Antigenic epitope of rice grain glutelin GluA2 subunit, antibody thereof and application

A technology for rice grains and antigenic epitopes, applied in the fields of molecular biology and immunology, can solve the problems of affecting antibody specificity, complexity and cumbersomeness, and achieve the effects of high specificity and sensitivity and simple preparation method.

Active Publication Date: 2019-04-16
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional separation and identification techniques are complicated and cumbersome, and in the process of polyclonal antibody preparation, due to the existence of multiple epitopes, some foreign proteins will inevitably be mixed in the expression and purification of recombinant proteins, and the prepared antibodies are still There may be cross-reactivity, thereby affecting the specificity of the antibody

Method used

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  • Antigenic epitope of rice grain glutelin GluA2 subunit, antibody thereof and application
  • Antigenic epitope of rice grain glutelin GluA2 subunit, antibody thereof and application
  • Antigenic epitope of rice grain glutelin GluA2 subunit, antibody thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Prediction of Candidate Epitopes

[0014] The gene ID of GluA2, the corresponding member of the rice grain Glu family, in GenBank is: Y00687.1, and the nucleotide sequence of the coding region is shown in SEQ ID NO:3.

[0015] The encoded full-length amino acid sequence of GluA2 is shown in SEQ ID NO:4.

[0016] The amino acid sequence thereof was subjected to sequence analysis by searching the protein database (UniProtKB; http: / / www.uniprot.org / ). Using the multiple sequence alignment tool (ClustalW2; http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ) to analyze the variable region, conserved region, hydrophilicity, and exposure of the amino acid sequences of these glutenin family members and flexibility etc. Use online analysis software (PREDICTED ANTIGENIC PEPTIDES; http: / / bio.dfci.harvard.edu / Tools / antigenic.html / ) to predict the antigenic region of the glutenin subunit, and determine that the polypeptide can uniquely recognize the corresponding glutenin subuni...

Embodiment 2

[0017] Example 2: Synthesis of a dedicated antigen for the preparation of GluA2 antibodies

[0018] According to the prediction results of the antigenic determinant of the above sequence analysis, a cysteine ​​residue (Cysteine ​​residue, Cys) is added to the C-terminus of the Glu subunit-specific antigenic determinant polypeptide sequence to carry out the synthesis of the antigenic sequence TSQWQSSRRGSPRGC (such as SEQ ID NO: 2), and coupled with a macromolecule carrier - Keyhole limpet hemocyanin (KLH) via SMCC to form the dedicated antigen of GluA2 subunit antibody, freeze-dried and stored at -80°C.

Embodiment 3

[0019] Embodiment 3: Preparation of polyclonal antibody serum

[0020] Animals to be immunized Healthy New Zealand white rabbits aged about three months and weighing more than 2 kg were selected, and the animals were immunized with the antigen prepared in Example 2. The specific steps are as follows: first, each rabbit was induced before immunization, and 0.5 mL of Freund's complete adjuvant was subcutaneously injected into the limbs, underarms and back to stimulate the local immune response, and the first immunization was performed one week later. Before the first immunization, blood was taken from the ear vein as a negative control. The antigen was washed with PBS (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 ,2mMKH 2 PO 4 ,pH 7.4) diluted to 1mg·mL -1 , stored at -20°C after aliquoting. Take 500μL of 1mg·mL -1 The antigen (0.5 mg) was diluted again with 300 μL PBS, and then an equal volume of Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant...

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Abstract

The invention discloses an antigenic epitope of a rice grain glutelin GluA2 subunit, an anti-rice grain glutelin GluA2 subunit polyclonal antibody and a preparation method and application thereof. Theantigenic epitope of the rice grain glutelin GluA2 subunit has the amino acid sequence shown as SEQ ID NO.1. antiserum is prepared through polypeptide synthesis, polypeptide and carrier protein coupling and animal immunizing, and a glutelin GluA2 subunit polyclonal antibody is obtained through affinity chromatography and purification. The prepared polyclonal antibody has high titer, strong affinity and good specificity and can produce specific binding reaction with the rice glutelin GluA2 subunit, and an important tool is provided for the basic research of gluten, such as analysis in specificity to its important members, expression profile and content and the study of its related physiological functions.

Description

technical field [0001] The invention belongs to the fields of molecular biology and immunology, and in particular relates to an antigenic epitope, a specific antibody, and a preparation method and application of rice grain glutenin family GluA2 subunit. Background technique [0002] According to the classic Osborne protein fractionation extraction theory, rice seed storage protein (SSP) can be divided into four categories: glutelin (Glu), gliadin, globulin and water-soluble albumin. Among them, glutenin is the main component of rice SSPs, accounting for about 80% of the total content of SSPs in the grain. The amino acid composition of SSPs in most cereals is deficient in lysine, so it is generally believed that lysine is the first limiting amino acid on the nutritional level of cereal SSPs. Rice grain protein is recognized as a high-quality plant protein, but there are relatively few studies on the structure and properties of rice grain protein. Therefore, the composition ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K16/16G01N33/68
CPCC07K14/415C07K16/16G01N33/68G01N2333/415
Inventor 朱岚何莹曾汉来罗雅
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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