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Flavanone-3-hydroxylase antigen epitope peptide, and antibody and application thereof

A technology of antigenic epitopes and flavanones, applied in the direction of oxidoreductases, antibody mimics/scaffolds, peptides, etc., can solve problems affecting antibody specificity, complex technical process, antibody cross-reaction, etc., to achieve strong specificity, Simple operation and high accuracy effect

Active Publication Date: 2020-09-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The identification equipment is expensive and the identification technology process is complex and cumbersome. 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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  • Flavanone-3-hydroxylase antigen epitope peptide, and antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Excavation of Candidate Flavanone-3-Hydroxylase F3H Epitope and Synthesis of Polypeptide Antigen

[0024] 1. Taking the special rice (Oryza sative L) "Luotian Red Rice" (RM1 for short) as the research object, its flavanone-3-hydroxylase F3H gene F3H-2 (Os10g0536400), the nucleotide sequence is as SEQ ID NO : 3, the encoded amino acid full-length sequence is shown in SEQ ID NO: 4. Analyzing the hydrophilicity, exposure and flexibility of the amino acid sequence, an amino acid sequence CRTHGFFQVVNHGI was selected at the N-terminus of the protein, as shown in SEQ ID NO: 1, which is located at No. 64 in SEQ ID NO: 4 -The amino acid sequence at position 77, after comparing the database by BLAST, it is determined that this polypeptide can represent the epitope of plant flavanone-3-hydroxylase (rice Os-F3H).

[0025] 2. According to the prediction results of the epitope of the above sequence analysis, a cysteine ​​residue (Cysteine ​​residue, Cys) is added to the C-...

Embodiment 2

[0026] Example 2 Preparation of flavanone-3-hydroxylase specific antibody anti-F3H

[0027] The healthy New Zealand white rabbits with an age of about 3 months and a body weight of more than 2 kg were selected as the immunized animals. Rabbits were induced before immunization, and 0.5 mL of Freund's complete adjuvant was subcutaneously injected into the limbs, armpit and back to stimulate the local immune response. The first immunization was carried out 7 days later, and blood was taken from the ear vein of the rabbit before the first immunization as a negative control. The first immunization reagent is the above antigen with PBS (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4 , pH7.4) diluted, take 500μL of 1mg·mL -1 Antigen (ie 0.5 mg), then add 1400 μL of complete Freund's adjuvant (first immunization) or 700 μL of Freund's incomplete adjuvant (second to fourth immunization), and mix and emulsify on a vortex. Rabbits were injected subcutaneously at multiple ...

Embodiment 3

[0030] Example 3 Application of flavanone-3-hydroxylase specific antibody anti-F3H

[0031] The total grain protein of red rice RM1 and white rice 9311 was extracted respectively. Weigh 0.2 g of the grain sample into a 2 mL sterilized centrifuge tube, add 800 μL of pre-cooled extraction buffer [62.5 mM tris-Hcl (pH 7.4), 10% (v / v) glycerol, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 5% (v / v) β-mercaptoethanol], mixed and vortexed and placed on ice for 30 min. The sample was centrifuged at 12000rpm / min at 4°C for 20min, and the supernatant was taken and stored in a -80°C refrigerator for later use.

[0032] When the antibody anti-F3H detects the content of the key enzyme F3H, Western blot can be used. The protein extract was separated by SDS-PAGE electrophoresis and quickly transferred to a nitrocellulose membrane by a tank transfer system. The specific antibody anti-F3H prepared in Example 2 was used, diluted with blocking solution containing 0.05% Tween-20 at a ratio of 1:2000, and t...

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Abstract

The invention discloses a flavanone-3-hydroxylase antigen epitope peptide, a fusion protein and a specific antibody of the flavanone-3-hydroxylase antigen epitope peptide, and a preparation method andapplication of the antibody. The amino acid sequence of the flavanone-3-hydroxylase antigen epitope peptide is as shown as SEQ ID NO:1. After being modified, the antigen epitope peptide is subjectedto solid-phase synthesis and is coupled with a carrier protein to obtain the fusion protein used as an artificially synthetic antigen, and the amino acid sequence is as shown as SEQ ID NO:2. The specific antibody of the invention is obtained by immunizing animals by the artificially synthetic antigen and performing separation and purification. The prepared polyclonal antibody has the advantages ofhigh affinity, high specificity, high sensitivity, operation simplicity and convenience, and can perform specific binding reaction with rice flavanone-3-hydroxylase; in addition, the preparation costis low; the yield is high; and an important tool is provided for the relevant physiological function study and identification of the rice flavanone 3-hydroxylase.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a flavanone-3-hydroxylase antigen epitope peptide, a fusion protein, a flavanone-3-hydroxylase specific antibody, a preparation method and application thereof. Background technique [0002] Flavonoids in plants have attracted the attention of scientists because of their medicinal value. Anthocyanins are water-soluble flavonoids, which belong to secondary metabolites of flavonoids, and have different contents in various organs and tissues of plants. It plays a key role in the process of flower color formation and fruit coloration. A large number of studies have confirmed that flavonoids have various medicinal values ​​such as anti-oxidation, free radical scavenging, cardiovascular and cerebrovascular protection, anti-tumor and anti-inflammation. The synthesis of anthocyanins is a multi-enzyme pathway, in which flavanone 3-hydroxylase (F3H) is one of the key enzymes i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C07K19/00C07K16/40G01N33/573
CPCC07K16/40C07K2319/00C12N9/0071C12Y114/11009G01N33/573
Inventor 何莹孙玉珺罗雅汪倩曾汉来
Owner HUAZHONG AGRI UNIV
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