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Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies

An allergen-like, allergen-like technology, applied in the directions of allergen antigen components, gene therapy, fusion polypeptides, etc.

Inactive Publication Date: 2011-02-16
BIAL IND FARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It thus appears that the currently known allergen extracts have significant disadvantages in achieving an optimal treatment of mite allergy

Method used

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  • Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies
  • Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies
  • Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Example 1: Purification of natural allergens Der p 1 and Der p 2 from the body of mites

[0195] A mixture of lyophilized body and feces of D. dust mites (Laboratorios Leti, Madrid, Spain) was used as starting material in 10 volumes (p / v) supplemented with 1 mm PMSF (phenylmethanesulfonyl fluoride) in PBS (phosphate buffered). brine) was extracted with rapid stirring for 15 minutes at 4°C. It was then centrifuged at 3,800xg for 15 minutes at 4°C. The extraction supernatant was filtered through AP (Millipore) and 60% ammonium sulfate 361 g / l) was added slowly for 30 min. After stirring for 1 hour at 4°C, it was centrifuged at 17,000 xg for 15 minutes at 4°C.

[0196] · Purification of native Der p 1

[0197]The pellet obtained after centrifugation was resuspended in 2 ml of 20 mM Tris pH 8.0 and filtered through 0.22 μm. Molecular sieve chromatography was performed on a Superdex S20016 / 60 column (GE-Healthcare, Uppsala, Sweden) equilibrated with PBS and injected wi...

Embodiment 2

[0203] Example 2: Cloning of Der p 1 and Der p 2 allergens

[0204] Complementary DNA (cDNA) encoding the allergens Der p 1 and Der p 2, in each case cloned by reverse transcription using mRNA isolated from dust mites as template and specific primers, followed by PCR amplification . mRNA was isolated from 100 mg of D. dust mite bodies (Laboratiorios Leti, Madrid, Spain) using the Quick Prep MicroRNA Purification Kit (GE-Healthcare). cDNA was obtained by reverse transcription of mRNA using the First Strand cDNA Synthesis Kit (GE-Healthcare).

[0205] Primers consist of hybridization regions, various cleavage sites for different restriction enzymes (underlined below) and anchor nucleotides. The PCR amplification reaction had the following components in a reaction volume of 50 μl: Amplification buffer x 10, 5 μl; 200 μm dNTPs; 100 μm various oligonucleotide primers; 2.5 units of Taq polymerase (PfxDNA polymerase, Invitrogen); 1 ng DNA Template and make up to 50 μl of sterile...

Embodiment 3

[0212] Example 3: Expression and purification of recombinant Der p 2

[0213] The corresponding plasmids were transformed by the Hanahan method [(21) Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580] E. coli BL21(DE3) cells were plated on Petri plates containing LB medium supplemented with 200 μg / ml ampicillin. 50 ml of the same medium were pre-seeded from the cell colonies and incubated overnight at 37°C with stirring (260 rpm). The pre-inoculum was used to inoculate 1 liter of the same medium, starting from an optical density of 0.2 (600 nm). It was incubated at 37°C with stirring until an optical density (600 nm) of 0.6 was reached (approximately 90 minutes), at which point induction was performed with isopropyl-thio-β-galactoside (IPTG) at a final concentration of 0.6 mM . After an induction period of 3 hours, cells were collected by centrifugation.

[0214]Cells were centrifuged at 10000 rpm for 15 minutes a...

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Abstract

The present invention refers to recombinant ADN molecules coding to hybrids polypeptides of different allergens from D. pteronyssinus useful for the prevention and treatment of allergies, particularly allergies caused by mites. Specifically, the invention describes hybrid proteins composed of fragments of allergens Derp p l y Derp p 2 with hypoallergenic characteristics and maintain their immunogenic capacity, being particularly useful for the treatment of allergy. The invention also describes the production methods of these polypeptides in heterologous expression systems. Besides, the invention describes efficient purification methods of these hybrid proteins.

Description

Field of Invention [0001] The present invention relates to the production of products for the prevention and treatment of allergies, particularly those caused by house dust mites, and more particularly those caused by mites of the genus Dermatophagoides and more particularly due to resistance to Type 1 and A domain of hybrid proteins of allergy induced by sensitization to class 2 allergens. Background technique [0002] Allergy is a specific inherited or acquired disorder of the ability to react to foreign bodies, which are often harmful (allergens). Allergies are associated with inflammatory reactions in the affected organs (skin, conjunctiva, nose, pharynx, bronchial mucosa, gastrointestinal tract). Immediate symptoms of the disease include rhinitis, conjunctivitis, dermatitis, asthma, and anaphylactic shock; while chronic manifestations of the disease include delayed-type asthma and atopic dermatitis. Type I allergy is an important health problem in developed countries....

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K39/35
CPCC07K14/43531A61K39/35C12N15/70A61K39/00A61P37/00A61P37/08A61K48/00C07K2319/00
Inventor 胡安安德烈斯·阿斯图里亚斯奥特加伊纳吉·伊巴罗拉洛佩兹德达瓦里洛玛利亚卡门·阿瑞拉罗德里格斯阿尔贝托·马丁尼兹葛雷特
Owner BIAL IND FARM
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