梭子蟹变应原的分离、纯化与鉴定

目的：对梭子蟹（Portunus pelogicus（Linnaeus））变应原进行分离,鉴定其主要及次要变应原,采用蛋白纯化技术获取梭子蟹天然的主要变应原并进行鉴定,为标准化变应原疫苗的研制提供理论依据.方法：取常规方法制备梭子蟹浸出液,经SDS-PAGE分离,测定各组分的相对分子量;同时用26例对蟹过敏的病人血清进行Western blot,鉴定其主要及次要变应原;利用快速制备液相色谱（FPLC）纯化技术（凝胶过滤层析和离子交换层析）获取主要变应原并作鉴定.结果：SDS-PAGE显示梭子蟹有19条可辨蛋白带,分子量在13 000～90 000之间,其中主带有9条,分子量是20 900、24 200、27 100、29 200、33 700、38 900、48 700、74 700、89 100;Western blot结果表明,26例蟹过敏患者血清全部呈阳性反应,浸出液中共有5条致敏条带,其中分子量在74 400、48 700的是主要变应原,阳性反应率均为100%;纯化后获取了74400、48700的主要变应原;经过免疫鉴定证实其具有免疫活性.结论：梭子蟹74 400和48 700的变应原为主要变应原,层析技术可以对分子量为74400和48700的主要变应原成分进行纯化.     

Purification and characterisation of a major allergen of Alternaria alternata

A major allergen of Alternaria (Alt a Bd29K) was identified by SDS-PAGE and immunoblotting. It was purified by gel-permeation and ion-exchange HPLC. The apparent molecular mass was 29,000, with a cysteine linked sub-unit of 15,000 Mr. The apparent isoelectric point was 4.2. It was found to be a constituent of the Alt I allergen group.     

Purification and characterization of the major dog allergen, Can f I

An important dog-hair and dander-specific allergen Ag13 has been purified by means of immunoaffinity chromatography utilizing rabbit antibody specific for Ag13. Purity was judged to be very high as detected by crossed immunoelectrophoresis and SDS-PAGE. The purified allergen was subjected to amino acid analyses. Molecular weight was about 22 kD in HPLC-gel filtration and 25 kD in SDS-PAGE with an additional band at 18 kD. In vitro IgE binding of the allergen was investigated by luminescence immunoassay (LIA) inhibition. Removal of Ag13 from dog hair and dander extract (DHD) removed 50 +/- 1.5% of the IgE binding capacity. The purified allergen inhibited up to 56.5% of the IgE activity to DHD as measured with a pool of serum from dog-allergic patients. Out of 26 dog-allergic patients, 24 had a positive skin-prick test to the allergen. Out of 23 dog-allergic patients, 16 reacted with the allergen in IgE immunoblotting. We suggest that Ag13 be termed Can f I. The allergen will be a marker allergen for environmental dog hair and dander exposure.     

Purification and characterization of an agglutinin of the soft coral Sinularia species.

A D-galactose-specific agglutinin, named sinularian, has been isolated from the soft coral Sinularia sp. by affinity chromatography on acid-treated Sepharose 4B and by gel filtration on HPLC. Sinularian was a glycoprotein containing 11% sugar. It gave a single band corresponding to 78 kDa in SDS-PAGE, irrespective of a treatment with 2-mercaptoethanol. Sinularian agglutinated rabbit erythrocytes and murine leukemia cells but not sheep or human ABO erythrocytes. Its hemagglutinating activity was Ca(++)-independent. Sinularian promoted binding of macrophages to tumor cells.     

New occupational allergen in citrus farmers: citrus red mite (Panonychus citri).

BACKGROUND: There have been several reports of occupational allergy to spider mites (Tetranychidae), but no published report has described citrus red mite (CRM, Panonychus citri)-induced occupational asthma confirmed by specific bronchial challenge. OBJECTIVE: The purpose of this study was to evaluate clinical and immunologic characteristics of CRM-induced occupational asthma. METHODS AND RESULTS: We encountered 16 cases of CRM-induced occupational asthma among farmers cultivating citrus fruits. Asthmatic attacks corresponded closely with their work on citrus farms. The mean duration of the latent period was 12.9 (range 7 to 20) years. During their first visit to our clinic, nine patients with FEV1 lower than 70% of predictive value showed reversible airway obstruction after inhalation of bronchodilator, and seven with FEV1 greater than 70% of predictive value showed airway hyperresponsiveness to methacholine. Fifteen of the 16 also complained of recurrent nasal symptoms, which had developed at an earlier time than the asthmatic symptoms. They showed strong positive reactions to CRM extract on skin prick test (A/H ratio > or = 1.0) and had high serum specific IgE antibody against CRM which was detected by ELISA. Skin prick test with common inhalant allergens revealed that 10 had an isolated positive response to CRM with negative results to common inhalant allergens in their environment. The ELISA inhibition tests with CRM demonstrated significant inhibitions by CRM in a dose-dependent manner, while minimal inhibitions were noted by D. pteronyssinus and mugwort allergens. CONCLUSION: These findings suggest that CRM could induce IgE-mediated bronchoconstriction in exposed workers on citrus farm.     

Purification and molecular cloning of a major allergen from Anisakis simplex

A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.     

Purification and characterization of a major cross-reactive allergen from Epicoccum purpurascens

BACKGROUND: Epicoccum purpurascens (formerly nigrum) (EP), is a ubiquitous saprophytic mould found both indoors and outdoors. Several studies have reported sensitization to EP in 5-7% of different populations worldwide. The diagnosis of mould allergy requires a standardized fungal extract that contains all its important allergenic proteins. The crude allergen extract from EP was standardized earlier, however none of its allergens have been purified. METHODS: A major allergen from spore-mycelia extract of EP was purified using concanavalin A (Con A) Sepharose chromatography, gel filtration and electro-elution. The allergen isolated was characterized for its IgE-binding ability and cross-reactivity with five well-known allergenic fungi by ELISA and immunoblot. RESULTS: A 33.5-kD glycoprotein allergen of EP, Epi p 1, was purified to homogeneity. All the EP allergic patients' sera tested recognized this protein. Periodate modification of Epi p 1 showed partial loss in IgE binding while proteinase K treatment caused complete loss in binding to IgE. Dose-dependent inhibition in binding of rabbit anti Epi p 1 antibodies was obtained with Epi p 1, Aspergillus fumigatus, Alternaria alternata, Curvularia lunata, Cladosporium herbarum and Fusarium solani in ELISA. Rabbit antibodies to all the above five fungi recognized Epi p 1 in immunoblot, confirming that Epi p 1 shares common epitopes with the fungi tested. CONCLUSION: A major glycoprotein allergen of 33.5 kD was purified from EP which cross-reacts with other fungi. Hence this glycoprotein can be exploited to reduce the panel of allergen extracts used for therapy of mould allergy.     

Purification of Par j I, the major allergen of Parietaria judaica pollen

A component of Parietaria judaica pollen extract, previously identified as the major allergen, then reported as Pj10 and hereafter denominated Par j I has been isolated by a combination of 65% ammonium sulphate salt precipitation and gel filtration and an Ultrogel AcA54 column. The purified allergen appeared essentially homogeneous on gel filtration HPLC. The mol. wt of Par j I was estimated by electrophoretic and chromatographic techniques. All results gave values in a range from 13 K to 25 K. Analysis in SDS-PAGE under reducing conditions revealed a broad band corresponding to a mol. wt of 10 K, which retained allergenicity when tested with a patients serum pool. CIE and CRIE patterns of the isolated Par j I displayed the two precipitating lines already reported as those exhibiting the highest IgE-binding ability. Par j I showed a specific allergenic activity about 10-fold higher than that of the whole extract and was demonstrated to be the major allergen of Parietaria judaica as assessed in 25 sensitive human sera.     

Purification and characterization of a cross-reactive 45-kD major allergen of Fusarium solani

BACKGROUND: Fusarium solani (FS) is an important source of fungal allergen. A 45-kD major allergen of FS showed reactivity with patients' sera sensitive to many fungi. OBJECTIVES: To purify and characterize a 45-kD common allergenic protein from FS, which may be useful for the diagnosis of and therapy for fungal allergy. METHODS: FS culture filtrate extract was seperated on SDS-PAGE; 45-kD protein was electroeluted and purified on C18 column using reverse-phase high-pressure liquid chromatography (rpHPLC). The purified protein was functionally and biochemically characterized by in vitro and in vivo methods. RESULTS: The 45-kD protein showed a single peak on rpHPLC. The N-terminal amino acid sequence of this protein did not show homology to enolase or known fungal proteins. It showed cross-reactivity with Epicoccum nigrum, Curvularia lunata, Cladosporium herbarum and Alternaria alternata by ELISA and ELISA inhibition using rabbit antibodies raised against these fungi. IgE ELISA inhibition with patients' sera positive to different fungi demonstrated allergenic cross-reactivity of the 45-kD protein with other fungal extracts. This 45-kD protein released a significant amount of histamine in FS-allergenic patients. CONCLUSION: A cross-reactive 45-kD allergenic/antigenic protein was purified to homogeneity and characterized. It has prospects for use in allergen therapy.     

Purification of Parj I, a major allergen from Parietaria, judaica pollen

A major allergen, the Parj I, was purified to homogeneity from Parietaria judaica pollen by means of ultrafiltration dialysis, preparative polyacrylamide gel chromatography and affinity chromatography through a column of Sepharose-monoclonal antibody specific for Parj I. The homogeneity of the Parj I was assessed by one single arc of immunoprecipitation both in cross immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis, by one single band of radiostaining after a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose and by one single peak after a size exclusion chromatography on high-performance liquid chromatography (HPLC). The homogeneity was further supported by crossed Laurell immunoelectrophoretic analysis, in that only one arc of precipitation was magnified in CIE after addition of the purified allergen. The purified Parj I allergen was capable of interacting in vitro with 70% of the human IgE specific for a crude P. judaica extract, as determined by radioallergosorbent test inhibition. The purified Parj I was capable of inducing positive reactions in vivo in skin prick tests, and of inducing release of histamine from blood containing basophils as determined by a histamine release assay. The amino acid analysis of the Parj I showed 118 amino acid residues per monomer analyzed and, among other residues, three methionine residues were detected. The molecular weight of the Parj I estimated by HPLC and amino acid composition was 26 kilodaltons.     

Purification and fine characterization of a major allergen from Olea europaea pollen extract

Olea europaea (olive) pollen extract was prepared by aqueous extraction and characterized by biochemical and immunochemical methods. Two components, displaying respective mol. wt. of 17000 and 19000, were the most reactive allergens, being the doublet (designated Ole e I) recognized by most sera tested. The 19000 mol. wt. component, purified by conventional biochemical procedure and lectin-affinity chromatography from the Ole e I doublet, was deglycosylated and analyzed by SDS–PAGE and by ELISA inhibition. The results obtained suggest that the 19000 mol. wt. component represents the glycosylated form of the 17000 component.     

Purification and immunobiochemical characterization of folding variants of the recombinant major wasp allergen Ves v 5 (antigen 5)

Background: Antigen 5 is one of three major allergens in wasp venoms, but unlike phospholipase A(1) and hyaluronidase, both of which are enzymes, its biological function is unknown. The cDNA coding for this allergen has been isolated and used for recombinant expression. Thorough analysis of the expression product is essential in order to evaluate the usefulness for in vivo or in vitro application. Objective: In this study, folding variants of the recombinant major allergen Ves v 5 from Vespula vulgaris were immunologically and biochemically investigated in order to determine their possible applicability for diagnostic or therapeutic purposes. Method: The cDNA encoding Ves v 5 was cloned into the expression vector pSE420 which generates recombinant products lacking a tag sequence. After expression, inclusion bodies were purified, subsequently denatured and dialyzed against different solutions. The structural properties of soluble proteins were analyzed by size exclusion chromatography, non-reducing SDS-PAGE, native PAGE, N-terminal sequencing, proteolytic digestion and ion exchange chromatography. Immunological investigations were performed by using different monoclonal antibodies (mAbs) specific for Ves v 5 and IgE from patients allergic to wasp venom allergens. Results: After dialysis, soluble monomeric recombinant Ves v 5 was more than 95% pure in each case. Using different dialysis solutions, clearly distinguishable folding variants were obtained. In one case, the recombinant allergen was comparable with the natural counterpart in respect of migration in non-reducing SDS-PAGE, native PAGE and IgE reactivity. This variant reacted with two different Ves v 5-specific mAbs and produced a stable fragment after proteolytic digestion. Elution from a cation exchange chromatography column was achieved with 320 mM NaCl. In two other cases, folding variants exhibited a different migration behavior in SDS-PAGE and native PAGE compared with the natural allergen. Also, the mAb 1E11 recognized none of these variants since it presumably detected a conformational epitope. Moreover, the IgE reactivity was clearly reduced and proteolytic digestion effected almost complete degradation. These variants eluted from the cation exchange column with 400 mM NaCl. Conclusion: Defined folding strategies resulted in both soluble misfolded variants with reduced IgE reactivity, potentially suitable for immunotherapy, and natural-like folded variants for diagnosis.     

Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor

A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.     

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I)

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith apples were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients’ sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Fürthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity. Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.     

Purification and characterization of Par o I, major allergen of Parietaria officinalis pollen.

Par o I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par o I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par o I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par o I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.     

Purification and characterization of Pla a 1, a major allergen from Platanus acerifolia pollen

BACKGROUND: Plane trees, as Platanus acerifolia, are an important source of airborne allergens in cities of the United States and Western Europe. Little is known about the relevant allergens of this pollen. The aim of this study was to identify relevant allergens from P. acerifolia pollen and purify and characterize a major allergen of 18 kDa. METHODS: P. acerifolia pollen extract was fractionated using ion exchange, gel filtration, and reverse-phase chromatography. Analyzes were carried out by EAST, SDS-PAGE, isoelectric focusing, immunoblotting and amino-acid sequencing. RESULTS: An 18-kDa protein from the P. acerifolia pollen extract, which we named Pla a 1, was purified. This nonglycosylated protein had an isoelectric point value higher than 9.3 and was recognized by up to 92% of monosensitized Platanus allergic patients and 83% of polyzensitized patients. Sequencing of its N-terminal yielded an amino acid sequence which showed no homology to the known proteins in the databases. Other relevant allergens detected in monosensitized patients were proteins of 43 and 52 kDa, with immunoglobulin (Ig)E-binding prevalences of 83 and 42%, respectively. Profilin was an important allergen in polyzensitized patients. CONCLUSIONS: The most relevant allergens from the P. acerifolia pollen have been determined. A major allergen, specific of this pollen, and named Pla a 1, has been purified and characterized.     

Purification and IgE binding capacity of Der s 3, a major allergen from Dermatophagoides siboney

Background Sensitization to the house dust mite Dermatophagoides siboney has been demonstrated in asthmatic patients. Previously, Dermatophagoides siboney group 1 and group 2 allergens, named Der s 1 and Der s 2, respeetively, have been purified. Objectives The aim of this study was to purify and to study the IgE reactivity of a 30 kDa component, suspected to correspond to group 3 allergens. Methods The protein was purified by affinity chromatography using anti-Der f 3 monoclonal antibodies and semi-preparative SDS-PAGE. The IgE binding capacity of the purified fractions was tested with sera from 106 mite- sensitive asthmatic patients using a modified chemiluminiscent method. Results Affinity chromatography resulted in fractions containing the 30 kDa component which was further purified to homogeneity by SDS-PAGE. Seventythree per cent of the sera showed IgE reactivity to this protein, indicating that it is a major allergen. The protein also reacted with anti Der f 3 polyclonal antibodies and had tryptic activity. There were differences in the reaetivity to Der s 3 according to the age of the patients. Conclusion Based on the frequency of IgE reactions and the reactivity with antibodies directed to Der f 3, it is proposed to name this 30 kDa allergen from D. siboney, Der s 3.