Hypoallergenic hybrid proteins of major class 1 and class 2 mite allergens for the treatment of allergy

A technology of host cells and sequences, applied in the directions of allergen antigen components, gene therapy, fusion polypeptides, etc.

Inactive Publication Date: 2016-10-19
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 class 1 and class 2 mite allergens for the treatment of allergy
  • Hypoallergenic hybrid proteins of major class 1 and class 2 mite allergens for the treatment of allergy
  • Hypoallergenic hybrid proteins of major class 1 and class 2 mite allergens for the treatment of allergy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Example 1: Purification of natural allergens Der p 1 and Der p 2 from mite bodies

[0195] Using a mixture of freeze-dried body and feces of D. dust mite (Laboratorios Leti, Madrid, Spain) as starting material, 10 volumes (p / v) of PBS (phosphate-buffered saline) supplemented with 1 mm PMSF (phenylmethylsulfonyl fluoride) brine) at 4°C for 15 minutes with rapid agitation. Then centrifuge at 3,800 xg 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 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 with 2 ml of the sample from t...

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, were cloned by reverse transcription using mRNA isolated from dust mite as template and specific primers, followed by amplification using PCR . mRNA was isolated from 100 mg of D. dust mite body (Laboratiorios Leti, Madrid, Spain) using the Quick Prep MicroRNA Purification Kit (GE-Healthcare). cDNA was obtained by reverse transcription of mRNA using First Strand cDNA Synthesis Kit (GE-Healthcare).

[0205] Primers consist of a hybridizing region, various cleavage sites (underlined below) for different restriction enzymes, 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 of various oligonucleotide primers; 2.5 units Taq polymerase (Pfx DNA polymerase, Invitrogen); 1 ng DNA template and make up to 50 μl of s...

Embodiment 3

[0212] Example 3: Expression and Purification of Recombinant Der p 2

[0213] Transformed with the corresponding plasmid by the Hanahan method [(21) Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids (research on transforming E. coli with plasmids). Escherichia 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-inoculated from cell colonies and incubated overnight at 37°C with agitation (260 rpm). Inoculate 1 liter of the same medium with the pre-inoculum, starting from an optical density (600 nm) of 0.2. It was incubated at 37°C with agitation until an optical density (600 nm) of 0.6 was reached (approximately 90 minutes), at which point induction was performed using isopropyl-thio-β-galactoside (IPTG) at a final concentration of 0.6 mM . After a 3 hour induction period, cells were collected by centrifugation.

[0214] Cells were centrifuged at 10000...

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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 p1 y Derp p2 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 manufacture of products for the prophylaxis and treatment of allergies, in particular allergies caused by house dust mites, and more particularly by mites of the genus Dermatophagoides and more particularly due to the sensitivity to class 1 and Class 2 allergen sensitization caused by the field of hybrid protein of allergic reaction. Background technique [0002] Allergy is a specific hereditary or acquired disorder of the ability to react to a foreign body, usually harmful (allergen). Allergies are associated with inflammatory reactions in affected organs (skin, conjunctiva, nose, pharynx, bronchial mucosa, gastrointestinal tract). Immediate symptoms of the disease include rhinitis, conjunctivitis, dermatitis, asthma, and anaphylactic shock; whereas chronic manifestations of the disease include delayed reactions of asthma and atopic dermatitis. Type I allergy is an important health problem in developed countri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435A61K39/35
CPCA61K39/35C07K14/43531C12N15/70A61P37/00A61P37/08A61K39/00A61K48/00C07K2319/00
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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