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Method for identifying allergenic proteins and peptides

An allergenic, protein technology, applied in the field of identification of allergenic proteins and peptides, which can solve problems such as lower-level structure homology and functional properties

Inactive Publication Date: 2011-09-14
MJN U S HLDG LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The four casein molecules have very low primary structure homology and different functional properties, but they are all phosphorylated proteins with rheomorphic and highly hydrated quaternary structures that are easily degraded by some proteases

Method used

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  • Method for identifying allergenic proteins and peptides
  • Method for identifying allergenic proteins and peptides
  • Method for identifying allergenic proteins and peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Identification of cDNA sequences encoding IgE-reactive milk components and fragments thereof

[0085] In order to identify IgE-reactive proteins and IgE-reactive protein fragments contained in milk, an expression cDNA library was first constructed using mammary gland tissue of lactating cows. The library was screened with sera from cow's milk allergic patients. cDNAs encoding IgE-reactive full-length αS1-, αS2-, β-, κ-casein and β-lactoglobulin and their IgE-reactive fragments were identified. The results were rather surprising, mammalian proteins produced in bacterial systems did not add eukaryotic post-translational modifications that sometimes play a role in IgE binding, as reported in major house dust mite allergens (Jacquet et al. , 2002). The deduced amino acid sequence is shown in Figure 1A -C (SEQ ID No. 22-84). From the resulting IgE reactive allergen fragments conclusions can be drawn about the position of the IgE epitopes.

[0086] Experimenta...

Embodiment 2

[0089] Example 2: Differences in allergenic activity and IgE reactivity of milk components

[0090] In order to assess whether a molecule has allergenic potential, not only its IgE reactivity needs to be demonstrated, but also its biological activity. A scientifically accepted model for examining molecular biological activity is the induction of histamine release from human basophils (Purohit 2005). For this, a rat basophilic leukemia cell line expressing the α-chain of the human FcεRI receptor was used (Hoffmann et al., Int Arch Allergy Immunol, Vol 126(4), p. 277-285). Different milk components including whole milk extracts of different species, milk fractions, purified native and recombinant milk proteins and recombinant protein fragments were examined (n=33). The percentage of serum IgE antibodies triggering the release of β-hexosaminidase from humanized RBL cells in response to each milk component in 78 patients was determined ( image 3 ).

[0091] The most effective ...

Embodiment 3

[0102] Example 3: Identification of proteins and / or peptides with and without allergenic activity in milk samples and milk-derived products by mass spectrometry

[0103] The sequence database of allergenic milk proteins and peptides established by the combination of IgE reactivity and allergenic activity can be used as a basis for establishing reliable and reliable methods for assessing and predicting the allergenicity of milk samples and milk-derived products. Reproducible analysis methods. Detection of potentially allergenic components in compound milk samples using mass spectrometry. As an example, a commercially available highly hydrolyzed hypoallergenic milk formulation was analyzed using a combination of mass spectrometry and upstream high performance liquid chromatography (HPLC). Figure 5 Chromatographic analysis is shown, and extracted mass spectra demonstrate that all proteins have been hydrolyzed into small peptides with a maximum length of 14 amino acids.

[0104...

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Abstract

The present invention relates to a method for identifying allergenic proteins and peptides. More specifically, the present invention provides a method for identifying allergenic milk proteins and / or peptides comprising the steps of: providing at least one expression library comprising DNA or cDNA derived from the mammary gland tissue of a lactating mammal, preferably a lactating cow, expressing at least one protein or peptide encoded by said expression library, determining the binding capacity of said at least one protein or peptide to IgE of at least one serum of an individual who is sensitive to mammalian milk,preferably cow's milk, contacting the at least one protein or peptide exhibiting an IgE binding with basophil cells, eosinophil cells or mast cells and identifying the at least one protein or peptide as being allergenic when said basophil cells, eosinophil cells or mast cells release upon contact with at least one protein or peptide at least one mediator.

Description

technical field [0001] The present invention relates to methods of identifying allergenic proteins and peptides. More specifically, the present invention provides a method for identifying allergenic milk proteins and / or peptides, comprising the steps of: providing at least one expression library comprising DNA or cDNA from mammary gland tissue of a lactating mammal, preferably a lactating cow , expressing at least one protein or peptide encoded by the expression library, and determining the binding ability of the at least one protein or peptide to the IgE of at least one serum of an individual sensitive to mammalian milk, preferably milk, will be carried out as in step c ) wherein at least one protein or peptide exhibiting IgE binding capacity is contacted with basophils, eosinophils or mast cells, and when said basophils, eosinophils or mast cells are contacted with the step At least one protein or peptide in d) is identified as allergenic when at least one protein or peptid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68A61K38/08
CPCG01N33/6854G01N2500/00G01N2800/24A61P37/04A61P37/06G01N33/68
Inventor R·瓦伦塔U·赫尔斯M·富克-特克尔H·霍赫瓦尔纳U·舒尔梅斯特I·斯沃博达E·A·F·范托尔
Owner MJN U S HLDG LLC
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