Identification of an abundant allergen from the sheep louse, Bovicola ovis

Infestation of sheep with the louse Bovicola ovis is common worldwide and leads to an allergic dermatitis referred to as ‘scatter cockle’. IgE from an infested lamb was used in immunoaffinity chromatography to purify allergens from crude preparations of whole B. ovis and its faeces. SDS-PAGE of the affinity-purified eluates from both preparations showed a dominant band with M_r of 28.5 kDa. Spleen cells from a mouse immunised with B. ovis faecal antigens were used to produce hybridomas which were screened by ELISA to identify those producing monoclonal antibodies (mAb) to the allergens purified by IgE immunoaffinity chromatography. Western blotting demonstrated that all of the mAbs examined recognised the 28.5 kDa allergen. The allergen, purified using immunoaffinity columns constructed with one of the specific mAbs, was shown to cause immediate and late-phase responses on intradermal skin testing in B. ovis-infested but not in naïve lambs. Levels of serum IgE specific for the purified allergen were significantly higher in infested than in naïve lambs (P ? 0.0025). N-terminal and internal amino acid (aa) sequences obtained from the purified 28.5 kDa allergen were used to design primers to amplify a partial cDNA probe from B. ovis cDNA by PCR. The amplified probe was radiolabeled and used to screen a B. ovis cDNA library. The complete nucleotide sequence of the allergen was determined from the sequences of the positive clones from the library. The full-length cDNA encodes a 255 aa protein including a secretory leader sequence of 26 aas and a mature protein of 229 aas. The encoded protein showed strong homology to several hypothetical proteins of unknown function from diverse species and weak homology with lipid-binding proteins of Xenopus tropicalis and Galleria mellonella. This is the first allergen to be identified from a louse and it has been designated Bov o 1 in accordance with the criteria of the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee.     

Oligoclonal Enzyme-linked Immunosorbent Assay Capable of Determining the Major Food Allergen, Ovomucoid, Irrespective of the Degree of Heat Denaturation

We have recently established an enzyme-linked immunosorbent assay (ELISA) for total ovomucoid determination, irrespective of the degree of its heat denaturation, by using a monoclonal antibody (mAb) 7D specific to the carbohydrate moiety of ovomucoid (Biosci Biothechnol Biochem, 68, 2490–2497, 2004). Two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAb 7D and two other mAbs with different epitopes as a second antibody. Second, it was shown that usage of denaturing reagents such as SDS and β-mercaptoethanol for extraction was acceptable for ELISA within a range of stability of a first antibody on a solid phase. Properties of the oligoclonal sandwich ELISA system thus constructed were discussed in connection with allergen labeling.     

肾综合征出血热双价纯化疫苗（Vero细胞）安全性的研究

在肾综合征出血热（HFRS）双价纯化疫苗（Vero细胞）的研制中,对病毒培养用Vero细胞致瘤性和试制疫苗的过敏原性进行了动物实验研究,结果表明,病毒培养用Vero细胞无致瘤性,三批试制疫苗无过敏原性。说明用Vero细胞为培养介质的HFRS双价纯化疫苗是安全的。     

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.     

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.     

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.     

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.     

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.