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Anthocyanin synthase epitope peptide and its antibody and application

An anthocyanin synthase and antigenic epitope technology, applied in hybrid peptides, fusion polypeptides, antibody mimics/scaffolds, etc., can solve problems affecting antibody specificity, complex technical process, antibody cross-reaction, etc., to achieve specificity Strong, simple operation, high accuracy effect

Active Publication Date: 2021-03-23
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

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  • Anthocyanin synthase epitope peptide and its antibody and application

Examples

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Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Excavation of Candidate Anthocyanin Synthase ANS Epitope and Synthesis of Polypeptide Antigen

[0022] 1. Taking purple potato (Ipomoea batatas (L) Lam.) as the research object, the gene ID of its anthocyanin synthase Lb-ANS is: FJ478179, the nucleotide sequence is shown in SEQ ID NO: 4, and the encoded amino acid The full-length sequence is shown in SEQ ID NO:3. Analyzing the hydrophilicity, exposure and flexibility of the amino acid sequence, an amino acid sequence AGNVQGYGSKLANASG was selected at the N-terminus of the protein, as shown in SEQ ID NO: 1, which is located at the 123rd- The amino acid sequence at position 139 is compared with the database by BLAST, and it is confirmed that this polypeptide can represent the epitope of plant anthocyanin synthase ANS (sweet potato Lb-ANS and rice Os-ANS).

[0023] 2. According to the prediction result of the antigenic determinant of the above sequence analysis, a cysteine ​​residue (Cysteine ​​residue, Cys) is a...

Embodiment 2

[0024] Example 2 Preparation of anthocyanin synthase-specific antibody anti-ANS

[0025] 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 poi...

Embodiment 3

[0028] Example 3 Application of anthocyanin synthase-specific antibody anti-ANS

[0029] The total protein of special crops such as rice grains, purple potato tubers, rice leaves and purple sweet potato leaves were extracted respectively. Taking purple sweet potato as an example, take the purple sweet potato tissue material in a pre-cooled porcelain mortar, and grind it into powder with a mortar under liquid nitrogen. Take 0.5g powder and 1mL lysate (containing 7.0M urea, 4% CHAPS, 2.0M thiourea, 65mM DTT) and mix well, centrifuge at 16000×g for 1h, and take the supernatant as protein extract.

[0030]When the antibody anti-ANS is used to detect the content of the key enzyme ANS, the western blot method can be used. The protein extract was separated by SDS-PAGE electrophoresis and quickly transferred to a nitrocellulose membrane by a tank transfer system. Using the specific antibody anti-ANS prepared in Example 2, after diluting with blocking solution containing 0.05% Tween-...

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Abstract

The invention provides an anthocyanin synthase epitope peptide, a fusion protein and a specific antibody thereof, and a preparation method and application of the antibody. The amino acid sequence of the anthocyanin synthase epitope peptide is shown as SEQ ID NO: 1. The invention discloses the ANS (Anthocyanidin Synthase) epitope peptide. The antigen epitope peptide is modified, subjected to solid-phase synthesis and coupled with a carrier protein to obtain a fusion protein as an artificially synthesized antigen, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 2. The artificially synthesized antigen immunes an animal, and the anthocyanin synthetase specific antibody is obtained through separation and purification. The antibody has the advantages of strong specificity, high sensitivity, high accuracy, simplicity and convenient operation, intuitive result, low cost and the like. The preparation method is simple to operate and has feasibility of industrial production.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an anthocyanin synthase antigenic epitope peptide, a fusion protein, an anthocyanin synthase specific antibody, a preparation method and application thereof. Background technique [0002] Because of its high anthocyanin characteristics, special colored crops are rich in nutrition and have health care functions, which just meet the requirements of "green food" pursued by the public. Therefore, the study of key enzymes in the phytopigment synthesis pathway can provide basic physiological and molecular research support for the mechanism of phytopigment accumulation, and lay the foundation for the development of germplasm resources of high-anthocyanin-type crops. [0003] Structural genes for enzymes involved in the flavonoid pathway are required for phytochrome biosynthesis. Among them, anthocyanin synthase (ANS) is the key enzyme in the process from "colorless to colo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C07K19/00C07K16/40C07K16/06G01N33/573
CPCC07K14/795C07K16/06C07K16/40C07K2317/33C07K2317/92C07K2319/00C12N9/0071C12Y114/11019G01N33/573G01N2333/90245
Inventor 何莹曾汉来汪倩罗雅熊信果
Owner HUAZHONG AGRI UNIV
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