Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens

BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, contains some acidic/basic isoforms. The N-terminal amino acid sequences of some acidic Cyn d 1 isoforms were found to be different from those of Cyn d 1 cDNA clones identified previously. METHODS: A predicted 17-meric oligonucleotide probe was designed to fish the unidentified isoallergen cDNAs out of BGP cDNA library. The reactive clones were isolated and verified by sequencing. Two of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. RESULTS: All four cDNA clones encode the full-length Cyn d 1 with mature proteins of 244 amino acid residues. A 97-99% identity was found among the deduced amino acids of these four clones while an 86% identity was elicited between the four clones and the ones previously identified. The predicted isoelectric focusing (pI) values of the newly identified Cyn d 1s are acidic while pIs of the previously identified Cyn d 1s are basic. The two recombinant acidic Cyn d 1 proteins possess the epitopes recognized by mouse and rabbit polyclonal anti-Cyn d 1 antibodies, and have human IgE-binding capacity as revealed by immunodot assay. CONCLUSIONS: The present study identified full-length cDNAs encoding new isoallergens of Cyn d 1, and separated Cyn d 1 gene into an acidic group and a basic group.     

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen

Background Profilin, an actin-binding protein, was previously described as a panallergen which is involved in about 20% of Ihe crossreactivity found among pollen and food allergic patients. This allergen is usually under-represented in natural extracts used for allergy diagnosis. Objectives To obtain an immunologically active and soluble recombinant profilin from Cynodon dactylon pollen which could be used for diagnostic and therapy. Methods Isolation of cDNA clones was performed by polymerase chain reaction amplification using degenerate primers. Expression in Eschehchia coli BL21 (DE3) was carried out using vector pKN172, and the expressed product was isolated by affinity chromatography on poly L-proline-Sepharose. Results Four cDNA inserts coding for Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) were cloned and sequenced. Full-length C. dactylon profilin gene was expressed in Escherichia coli as non fusion protein. Induced cells could produce high amounts of recombinant Cyn d 12, and after a single purification step on poly (L-proline)-Sepharose, up to 45 mg of pure allergen per litre culture could be obtained. The reactivity of recombinant Cyn d 12 with IgE antibodies present in sera from Bermuda grass-allergic patients is comparable to that of the natural Bermuda grass allergen. Recombinant Bermuda grass pollen profilin was shown to share B-epitopes with sunflower prolilin. Conclusions Our results showed that this heterologous expression system and purification procedure are suitable for the production of large amounts of pure allergen which can be used for the characterization of allergenic epitopes recognized by T and B cells and finally for diagnostic and therapeutic purposes.     

Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1

BACKGROUND: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. METHODS: After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. RESULTS: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. CONCLUSIONS: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.     

Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen

Background: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. Objective: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. Methods: A canary grass pollen λgtl 1 cDNA expression library was constructed and screened with sera of grass-pollen-sensitive patients. IgE-reactive clones were isolated, sub-cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. Results: One of the clones encoded the group 1 allergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol 1 1, from velvet grass pollen and 86.6% with Phi p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6–95.5% nucleotide and 40.1–81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. Conclusions: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens.     

Characterization of the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d I

An allergen from Cynodon dactylon (Bermuda grass) pollen, Cyn d I, has been purified by a combination of concanavalin A-Sepharose affinity chromatography, and carboxymethyl-Sepharose chromatography. The allergen constitutes the major allergenic component of the pollen extract as observed by immunoelectrophoretic techniques, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an IgE-inhibition experiment, and skin testing. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Cyn d I is presented as a dominant 32 kd band and a minor 29 kd band, both binding IgE. Both bands are identified by monospecific rabbit antibodies (Abs) raised against Cyn d I. These Abs only weakly precipitate allergens from other grass species, indicating that Cyn d I possesses some unique immunochemical properties. Two of four purified murine monoclonal Abs raised against Cyn d I also bind to both bands of Cyn d I, indicating that the bands represent isoallergens with slightly different immunochemical properties. All four monoclonal Abs cross-react with pollen components from other grass species, especially Poa pratensis and Dactylis glomerata. The NH2-terminal sequence corresponding to approximately 10% of the complete sequence was determined, and it revealed high homology to the corresponding sequence of the major allergen of Lolium perenne, Lol p I. From the amino acid composition determination and immunoelectrophoretic comparison, the amount of Cyn d I in the source whole-pollen extract was estimated to be 15% wt/wt.     

Oligoclonal analysis of the atopic T cell response to the group 1 allergen of Cynodon dactylon (bermuda grass) pollen: pre- and post-allergen-specific immunotherapy

BACKGROUND: Bermuda grass pollen (BGP) is an increasingly important seasonal aeroallergen in Australia and other subtropical and temperate regions. BGP shares minimal allergenic cross-reactivity with pollens of rye grass or other Pooideae grasses often used for desensitization regimens in grass pollen allergy. Current allergen immunotherapy is seldom used in asthmatic patients due to IgE-mediated side effects. Since clinically effective immunotherapy is linked with altered allergen-specific T cell response, characterisation of human T cell reactivity to Cyn d 1, the major B cell allergen of BGP, should permit the design of effective and safe immunotherapy for BGP allergy. METHODS: Short-term BGP-specific CD4+ T cell lines were established from peripheral blood of 14 BGP-sensitive patients before and after conventional 50% BGP and 50% 7-grass mix subcutaneous specific allergen immunotherapy (SIT). T cell diversity of antigen specificity and function was assessed by proliferation and cytokine production to BGP, Cyn d 1 and Cyn d 1 peptides. RESULTS: Three highly immunogenic regions of Cyn d 1 were identified in 13/14 patients pre-SIT: Cyn d 1 (109-128), (181-209) and (217-241). The SIT regimen was clinically efficacious. Following SIT, decreased proliferation to BGP, Cyn d 1 and Cyn d 1 peptides was observed with a marked decrease in the IL-5:IFN-gamma ratio. CONCLUSIONS: Cyn d 1 is a major T cell allergen of BGP. Decreased Cyn d 1-specific IL-5 dominant T cell responses were observed in association with clinically effective treatment with the 50% BGP and 50% 7-grass mix. Identified dominant T cell regions of Cyn d 1 should facilitate safer vaccine development for BGP-induced asthma in addition to rhinitis.     

Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein. OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4. METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies. RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner. CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.     

Unique and Cross-Reactive T Cell Epitope Peptides of the Major Bahia Grass Pollen Allergen, Pas n 1

Background: Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4(+) T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods: Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results: Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions: The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.     

Analysis of allergenic components of Bermuda grass pollen by monoclonal antibodies

A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens (Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP-allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP-allergic patients. Among those human IgE-binding molecules, Cyn d Bd35K reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purification of these allergens.     

Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11

BACKGROUND: Allergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n = 184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. CONCLUSION: As the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.     

Characterization of api g 1.0201, a new member of the Api g 1 family of celery allergens

BACKGROUND: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients. In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level. Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1. Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features. METHODS: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced. The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots. RESULTS: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues. The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101. Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody. In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments. Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a. Total inhibition was only achieved when using purified natural Bet v 1. CONCLUSIONS: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens. Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy.     

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen Phl p 13 from timothy grass pollen (Phleum pratense)

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.     

The dominant 55 kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55 kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55 kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55 kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.     

Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.     

Recombinant pollen allergens from Dactylis glomerata: preliminary evidence that human IgE cross-reactivity between Dac g II and Lol p I/II is increased following grass pollen immunotherapy.

We previously described the isolation of three identical complementary DNA (cDNA) clones, constructed from Orchard/Cocksfoot grass (Dactylis glomerata) anther messenger RNA (mRNA), expressing a 140,000 MW beta-galactosidase fusion protein recognized by IgE antibodies in atopic sera. Partial nucleotide sequencing and inferred amino acid sequence showed greater than 90% homology with the group II allergen from Lolium perenne (Lol II) indicating they encode the group II equivalent, Dac g II. Western blot immunoprobing of recombinant lysates with rabbit polyclonal, mouse monoclonal and human polyclonal antisera demonstrates immunological identity between recombinant Dac g II, Lol p I and Lol p II. Similar cross-identity is observed with pollen extracts from three other grass species: Festuca rubra, Phleum pratense and Anthoxanthum odoratum. Recombinant Dac g II was recognized by species- and group-cross-reactive human IgE antibodies in 33% (4/12) of sera randomly selected from grass-sensitive individuals and in 67% (14/21) of sera from patients receiving grass pollen immunotherapy, whilst 0/4 sera from patients receiving venom immunotherapy alone contained Dac g II cross-reactive IgE. Cross-reactive IgG4 antibodies were detectable in 95% of sera from grass pollen immunotherapy patients. These preliminary data suggest that conventional grass pollen allergoid desensitization immunotherapy may induce IgE responses to a cross-reactive epitope(s) co-expressed by grass pollen groups I and II (and possibly group III) allergens.     

Conversion of grass pollen allergen-specific human IgE into a protective IgG(1) antibody

More than 100 million individuals exhibit IgE-mediated allergic reactions against Phl p 2, a major allergen from timothy grass pollen. We isolated cDNA coding for three Phl p 2-specific human IgE antibodies from a combinatorial library, which was constructed from lymphocytes of a grass pollen-allergic patient. Recombinant Phl p 2-specific IgE antibody fragments (Fab) recognized a fragment comprising the 64 N-terminal amino acids of Phl p 2 and cross-reacted with group 2 allergens from seven grass species. cDNA coding for the variable regions of one of the IgE Fab were cloned into aplasmid vector expressing the constant region of human IgG(1) to obtain a complete, recombinant Phl p 2-specific human IgG(1). This antibody blocked the binding of grass pollen-allergic patients IgE (n=26; mean inhibition: 58%) to Phl p 2 and caused a 100-fold reduction of Phl p 2-induced basophil histamine release. The recombinant human Phl p 2-specific IgG(1) may be used for environmental allergen detection, for standardization of diagnostic as well as therapeutic grass pollen allergen preparations and for passive therapy of grass pollen allergy.     

Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen

BACKGROUND: The identity of allergenic almond proteins is incomplete. OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.     

芒果果实中泛变应原profilin的克隆、表达及免疫学活性鉴定

目的　克隆并表达芒果果实中泛变应原肌动蛋白抑制蛋白(profilin)并了解其免疫学活性。方法利用RT-PCR结合:RACE技术克隆芒果果实中泛变应原profilin的全长基因,并进行序列分析。然后设计带有酶切位点的特异性引物,采用RT-PCR获得整个芒果profilin的开放阅读框,将其与pET-28a载体连接并转化大肠杆菌Escherichia coli BL21(DE3)进行诱导表达,通过Ni2 亲和层析柱对重组蛋白进行纯化,采用Western blot检测其IgE结合活性。结果克隆获得了芒果profilin的2个型。每个型的cDNA都包括一个编码131个氨基酸的开放阅读框。序列分析结果显示所克隆到的基因与许多水果和花粉的泛变应原profilin基因有很高的同源性(>70%)。根据变应原的命名规则,将它们分别命名为Man i 3.01和Man i 3.02,并将这两个基因提交到GenBank数据库中,其登录号分别为DQ270547和DQ400579。重组芒果profilin在大肠杆菌中高效的表达,进一步经Ni2 亲和层析柱纯化后经Western blot检测具有良好的免疫学活性。结论成功地克隆和表达了芒果profilin,并证明与芒果过敏者血清有特异IgE应答。     

Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids. The coding regions for the three cDNA clones share a common amino-terminus of 149 amino acids and a common carboxyl-terminus of 75 amino acids including a membrane spanning region. In addition, we isolated a genomic clone of human M-CSF. When each of the cDNA clones or the genomic clone were transfected into COS-7 monkey kidney cells, biologically active M-CSF was expressed as judged by the ability of transfected cell supernatants to stimulate proliferation and colony formation of murine bone marrow cells, as well as formation of monocytic colonies from human bone marrow cells. Surprisingly, proliferation of human bone marrow cells was not induced by recombinant human M-CSF. Analysis of the M-CSF proteins released by COS-7 cells revealed that monomer subunit proteins of 44 or 28 kDa were produced. In addition, we found that the membrane spanning region, present in all three forms of M-CSF cDNA, was not required for the synthesis of a biologically active protein. However, when the membrane spanning region was present in the three M-CSF cDNAs, cell surface associated forms of M-CSF could be readily detected.