实时荧光PCR检测食品中致敏原芥末成分

对植物原料产品和植物源性食品中芥末成分的快速鉴定是避免过敏性疾病发生的重要措施。依据芥末Sin A1管家基因的核酸序列设计特异性引物和探针,对3种芥末样品和21种非芥末植物样品进行实时荧光PCR检测,结果显示,过敏原芥末管家基因的样品FAM通道有荧光信号检出,非芥末样品FAM通道均无荧光信号检出。灵敏度实验表明,植物原料产品中对芥末的检测低限可达到1 mg/kg。此外对市售的芥菜籽等样品和深加工的芥末致敏原参考物质(葡萄糖)进行实际样品的检测,均能很好检出致敏原芥末成分。     

婴儿双歧杆菌对花生过敏小鼠模型肠道Th2反应的调节

目的研究婴儿型双歧杆菌对花生过敏小鼠肠道Th2型反应的调节作用。方法通过应用花生蛋白诱导肠道的Th2型反应,建立食物过敏小鼠模型。过敏小鼠灌胃给予婴儿型双歧杆菌（ATCC菌或CGMCC0313-2）或不做处理。然后分离小鼠小肠黏膜CD4＋T细胞或DC,另取肠黏膜组织进行石蜡包埋甲苯胺蓝染色肥大细胞计数,HE染色进行嗜酸细胞和单个核细胞计数,流式细胞检测CD4＋T中Th2（CD4＋IL4＋T）细胞和Treg（CD4＋CD25＋Foxp3＋T）比例,另取CD4＋T进行CFSE标记,与DC共培养4d后流式细胞检测CD4＋T增殖反应,收集细胞培养液ELISA检测IL-4、IL-5和IL—13分泌水平。结果过敏组小鼠Th2型细胞数,CD4＋T细胞增殖反应,IL4、IL-5和IL-13水平,肠黏膜中肥大细胞、嗜酸性细胞和单个核细胞数均明显高于对照组（P〈0．01）,而Treg数目低于对照组（P〈0．01）,婴儿双歧杆菌干预后,婴儿双歧杆菌组Th2型细胞数,IL4、IL-5和IL-13水平,肠黏膜中肥大细胞、嗜酸性细胞和单个核细胞数均明显低于过敏组（P〈0．01）,而Treg数目高于过敏组（P〈0．01）。结论口服婴儿型双歧杆菌可以抑制花生过敏导致的肠道Th2型反应。     

Purification and characterization of a novel isoallergen of a major Bermuda grass pollen allergen,Cyn d 1

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.     

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.     

Expression of the major olive pollen allergen Ole e 10 in the yeast Pichia pastoris: evidence of post-translational modifications

Olive pollen allergy is a clinical disorder that affects around 20% of the population in Mediterranean areas. The major olive pollen allergen, Ole e 10, is involved in cross-reactivity phenomena and asthma induction in allergic patients, and, besides its clinical interest, Ole e 10 is the first member of a new family of plant proteins. Ole e 10-specific cDNA has been cloned in the plasmid pPICZalphaA and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has been purified in a two chromatographic-step procedure. N-Terminal sequencing, mass spectrometry, IgG, and IgE binding assays were employed to characterize the recombinant allergen. These analyses revealed that the product undergoes a proteolytic cleavage in the N-terminal end with the loss of the first six residues. Different strategies were used to solve this problem, such as changes in the fermentation conditions and the employment of protease-deficient yeast strains. Proteolytic cleavage was minimized and about 51% of rOle e 10 was obtained as a full-length protein. Moreover, a covalent modification was found in the N-terminal end of the full-length rOle e 10. Peptide mapping and mass spectrometry analyses pointed to the existence of a phosphorylation located in a serine residue of the N-terminal segment of rOle e 10 and it was confirmed after treatment of the sample with alkaline phosphatase. Finally, both full-length and truncated rOle e 10 retained most of the IgG- and IgE-binding capabilities of the natural protein isolated from the pollen.     

Kiwellin, a Novel Protein from Kiwi Fruit. Purification, Biochemical Characterization and Identification as an Allergen*

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit. *The protein sequence data reported in this paper will appear in the Swiss-Prot and TrEMBL knowledgebase under the accessionnumber P84527.     

Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen

Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the most common causes of airway allergic disease, and has been shown to contain over 12 allergenic proteins on 1-D immunoglobulin E (IgE) immunoblots. However, only a few allergens have been identified and characterized. Cyn d 1 is a major allergen and the most abundant protein in BGP, representing 15% of the whole-pollen extract. To investigate variability in the IgE-reactive patterns of BGP-sensitized patients and to identify other prevalent allergens, a BGP extract was passed through an affinity column to remove Cyn d 1, and the non-bound material was collected and analyzed by 2-DE. IgE-reactive proteins were subsequently characterized by immunoblotting using serum samples from ten BGP-allergic patients. The prevalent IgE-reactive proteins were identified by MALDI-TOF MS, N-terminal sequence similarity, and LC-MS/MS. Here, we present a sub-proteome approach for allergen investigation and its use for determining BGP 2-DE profiles and identifying six novel allergens.     

Chemical modification studies onCocos nucifera pollen allergens

Two highly active allergens C_n II (M _r 158,000) and C_nVII (M _r 2900) isolated fromCocos nucifera pollen extract were treated with various protein modifying reagents in order to ascertain the amino acid residues responsible for their allergenicity, In C_n II modification of carboxy group and tryptophan residue led to 30 and ≽ 75% loss in allergenicity and those of lysine and tyrosine reduced 62 and 38% activity, Lysine, tyrosine, tryptophan and carboxy group of C_nVII were also modified causing 81, 17, ≽ 70 and 26% loss of allergenicity respectively, Allergenicity of both was highly affected by pronase and moderately affected by heat, Periodate destroyed about 50% of their allergenicity and other chemical reagents except urea had no remarkable effect     

A comparative biochemical study of conifer pollen allergens

In the order Coniferales, only the family Cupressaceae is regarded as being a significant source of airborne allergens, withJuniperus ashei characterized as the most significat aeroallergen. Pollen of the closely related speciesJ. virginiana has been shown to cross-react withJ. ashei pollen, however,J. virginiana pollen is not considered an important aeroallergen. Although there have been several reports of allergies toPinus pollen, the pollen of this genus is regarded as hypoallergenic. Our previous studies have shown that pollen extracts ofJ. ashei, J. virginiana, J. pinchotii, Cupressus macrocarpa, Pinus echinata andP. taeda all contained several proteins with the same molecular weights including the reported allergen ofJ. ashei. The present study compared the biochemistry ofJ. ashei, J. virginiana andP. echinata pollen. A time course experiment ofJ. ashei, J. virginiana andP. echinata showed thatJ. ashei released a greater quantity of protein within the first minute of moistening. SDS-PAGE analyses showed that the reported allergen ofJ. ashei pollen extracts was released in large quantities within the first minute of extraction. It was also determined that individual pollen grains ofP. echinata contained a greater quantity of protein than the pollen ofJ. ashei andJ. virginiana, but due to the large size of pine pollen there was less protein per gram of pollen. Lipid analysis of these three taxa showed that the pollen ofP. echinata contained more lipid per grain and per gram of pollen. Results indicate that the rapid release of the reported allergen fromJ. ashei pollen contributes to the allergenicity of this species compared to bothJ. virginiana andP. echinata.