Characteristic peptide of peanut allergen protein and application thereof

A technology of characteristic peptides and allergens, applied in the field of food testing, can solve the problems of difficult antibody identification, affect test results, false positives, etc., and achieve the effect of eliminating trypsin inhibitor activity, improving enzymatic hydrolysis efficiency, and achieving good linearity.

Inactive Publication Date: 2015-10-21
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method also has the characteristics of high sensitivity and high specificity, but the detection results of this application method in heat-processed foods are usually lower than the true value, mainly because heat treatment can denature proteins, reduce protein solubility, and change protein surface sites , making it less likely to be correctly recognized by antibodies
In addition, the ELISA method using antigen-antibody technology will inevitably produce cross-reactions, resulting in false positives and affecting the final test results.

Method used

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  • Characteristic peptide of peanut allergen protein and application thereof
  • Characteristic peptide of peanut allergen protein and application thereof
  • Characteristic peptide of peanut allergen protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Screening and determination of embodiment 1 characteristic peptide

[0047] There are 13 known peanut allergenic proteins, and the two with the highest content are Ara h1 and Ara h2, accounting for about 12-16% and 5.9-9.3% of the total peanut protein. Compared with the above two proteins, the content of other allergenic proteins in peanuts is relatively small, and may not provide satisfactory sensitivity in samples containing low concentration levels of peanut proteins, so the present invention can be used in Ara h1 and Ara h2 Appropriate peptides were selected as markers without further investigation of the remaining allergenic proteins.

[0048] According to the PeptideMass function provided by Uniprot, the trypsin digestion results show that Ara h1 and Ara h2 can theoretically form 89 and 23 digestion products, respectively. According to the UPLC-Q-ToF detection method provided above, 22 enzymatic polypeptides from Ara h1 and 11 from Ara h2 could be detected, and t...

Embodiment 2

[0068] Example 2 Sample pretreatment (optimization of enzyme digestion conditions)

[0069] Studies have shown that Ara h2 has a typical trypsin inhibitor structure. Destroying the spatial structure of the protein by hydrolyzing the disulfide bond can reduce its trypsin inhibitor activity and improve the efficiency of enzymatic hydrolysis. In this embodiment, the degree of protein disulfide bond destruction is increased by increasing the concentration of DTT and optimizing the heating process.

[0070] Prepare DTT with a concentration of 50, 100, 200, 300, 400, 500, 600, 700, 800 and 900mmol / L. In order to fully react the excess DTT and free disulfide bonds, the concentration of the IAA solution is consistent with DTT. And the amount added is three times that of DTT. According to the sample pretreatment method described above, the influence of different DTT concentrations on the digestion efficiency was obtained, and the peak areas of DLAFPGSGEQVEK and NLPQQCGLR were as foll...

Embodiment 3

[0072] Example 3 Detection of Allergen Content in Peanuts of Different Varieties

[0073] In the history of peanut planting for more than a thousand years, peanuts have undergone variety improvement, and a large number of peanut varieties have been derived. There are more than 500 varieties of peanuts that can be found only in China. So protein composition may vary between different peanut varieties. In this paper, with the help of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, the 20 most common types of peanuts in China were collected, and the above samples were detected according to the sample pretreatment method, UPLC-TQ-MS analysis method and Kjeldahl method as described above Ara h1, Ara h2 and total protein in peanuts, calculate the percentage of Ara h1 and Ara h2 in total protein in different kinds of peanuts, the results are shown in Table 5.

[0074] Table 5: Detection results of Ara h1 and Ara h2 contents of different varieties of...

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Abstract

The invention discloses characteristic peptide of peanut allergen protein and application thereof. The amino acid sequence of the characteristic peptide is DLAFPGSGEQVEK or NLPQQCGLR. Allergens which are the highest in content in peanuts, namely Arah1 and Arah2, are selected as candidate markers, the characteristic peptide of Arah1 and the characteristic peptide of Arh2 are screened out, corresponding internal standard substances are designed and synthesized, the analysis technology in which the high performance liquid chromatography and mass spectrum are used in a combined mode is adopted, and the content of the peanut allergen protein in food is calculated and obtained. A method of the application has good linear property and high sensitivity, recovery rate and precision degree.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a characteristic peptide of peanut allergen protein and its application. Background technique [0002] Peanut is also a common allergen food, and the allergic reaction caused by it accounts for 1% to 20% of food allergies. Currently, there are 13 recognized peanut allergens. Among them, Ara h1 and Ara h2 are the main peanut allergens, accounting for 12-16% and 5.9-9.3% of the total peanut protein respectively, and can be recognized by more than 90% of the serum of peanut allergic patients. In the natural state, Ara h1 is mainly a water-soluble protein that exists in the form of monomers and trimers, and contains 23 linear IgE binding sites. Ara h2 exists in two isoforms and contains 10 linear IgE binding sites. Ara h2 has high stability and resistance to enzymolysis, mainly because Ara h2 contains a large number of disulfide bonds to keep its structure stable. Ara h2 h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K7/06C07K14/415G01N30/02G01N30/72
Inventor 任一平张京顺洪宇伟陈启陈苘
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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