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Antigen epitope peptide of beta-lactoglobulin, complete antigen, antibody and method for determining residual quantity of beta-lactoglobulin

A technology of lactoglobulin and complete antigen, which is applied in the field of biotechnology and food, can solve the problems such as the difficulty of residual detection of β-lactoglobulin antigen, and achieve the effect of solving the problem of sensitization, accurate detection and high specificity

Active Publication Date: 2021-10-26
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In order to solve the problem in the prior art that the residual detection of β-lactoglobulin antigen in the hydrolyzed milk is difficult, the present invention provides a kind of β-lactoglobulin Protein antigenic epitope peptide, complete antigen, antibody and method for determining residual amount of β-lactoglobulin

Method used

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  • Antigen epitope peptide of beta-lactoglobulin, complete antigen, antibody and method for determining residual quantity of beta-lactoglobulin
  • Antigen epitope peptide of beta-lactoglobulin, complete antigen, antibody and method for determining residual quantity of beta-lactoglobulin
  • Antigen epitope peptide of beta-lactoglobulin, complete antigen, antibody and method for determining residual quantity of beta-lactoglobulin

Examples

Experimental program
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Effect test

Embodiment 1

[0064] This embodiment provides a hapten of β-lactoglobulin B cell action epitope AA12-37, and the amino acid sequence of the action epitope peptide is CGAQALIVTQTMKGLDIQKVAGTWYS.

[0065] In the second aspect, this embodiment provides a method for synthesizing the above-mentioned epitope peptide. Specifically, the solid-phase synthesis method is used to link the C-terminal amino acid and Wang resin, and condensation is carried out step by step. After synthesis, the sequence is cut off from the solid phase carrier with strong acid, and the synthesized epitope polypeptide is purified by high performance liquid chromatography, and freeze-dried for subsequent use.

[0066] like figure 1 As shown, the synthesized epitope polypeptide is purified by high performance liquid chromatography, and the purity reaches 92.750%. The relative molecular mass of the epitope peptide was determined by mass spectrometry to obtain figure 2 , the MS spectrum of the sequence CGAQALIVTQTMKGLDIQKVAG...

Embodiment 2

[0068] This example provides a method for synthesizing the complete antigen. Specifically, the purified epitope peptide in Example 1 is coupled with bovine serum albumin (BSA) using the glutaraldehyde method to finally obtain the complete antigen. Bovine serum albumin has stable physical and chemical properties, is not easy to change, has good immune activity, is cheap and easy to obtain, and contains many free amino groups, and has good solubility in different pH values, ionic strengths and organic solvents.

[0069] To identify the complete antigen, specifically, the synthetic epitope BSA and the complete antigen were scanned at the full wavelength of 190-1100nm respectively, and the carrier protein BSA and the synthetic polypeptide had maximum absorption peaks at 210nm and 200nm respectively. The complete antigen has the characteristic absorption peaks of the two at 190-1100nm and there is a certain spectral superposition phenomenon. Therefore, it can be judged that the pre...

Embodiment 3

[0071] This example provides a monoclonal antibody.

[0072] In this example, the complete antigen prepared in Example 2 was used to immunize BALB / c mice. After 4 times of immunization, cell fusion, cell selection, and subcloning were performed several times, and these cells with high activity were injected into BALB / c mice. Intraperitoneal culture of mice, followed by ascitic fluid collection, monoclonal antibody was obtained and purified.

[0073] After obtaining the above monoclonal antibody, in this embodiment, the titer of the monoclonal antibody was determined by an indirect enzyme-linked immunosorbent immunoassay (ELISA) method.

[0074] The antigen coating concentration is 5μg / mL, and the monoclonal antibody is 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000 multiple dilutions.

[0075] The specific determination method of monoclonal antibody potency is as follows:

[0076] ① Antigen coating: Add the diluted antigen to the microti...

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Abstract

The invention relates to an antigen epitope peptide of beta-lactoglobulin, a complete antigen, an antibody and a method for determining residual quantity of beta-lactoglobulin. The amino acid sequence of the antigen epitope peptide is CGAQALIVTQTMKGLDIQKVAGTWYS. The method for determining the residual quantity of the beta-lactoglobulin comprises the following steps: S1, synthesizing a beta-lactoglobulin acting epitope peptide; S2, preparing the beta-lactoglobulin acting epitope peptide into a complete antigen; S3, preparing a monoclonal antibody or polyclonal antibody; and S4, taking the monoclonal antibody or polyclonal antibody in the step S3 as a primary antibody, and detecting the residual quantity of the beta-lactoglobulin in the hydrolysate of the beta-lactoglobulin hydrolyzed by different proteases by adopting an indirect competitive ELISA method. According to the invention, the beta-lactoglobulin is determined by the indirect competitive ELISA method; AA12-37 antigen epitope peptide of the beta-lactoglobulin is used as an immunogen; and the immunogen is used for preparing an antibody required by determination. The prepared antibody has extremely high specificity on peptide fragments containing action epitopes remained in the beta-lactoglobulin hydrolysate; and the detection is more accurate.

Description

technical field [0001] The invention belongs to the fields of biotechnology and food technology, and specifically relates to an antigenic epitope peptide of β-lactoglobulin, a complete antigen, an antibody and a method for determining the residual amount of β-lactoglobulin. Background technique [0002] Cow's milk is a very high-quality protein source, and it is also one of the common allergens. Its sensitization has a serious impact on the health of infants and young children. Milk protein allergy is usually more severe in infancy, which can cause reactions including skin, gastrointestinal tract, respiratory tract, etc., and even more serious systemic allergic reactions or shock. [0003] Milk is rich in protein, with about 30-35 grams of protein per liter of milk, and more than 25 different types of protein. Theoretically, any protein has potential allergens. At present, it is generally believed that casein, β-lactoglobulin and α-lactalbumin are the main allergens in milk...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K16/18G01N33/68G01N33/577
CPCC07K14/4717C07K16/18G01N33/68G01N33/577G01N2333/4713
Inventor 丛艳君刘迪
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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