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The present invention is in the field of diagnosis and treatment of allergies, particularly pollen allergies. This family of trees and shrubs, especially olive, are widely distributed in all countries of the Basin Mediterra- nea.
An efficient method for the purification of recombinant allergen Ole e 1. This type of allergy is caused by IgE antibody formation against airborne antigens. The Parthenon Publishing Group-Carnforthpp. This method basically consists in modulating the immune response in the patient by regular administration and increasing concentrations of the proteins that cause allergy allergenic extracts.
Allergenic extracts isolated from natural sources are complex mixtures of proteins and other molecules. Composition, and therefore allergenicity, it depends on the material used, which varies according to environmental conditions, in the case of pollens, the maturation phase, in the case of fungi, the sal haloidea se combinance conditions for mites, etc. Even some extracts may contain an insufficient concentration of major allergens may be contaminated with undesirable components, against which sal haloidea se combinance patient is not allergic, or both.
Basic and practical aspeets or recombinant allergens. Purification is more efficient and cheaper, since it is part of a greater proportion of the protein of interest. Furthermore, by these techniques they can be sal haloidea se combinance easily specific fragments of these proteins, which may serve for characterization of T cell epitopes The knowledge of these epitopes could improve specific forms of therapy using synthetic peptides or sal haloidea se combinance combinant sal haloidea se combinance.
T-cell responses to allergens: Immunol Today 17, The application of recombinant DNA techniques has led to the isolation of a large number of proteins with allergenic characteristics [ 4 Stewart, G. The molecular biology of allergens. In Asthma and Rhinitis. Busse and Holgate S. Bac ell Scientifics Publicationspp. The allergens of Olea europaea and Parietaria spp.
This high percentage of asthmatic responses is compounded because asthma symptoms are more severe than in other forms of hay fever. These symptoms sometimes do not diminish anything past several months or even all year [ 5 ]. The major pollen allergen 0. Immu- nochemical characterization by immunoblotting, CRIE and immunodetection by a monoclonal sal haloidea se combinance.
Ole e 1 is an acidic protein having two variants of amino acid residues, one of which is the glycosyl cosilada form with a molecular weight of 20 kDa, and another This allergen has microheterogeneity in different positions resulting in different detected isoforms [ 89 Lombardero J. Allergy 24, ] and 10 Villalba, M. Cloning and expression of Ole and I, the major allergen from olive tree pollen.
This allergen sample as Ole e 1, a certain heterogeneity in its sequence [ 11 Batanero, E. Sequence analysis of three cDNA encoding protein isoforms. The biological role of Ole e 1 in olive tree pollen is unknown although it could be related to germination, since it has some similarity with other proteins such as Zml3 pollen protein maize [ 12 Heiss, S.
The Zml3 protein has recently been described as a potential allergen, since it sal haloidea se combinance ability to bind IgE [ 12 ]. Gene cloning can be performed using various methods [ 13 Sambrook, J. Similarly detection of the clones carrying the gene of interest can be done in several ways: Sal haloidea se combinance the pre I sat invention have combined the two most direct methods sal haloidea se combinance both steps: These primers were added at their ends recognition sequences various restriction endonucleases.
DNA inserted, which matched the expected size was sequenced by the Sanger method. Computer analysis of the sequences showed that all sequences coding for the allergen Ole e 1. Expression of cloned genes. There is a wide variety of systems expression of heterologous proteins.
In the present invention has been overexpressed gene Ole e 1 in two different systems that allow the production of a non-fused protein using the expression vector pKN [ 15 Way, M. Identification of a region in segment 1 of gelso- lin critical for actin binding.
Use of T7 phage polymerase Bacteriological to direct selective high-level ex- pression of cloned genes. IPTG was added at concentrations between 0. The culture was harvested hours after induction. Purification of recombinant allergen Purification of recombinant allergen was performed by two single chromatographic steps: The chromatography gel filtration Sephadex G was performed to remove the urea, added to the sample to obtain a complete solubilization of the inclusion bodies where the fusion protein was.
Then the sample was passed through a column bound to glutathione agarose. The GST enzyme specifically binds glutathione, and after a series of washings, can be eluted by the addition of free glutathione. The fusion protein thus obtained was digested with thrombin to release the recombinant allergen as between both proteins there is a cleavage site for this enzyme artificially introduced. Ole e 1 recombinant can be separated from the GST protein by passing the mixture again by an affinity column, as described above, thereby obtaining pure recombinant allergen.
Alternatively, one can treat the column containing the fusion protein with thrombin, so that the recombinant Ole e 1 is freed, while the protein is retained on the column. This expression can be accomplished in other microbial systems Pseudomonas, Bacillus or eukaryotic yeast, Saccharo yces cerevisiae or as Pichia pastoris or insect cells.
The allergen produced can be purified by a simple process comprising two chromatographic steps, one gel filtration and one affinity. The recombinant allergen produced by this method does not differ in their binding properties of natural IgE filinas proposed.
The invention also describes diagnostic methods using this recombinant allergen. Diagnostic methods, both in vi tro and in vivo, currently used are based on the use of allergen extracts. Due to the difficulty of obtaining reproducible batches of allergenic extracts interesting reproducible using pure preparations of allergen obtained by recombinant DNA technology [ 2 ]. Characterizing the reactivity pattern of each patient can lead in the near future, sal haloidea se combinance development of a more individualized type of immunotherapy, and therefore effi- you.
Its application to sal haloidea se combinance treatment of Type I allergy can be established in two ways: Lanes 1 and 2, BL21 E. Immunodetection of recombinant Ole e 1. Comparison immunodetection of IgE reactivity of sera from patients allergic to Olea europaea. These membranes were incubated with a pool of sera of allergic patients Olea europaea lane l or preincubated with different amounts of protein per ml of serum: The plates sal haloidea se combinance glued 5 mg olive pollen extract per well.
Immunodetection of IgE reactivity from serum diluted 1: Since many of the recombinant DNA techniques employed in this invention are routinely used by skilled personnel in the field, it is better enter a short description of these more than describe each time used techniques.
Except where there is a specific indication, these protocols are described in reference Sambrook et al [ Modified protocol described in Sambrook et al, pages 1. A culture of E. Are added from 2 to 5 units of restriction endonuclease per ug DNA and the reaction is incubated for 1 to 3 hours at temperatures recommended by the manufacturer.
This technique is also described on pages 5. DNA fragments were stained using 0. DNA was visualized by ultraviolet illumination between and nra. Ligation of DNA fragments for ligation of DNA fragments with complementary cohesive ends, ng of each fragment are incubated in a reaction volume of 10 to The DNA was introduced by the protocol calcium chloride, as described in Sambrook, pages 1.
Studies on transformation of Escherichia coli tion With plasmids. Selection was performed on solid LB with ampicillin Sampling plasmids in E. This protocol this described on pages 1. Basically it followed the technique described by Laemmli [ 18 Laemmli, UK Cleavage of structural proteins During the assambly of the head of bacteriophage T4.
Gels, 10 x 10 cm and a polyacrylamide concentration of The proteins used as standards were the Bio-Rad kit for low sal haloidea se combinance weights.
Sal haloidea se combinance calculation of molecular weights and densitometric analysis of the gels was performed using an image analyzer Bio Image, Millipore Corp.
The kilodalton sal haloidea se combinance of Candida albicans is an alcohol-dehydrogenase: Molecular cloning and immunological analysis using mono- clonal antibodies.
The membrane is then incubated with the antibodies of interest for several sal haloidea se combinance these antibodies may come from patients or from immunized animals. This commercial anti-IgG antibody will, if we have used an immunized animal, or anti-IgE, if the serum is derived from human. The membrane is again washed in PBS, and stained with 4-chloro-l-naphthol 0. The reaction is stopped with distilled water. The purified RNA sal haloidea se combinance resuspended in ul diethylpyrocarbonate treated distilled water, and its concentration was determined at nm.
From mg of pollen they were obtained about 8. Synthesis of first strand complementary DNA. Profilin gene amplification by PCR. The primers used were based PCR sequence allergen Ole e 1 published previously [ 10 ]:. The primers consist of the hybridization site in boldseveral cleavage sites for the restriction endonucleases, EcoRI, HindIII and Sal haloidea se combinance underlinedand a nucleotide anchor. The PCR amplification reaction of the gene coding for the allergen Ole e 1 had the following components in a reaction volume of 50 ul: XLO amplification buffer, 5.