the placebo group. In addition, clear decreases of the scores of swelling and color of the nasal mucosa were observed in the L-92 intervention group at 6 and 8 wk after the start of ingestion of fermented milk. There were no significant differences in serum antihouse dust mite immunoglobulin E levels nor in T helper type 1/T helper type 2 ratio between the 2 groups. These results suggest that oral administration of L-92 can alleviate the symptoms of perennial allergic rhinitis, however, statistically significant changes were not shown in blood parameters.     

过敏原分离纯化技术的研究进展

过敏原是引起过敏反应的抗原性物质,对它的检测是预防发生过敏反应的重要方法,而过敏原分离纯化是过敏原检测技术的基础。主要叙述了过敏原的致敏机理,同时详细叙述了沉淀法、疏水性相互作用色谱、离子交换色谱、亲和色谱、凝胶过滤色谱、膜分离、蛋白质组学和人工合成过敏原技术在过敏原分离纯化中的研究进展,重点介绍蛋白质组学和人工合成过敏原技术这两种分离纯化的新方法。     

Replicating the cross-sectional distribution of house-dust mite allergen found in carpet

With a view to producing carpets that could be used to determine the ease of particulate aerosolisation during domestic activity, we measured the cross-sectional distribution of dust-mite allergen, Der p 1, produced using American Society for Testing and Materials method (ASTM F608–89) for embedding house dust in carpets with that produced by several alternative protocols. Allergen concentrations produced at different levels within the pile using the different techniques were also compared with those in carpets from actual houses – in which the majority of allergen is typically found towards the base of the pile. To obtain profiles of allergen, horizontal sections, 2-mm thick, were taken from new carpets after they had been seeded with dust and embedded using one of four following techniques: (1) dragging a fixed roller across the surface of the carpet four times, (2) using the same roller but following it up with 200 revolutions in a hexapod wear simulator, (3) dragging the fixed roller across the carpet surface 30 times (the ASTM method), and (4) 2 minutes under a commercial plate compactor. Fibre from each 2-mm-thick section was collected and the Der p 1 content determined using enzyme linked immunosorbent assay and results expressed as ng Der p 1 per area in each section. Embedding with a fixed roller alone was not found to be particularly effective, resulting in roughly equal amounts of dust being apportioned within each pile layer, irrespective of the number of embedding passes used. In contrast, a distribution biased towards the base of the pile was found after roller-embedding/hexapod wear, although still to a lesser extent than has been observed in used carpets. Plate compaction gave a similar allergen distribution profile to combined roller/hexapod treatment but was considerably easier to perform. Thus, both techniques offer promise for researchers seeking to replicate the cross-sectional distribution of dust mite allergen found in carpets after actual use (and conceivably other particulate pollutants also).     

热加工花生的蛋白分离及其过敏原纯化方法研究进展

花生不仅本身是一种营养丰富的食品,而且作为原料或配料广泛应用于食品加工中。然而花生及其制品是FAO/WHO认定的八大类食物过敏原之一,可导致严重的过敏反应,通常伴随终身,甚至危及生命。不同地区的人们食用花生的加工方式不同,其花生过敏的患病率也有所不同,热加工是花生的主要加工方式,因此各类热加工导致的花生致敏性变化成为研究热点。过敏原蛋白的分离作为花生热加工研究中的重要步骤,也变得十分重要。本文主要对常见的3种热加工花生(水煮、油炸和烘烤)中的花生蛋白分离及其过敏原纯化的方法研究进行综述。现有的花生热加工研究中蛋白分离技术主要是通过溶剂浸提;而过敏原纯化技术主要是借助层析法,根据各组分在物理化学性质上的差异进行纯化;此外还可以根据最终研究目的的不同采用其他的辅助方法达到分离纯化效果。通过对现有分离纯化方法进行了解和比较,可为热加工花生过敏原蛋白的分离纯化甚至进一步的分析检测提供理论参考和指导。     

Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis

Peanut is a major cause of type 1 hypersensitive reactions including anaphylaxis. This results from the presence of a number of protein allergens, six of which are being studied as part of the EU FP6 EuroPrevall programme. These are Ara h 1 (7S globulin), Ara h 2, Ara h 6 (2S albumins), Ara h 3/4 (11S globulins) and Ara h 8 (Bet v 1 homologue). Methods for the purification of Ara h 1, Ara h 3/4, Ara h 2 and Ara h 6 from peanut seeds and for the production of recombinant Ara h 8 in Escherichia coli are described with spectroscopic analyses being used to confirm that they are authentically folded. N-terminal sequencing of the proteins purified from peanut seeds also revealed details of the differences between isoforms and their generation by proteolytic processing within the seed. Preliminary IgE binding studies of the purified allergens confirmed that they retained their immunological properties indicating their suitability for use in allergy diagnosis.     

Production and characterization of an allergen panel for component-resolved diagnosis of celery allergy

In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future.     

色谱法在鸡蛋清主要过敏原分离纯化中研究进展

鸡蛋营养丰富,同时也是引起人体过敏的主要食物之一。蛋清中含有4种主要过敏原,包括卵白蛋白、卵转铁蛋白、卵类黏蛋白和溶菌酶。概述了鸡蛋中4种主要过敏原的理化特性及色谱法纯化过敏原的研究进展,旨在为规模化分离得到高纯度和高活性的过敏原提供参考。     

The analysis of crude and purified locust bean gum: A comparison of samples from different carob tree populations in Tunisia

The crude and purified locust bean gum (LBG) from seven areas of the north and centre of Tunisia (Bouarada, Bargou, Kessra, Haffouz, Borj Toumi, Ben Arous and INRGREF) were analyzed for moisture, ash, protein, acid-insoluble matter and mannose/galactose ratio. The purified samples exhibited higher mannose/galactose ratios and lower amounts of ash, protein and acid-insoluble matter than the crude gum. The purified LBG from different regions had 3.43–6.99% moisture, 0.87–2.06% ash, 0.61–2.46% protein, 0.00–1.20% acid-insoluble matter and 3.55–4.32 mannose/galactose ratios. Statistical analysis revealed that purification significantly affected (P < 0.05) moisture, ash, protein, insoluble matter contents and mannose/galactose ratios of the crude LBG and purified LBG for all samples from different areas. The rheological properties of the different carob gum samples were determined, the best rheological properties are those of spontaneous carob trees of Bargou, Bouarada and Kessra areas. The climatic and geographic origin of carob and the cultivation mode influence the chemical and rheological properties. The purification of crude galactomannan samples by precipitation with isopropanol gave a clear and more stable solution, due to the elimination of impurities and endogenous enzymes.     

Peanut Allergy,Peanut Allergens,and Methods for the Detection of Peanut Contamination in Food Products

ABSTRACT: Attention to peanut allergy has been rising rapidly for the last 5 y, because it accounts for the majority of severe food‐related anaphylaxis, it tends to appear early in life, and it usually is not resolved. Low milligram amounts of peanut allergens can induce severe allergic reactions in highly sensitized individuals, and no cure is available for peanut allergy. This review presents updated information on peanut allergy, peanut allergens (Ara h1 to h8), and available methods for detecting peanuts in foods. These methods are based on the detection of either peanut proteins or a specific DNA fragment of peanut allergens. A summary of published methods for detecting peanut in foods is given with a comparison of assay formats, target analyte, and assay sensitivity. Moreover, a summary of the current availability of commercial peanut allergen kits is presented with information about assay format, target analyte, sensitivity, testing time, company/kit name, and AOAC validation.     

Purification and characterisation of an alkaliphilic esterase from a culinary medicinal mushroom, Sparassis crispa

In this study, we found that the fruiting body of the medicinal and edible mushroom Sparassis crispa produces an alkaliphilic esterase. The substrate specificity of this esterase was high for a p-nitrophenyl acetate substrate. The S. crispa esterase was purified using ammonium sulphate precipitation, anion exchange and gel filtration chromatography. The recovery and purification yields of the enzyme were 15–17% and 70–73 folds from six different strains of S. crispa, respectively. The molecular weight of the purified enzyme was approximately 60 kDa, as determined by SDS–PAGE. A zymogram analysis using a tributyrin substrate revealed that this enzyme is an esterase. The optimum pH and temperature were 8.0 and 50 °C, respectively. The pH and temperature stability profiles show that this enzyme is more stable under alkaline conditions and at 30–40 °C. K_m and V_max for this esterase enzyme acting on p-nitrophenyl acetate were 0.2 mM and 0.5 U/mg proteins, respectively.     

Recombinant tropomyosin from Penaeus aztecus (rPen a 1) for measurement of specific immunoglobulin E antibodies relevant in food allergy to crustaceans and other invertebrates

Immunoglobulin E (IgE)-mediated food allergy to crustaceans and mollusks is relatively common and affected individuals typically react to a range of different species. The only known major allergen of shrimp was first described over 20 years ago and later identified as the muscle protein tropomyosin. This protein may be useful as a defined and relevant diagnostic marker for allergic sensitization to invertebrate foods. In order to generate an assay reagent suitable for this purpose, tropomyosin from the shrimp Penaeus aztecus (Pen a 1) was produced as a recombinant protein in Escherichia coli and characterized with respect to IgE antibody binding properties in comparison to natural shrimp tropomyosin. Hexahistidine-tagged rPen a 1 accumulated as a predominantly soluble protein in the E. coli expression host and a two-step chromatographic procedure provided a high yield of pure and homogeneous protein. rPen a 1 displayed chromatographic and folding characteristics similar to those of purified natural shrimp tropomyosin. Serum preincubation with serial protein dilutions revealed similar capacity of recombinant and natural tropomyosin to compete with immobilized shrimp extract for IgE binding. rPen a 1 was further shown to extensively and specifically compete for IgE binding to extracts of other crustacean species, house dust mite and German cockroach.     

Isolation,cloning, and characterization of the 2S albumin: A new allergen from hazelnut

Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP‐HPLC. After N‐terminal sequencing, degenerated and poly‐d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut‐allergic patients. Results: N‐terminal sequencing of a ～10 kDa peak from size exclusion chromatography/RP‐HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N‐terminal and a 10aa internal peptide. The obtained clone (441 bp) encoded a 147aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.     

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.     

Analysis of free fatty acids in food substrates and in the dust and frass of stored-product pests: Potential for species discrimination?

The larger grain borer, Prostephanus truncatus, is a serious beetle pest that tunnels extensively to produce large quantities of dust and frass. The natural enemy Teretrius nigrescens is an important biological control beetle which is known to exploit at close-range solvent-extractable chemical cues in the dust and frass. The objective of the current study was to analyse quantitatively and qualitatively, the free fatty acid mixtures in different food-substrate materials both before and after insect attack by a range of stored-product pests in order to ascertain whether differences in these mixtures could explain the T. nigrescens selectivity to P. truncatus dust/frass over that of other species irrespective of food substrate. By TLC, GC and GC-MS we found triglyceride and five free fatty acids were the most abundant chemicals in dust/frass (palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3)). In maize flour, Sitophilus species did not significantly change free fatty acid concentrations whereas with P. truncatus, Rhyzopertha dominica and Dinoderus minutus there were 4-6-fold increases, and, for Tribolium species there were over 20-fold increases. These differences provide interesting insights to tunnelling/feeding habits and are correlated with known feeding preferences within grain. Principal component analysis (PCA) demonstrated that free fatty acid ratios in dust/frass of different species are most linked to the food substrate and confer little discriminatory information that could be used to distinguish between the different species. Although increases in free fatty acid concentrations are good indicators of pest infestation and this may contribute behaviourally in an additive or synergistic way, we conclude that other chemical(s) are present and are key to T. nigrescens recognition of P. truncatus on different substrates.     

Purification and characterisation of relevant natural and recombinant apple allergens

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purification.     

Efficacy of powder and essential oil from Chenopodium ambrosioides leaves as post-harvest grain protectants against six-stored product beetles

Powder and essential oil obtained from dry ground leaves of Chenopodium ambrosioides were tested under laboratory conditions (25±1°C, 70-75% r.h.) for their ability to protect grains from damage by six insect pests, Callosobruchus chinensis, C. maculatus, Acanthoscelides obtectus, Sitophilus granarius, S. zeamais and Prostephanus truncatus. The insects were reared and tested on whole maize grain for S. zeamais and P. truncatus, whole wheat for S. granarius, green peas for C. chinensis, mung bean for C. maculatus and white bean for A. obtectus. The powder prepared from dry leaves of C. ambrosioides was mixed with grains at different dosages ranging from 0.05-0.80% (wt/wt) for C. chinensis, C. maculatus and A. obtectus and from 0.8-6.4% (wt/wt) for S. granarius, S. zeamais and P. truncatus. The dosage of 0.4% killed more than 60% of all the bruchids 2 days after treatment, while a dosage of 6.4% induced total mortality of S. granarius and S. zeamais within the same exposure time. All levels of the dry ground leaf concentrations inhibited F1 progeny production and adult emergence of the tested insects. The dosage of 0.2 μl/cm² of the essential oil killed 80-100% of the beetles within 24 h except C. maculatus and S. zeamais, where this dosage induced only 20% and 5% mortality, respectively. These results indicate a scientific rationale for the use of this plant in grain protection by local communities in the western highlands of Cameroon.