重组人乳铁蛋白致敏性的初步研究

目的 初步评价牛乳腺生物反应器表达的重组人乳铁蛋白(rhLf)的致敏性.方法 通过生物信息学分析和消化稳定性试验了解rhLf致敏的可能性.结果 生物信息学分析显示rhLf与已知致敏原乳转铁蛋白和卵转铁蛋白有序列同源性,与Asp f2、Ole e 10和Ole e9存在结构相似性.消化稳定性试验显示rhLf在胃液中易被消化成小片段,在肠液中不易被消化.结论 rhLf具有一定的潜在致敏可能性.     

Identification of proteolytic bacteria from thai traditional fermented foods and their allergenic reducing potentials

ABSTRACT:  This study aimed to identify proteolytic bacteria from Thai traditional fermented foods and investigate their allergenic reducing potentials to wheat and milk allergens. Nine bacteria were isolated from fermented foods as follows: fermented soybean seeds (Thua Nao), fermented soybean paste (Thua Nao), wheat flour dough of steamed stuffed bun (Sa La Pao), and soaked rice from Thai fermented rice-noodle (Kha Nhom Jeen) processing. Both phenotypic and genotypic identifications were used in this study. It was found that all isolates were Gram-positive rods. Seven isolates were matched and identified as Bacillus subtilis by both techniques, and the remaining 2 isolates were phenotypically and genotypically identified as B. licheniformis and B. subtilis , respectively. The concentrated crude enzyme of B. subtilis DB and SR could reduce allergenicity of gliadin by hydrolyzing the allergenic gliadin fragments detected by immunoblotting. Furthermore, the enzyme of B. subtilis DB could also reduce allergenicity of β-lactoglobulin (β-LG) detected by hydrolyzing the major allergenic epitope of β-LG at Gln³⁵-Ser³⁶ position. B. subtilis DB and SR can be applied for the production of hypoallergenic wheat flour or milk food products.     

Effects of gamma radiation on the allergenic and antigenic properties of milk proteins

This study was carried out to evaluate the application of food irradiation technology as a method for reducing milk allergies. Bovine alpha-casein (ACA) and beta-lactoglobulin (BLG) were used as milk proteins. Using milk-hypersensitive patients' immunoglobulin E (IgE) and rabbit IgGs individually produced to ACA and BLG, the changes of allergenicity and antigenicity of irradiated proteins were observed by competitive indirect enzyme-linked immunosorbent assay. Allergenicity and antigenicity of the irradiated proteins were changed with different slopes of the inhibition curves. The disappearance of the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increase of the turbidity showed that solubility of the proteins decreased by radiation, and this decrease might be caused by agglomeration of the proteins. These results indicated that epitopes on milk allergens were structurally altered by gamma irradiation.     

中国典型水果过敏原蛋白研究进展

随着我国人民生活水平不断提高,水果消费量也在逐年增加,水果过敏问题因此也日益凸显。水果是十大食物过敏来源之一,水果过敏与自身遗传、环境和水果种类等多种因素相关,常和花粉、乳胶发生交叉过敏反应。水果过敏常见的症状有口腔过敏综合症和全身过敏反应。水果过敏发生的主要原因是由于水果中某些蛋白质使患者的免疫系统发生紊乱,属于I型速发型过敏反应。本文首先阐述水果过敏的发生机理、临床数据和症状、以及过敏原命名方式。其次以我国常见的引发过敏反应的特色水果为例,涵盖热带水果、蔷薇科水果和亚热带水果,总结过敏相关蛋白家族最新研究进展,并对现阶段常用的水果过敏原蛋白分子鉴定及检测技术进行综述。     

Comparative proteome analysis of seed storage and allergenic proteins among four narrow-leafed lupin cultivars

Lupin is an emerging crop worldwide due to its wide range of health benefits. In this study, a comprehensive proteome analysis was conducted using mature seed of four narrow leafed lupin cultivars, Uniharvest, Yorrel, Tanjil and Coromup, through two-dimensional gel electrophoresis followed by mass spectrometric protein sequencing. Two-dimensional gels recognised about 400 protein spots among the cultivars in the 10-100kDa molecular weight and 5.0-8.5 PI ranges. The results revealed a considerable variation of protein expression patterns with a total of 24 proteins showed differential expression among the cultivars, among which 19 were identified as β-conglutin, and 8 were identified as allergenic proteins. Most of the α, δ and γ-conglutins were showing similar expression among the cultivars. Overall, the differentially expressed proteins especially the cultivar specific proteins would be valuable markers for cultivar identification and for screening parental lines of low allergenicity in breeding process.     

典型热加工对花生致敏蛋白及其免疫反应性的影响

通过免疫学方法检测了花生制品加工过程中几种典型热加工方式对花生致敏蛋白的免疫反应性的影响。结果表明:烘焙、水煮、油炸和高温高压处理均使花生可溶性蛋白含量显著降低;随之,处理后样品中蛋白提取物的免疫反应性也显著下降,其中高温高压处理最为显著;热处理对花生致敏蛋白Ara h 2的溶解性及免疫反应性的影响较小,而对Ara h1和Ara h 3的影响则比较明显。      

Allergenicity of soybean: new developments in identification of allergenic proteins, cross-reactivities and hypoallergenization technologies

Soybean is considered one of the "big eight" foods that are believed to be responsible for 90% of all allergenic reactions. Soy allergy is of particular importance, because soybeans are widely used in processed foods and, therefore, represent a particularly insidious source of hidden allergens. Although significant advances have been made in the identification and characterization of soybean allergens, scientists are not completely certain about which proteins in soy cause allergic reactions. At least 16 allergens have been identified. Most of them, as with other plant food allergens, have a metabolic, storage, or protective function. These allergens belong to protein families which have conserved structural features in relation with their biological activity, which explains the wide immunochemical cross-recognition observed among members of the legume family. Detailed analysis of the structure-allergenicity relationships has been hampered by the complexity and heterogeneity of soybean proteins. A variety of technological approaches have been attempted to decrease soybean allergenicity. This paper provides a comprehensive review of the current body of knowledge on the identification and characterization of soybean allergens, as well as an update on current hypoallergenization techniques.     

典型热加工对花生致敏蛋白及其免疫反应性的影响

通过免疫学方法检测了花生制品加工过程中几种典型热加工方式对花生致敏蛋白的免疫反应性的影响。结果表明:烘焙、水煮、油炸和高温高压处理均使花生可溶性蛋白含量显著降低;随之,处理后样品中蛋白提取物的免疫反应性也显著下降,其中高温高压处理最为显著;热处理对花生致敏蛋白Ara h 2的溶解性及免疫反应性的影响较小,而对Ara h1和Ara h 3的影响则比较明显。     

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.     

Review of the development of methodology for evaluating the human allergenic potential of novel proteins

Safety assessment of novel proteins in genetic-engineered foods is a key component of the overall safety evaluation for these products. Since allergens are typically proteins, assessment of the potential allergenicity of the novel proteins in genetically engineered foods is critical. This article reviews methods available to assess the potential allergenicity of novel proteins, as well as problems and deficiencies in the existing methods. The role of bioinformatics and knowledge of allergenic epitopes in developing new approaches to this problem is discussed.     

Relative immunogenicity of commonly allergenic foods versus rarely allergenic and nonallergenic foods in mice

Food allergies affect 6 to 8% of children and 2% of adults in the United States. For reasons that are not clear, eight types of food account for a vast majority (approximately 90%) of food-induced hypersensitivity reactions. In this study, C57Bl/6 mice were used to test the hypothesis that commonly allergenic foods are intrinsically more immunogenic than rarely allergenic or nonallergenic foods in allergy-susceptible hosts. Groups of mice (n = 4 to 5) were injected intraperitoneally with the protein extracts (plus alum as an adjuvant) from chicken eggs, peanuts, almonds, filberts-hazelnuts, walnuts, soybeans, and wheat (commonly allergenic foods) and coffee, sweet potatoes, carrots, white potatoes, cherries, lettuce, and spinach (rarely allergenic and nonallergenic foods). Primary and secondary immune responses (as measured by specific IgG1 antibody serum levels) were measured by an enzyme-linked immunosorbent assay. Proteins from peanuts, almonds, filberts, sweet potatoes, cherries, and spinach elicited robust primary and/or secondary immune responses. Proteins from eggs, walnuts, and lettuce elicited poor primary responses but significant secondary responses. In contrast, wheat, soybeans, coffee, carrots, and white potatoes elicited barely detectable to poor primary and secondary immune responses. The order of the immunogenicity levels of these foods in mice is as follows: almonds = filberts > spinach (Rubisco) > peanuts > or = sweet potatoes > cherries > lettuce > walnuts > chicken eggs > carrots > or = white potatoes > wheat = coffee = soybeans. In summary, these data demonstrate for the first time that: (i) foods vary widely with regard to their relative immunogenicity in allergy-susceptible hosts and (ii) intrinsic immunogenicity in mice does not distinguish commonly allergenic foods from rarely allergenic or nonallergenic foods.     

Effects of twin-screw extrusion of peanut flour on in vitro digestion of potentially allergenic peanut proteins

Partially defatted peanut flour was processed in a twin-screw extruder. Resulting extrudates were dried, ground, and incubated with simulated gastric fluid for various time periods. Soluble protein content of the resulting digesta was measured after 10% trichloroacetic acid treatment to evaluate the digestibility. In vitro digestion using pepsin increased the solubility of peanut protein in 10% trichloroacetic acid solution from 2 to 6% to 65 to 75%. Four strong IgE-binding subunits (65, 22, 17, and 14 kDa) were found with immunoblotting in peanut proteins extracted from unextruded peanut flour; no IgE-binding bands were observed in extrudates. The 65-kDa (putative Ara h 1) subunit was insolubilized during extrusion, and its IgE-binding property was susceptible to in vitro digestion. Following extrusion cooking, no IgE-binding bands were detected by immunoblotting, including the strongly IgE-binding 14-kDa fraction, a strong IgE-binding band from native peanut protein that is stable in pepsin. The 22- and 17-kDa (putative Ara h 2) subunits retained a small amount of IgE-binding potential and became susceptible to pepsin hydrolysis after extrusion.     

Characterization of associated proteins and phospholipids in natural rubber latex

Non-rubber components present in natural rubber (NR) latex, such as proteins and phospholipids, are presumed to be distributed in the serum fraction as well as surrounding the rubber particle surface. The phospholipid-protein layers covering the rubber particle surface are especially interesting due to their ability to enhance the colloidal stability of NR latex. In this study, we have characterized the components surrounding the NR particle surface and investigated their role in the colloidal stability of NR particles. Proteins from the cream fraction were proteolytically removed from the NR latex and compare to those from the serum fractions using SDS-polyacrylamide gel electrophoresis revealing that both fractions contained similar proteins in certain molecular weights such as 14.5, 25 and 27 kDa. Phospholipids removed from latex by treatment with NaOH were analyzed using (1)H-NMR spectroscopy and several major signals were assignable to -(CH(2))(n)-, -CH(2)OP, -CH(2)OC═O and -OCH(2)CH(2)NH-. These signals are important evidence that indicates phospholipids associate with the rubber chain. The colloidal behavior of rubber lattices before and after removal of protein-lipid membrane was evaluated by zeta potential analysis and scanning electron microscope (SEM). The lowest zeta potential value of NR particles was observed at pH 10, consequently leading to the highest stability of rubber particles. Additionally, SEM micrographs clearly displayed a gray ring near the particle surface corresponding to the protein-lipid membrane layer.     

A novel extraction method for peanut allergenic proteins in chocolate and their detection by a liposome-based lateral flow assay

In this study, conditions for extracting the major peanut allergen (Ara h1) from chocolate were optimized, and the extracted samples were analyzed by a lateral flow assay (LFA) using liposomal nanovesicles. The optimal conditions using peanut-spiked chocolate were found to be extraction with a mixture of phosphate buffered saline and hexane for 30 min at 35 °C. After centrifugation, the buffer portion was treated with insoluble poly(vinylpolypyrrolidone) to remove phenolic compounds, and then analyzed by the LFA. The entire analysis, including sample preparation and LFA, could be easily completed within 2 h, and the detection limit was 158 g of peanuts/g of chocolate.     

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.