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Diethylstilbestrol mimic antigenic epitope peptide, and preparation method and application thereof

An antigenic epitope, diethylstilbestrol technology, applied in the direction of peptides containing affinity tags, peptides containing MBP tags, peptides, etc., can solve the adaptive matching of the immunological properties of epitope peptides, difficult to obtain detection effects, and inconsistent preparation methods. To clarify and other issues, to achieve the effect of high sensitivity, highlighting application value, and reducing harm to human health

Active Publication Date: 2018-08-03
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to address the technical defects of the prior art and provide an epitope peptide that mimics diethylstilbestrol to solve the lack of a DES in the prior art that has similar immune response characteristics and can replace it as a sensitive immune detection element. technical problem
[0005] The present invention also provides a method for preparing the above-mentioned antigenic epitope peptide simulating diethylstilbestrol, in order to solve the technical problem that its preparation method is not clear
[0006] The present invention also provides the application of the above-mentioned epitope peptide simulating diethylstilbestrol in a specific immunoassay method, so as to solve the problem that conventional detection methods in the prior art do not perform adaptive matching on the immunological properties of the epitope peptide, thus making it difficult to Technical Issues for Optimum Detection Results

Method used

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  • Diethylstilbestrol mimic antigenic epitope peptide, and preparation method and application thereof
  • Diethylstilbestrol mimic antigenic epitope peptide, and preparation method and application thereof
  • Diethylstilbestrol mimic antigenic epitope peptide, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1, panning and identification of DES antigen mimic epitope

[0041] (1) Affinity panning of DES antigen mimotope: the specific method is: use 0.1M NaHCO 3 (pH 8.6) dilute the anti-DES monoclonal antibody, and add it to a 96-well enzyme-labeled single well at a final concentration of 80 μg / ml, and coat overnight at 4°C. The next day, after rapid washing 6 times with TBST [50mM Tris-HCl (pH7.5), 150mM NaCl, 0.1% Tween-20 (v / v)], 350μl of 1% OVA blocking solution was added, and blocked at 4°C for 2h. Discard the blocking solution, wash 6 times with TBST, add 120 μl phage peptide library (phage display heptapeptide library, purchased from NEB Company, dilute the phage with TBST, the addition amount is 2.0×10 11 pfu), react at 25°C for 50min. The phage in the wells were discarded, quickly washed 10 times with TBST, patted dry, and eluted with 0.2M Glycine-HCl (pH 2.2) for 8 min, and then neutralized with 15 μl 1MTris-HCl (pH 9.1). Take 5 μl of the eluted phage to ...

Embodiment 2

[0045] Example 2, Sequencing of DES Antigen Mimotope Encoding Gene and Determination of its Amino Acid Sequence

[0046] The phage identified as DES antigen mimic epitope by indirect competitive enzyme-linked immunosorbent assay was amplified, and the DNA of the phage was extracted. The operation process is as follows: amplify the target phage, centrifuge the amplified product, transfer 500 μl phage supernatant to a new centrifuge tube; add 200 μl PEG / NaCl to precipitate the phage, let stand for 20 minutes, and centrifuge at 14000 rpm for 10 minutes. Discard the supernatant, resuspend the pellet in 100 μl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4M NaI), add 250 μl absolute ethanol to precipitate, let stand for 15 min, and centrifuge at 14000 rpm for 10 min. After the supernatant was discarded, the precipitate (DNA sequencing template) was washed with 70% ethanol, centrifuged at 14000 rpm for 10 min, the supernatant was discarded, and briefly dried in vacuum. Resusp...

Embodiment 3

[0047] Example 3, DES antigen mimotope tolerance to the organic solvent methanol

[0048] The phage particles displaying the DES antigen mimotope were subjected to methanol tolerance verification, the specific process was as follows: use 0.1M NaHCO 3 Dilute anti-DES monoclonal antibody (pH 8.6), coat 96-well microtiter plate, and incubate overnight at 4°C. The next day, wash with PBST [10mM PBS, pH7.4, 0.05% Tween-20 (v / v)] for 3 times, then block with 1% BSA, and block for 2 hours at 4°C; discard the blocking solution, wash with PBST for 3 times, add 50 μl of positive phage clones (approximately 2.0×10 10 pfu) and 50 μl (0%, 5%, 10%, 15%, 20%, 30%, 40%) of a series of concentrations of DES standard prepared in PBS buffer solution of methanol, incubated at 37°C for 45min, washed 6 times with PBST; Add 100 μl of HRP-labeled anti-M13 phage secondary antibody diluted 1:5000, incubate at 37°C for 45 minutes, wash 6 times with PBST; add 100 μl of TMB substrate solution, develop c...

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Abstract

The invention provides a diethylstilbestrol (DES) mimic antigenic epitope peptide, and a preparation method and an application thereof. Firstly, based on a phage display peptide library technology, ananti-DES monoclonal antibody is used as a target, and a DES antigen mimic epitope having the sequence of YVYPQQK, YVYPPGP or YVYPQSR is selected from a phage random display heptapeptide library. Centering on immune binding characteristics of the antigen mimic epitope, a specific detection method is designed based on enzyme-linked immunosorbent assay, colloidal gold immunochromatography and fluorescent microsphere immunochromatography. The antigenic mimic epitope has similar immunoreactivity characteristics to those of natural DES molecules, can replace an expensive DES standard substance having teratogenic and carcinogenic effects to be used in immunological detection of DES; the detection results are accurate and the sensitivity is high. The harm to human body health in the DES detectionprocess is effectively reduced, and meanwhile, the cost is saved.

Description

technical field [0001] The invention relates to the technical field of phage display peptide library, in particular to an epitope peptide simulating diethylstilbestrol, its preparation method and application. Background technique [0002] Diethylstilbestrol (DES) is a synthetic non-steroidal estrogen. It has been widely used as a growth promoter in animal husbandry because of its functions of promoting protein deposition, increasing animal daily gain and reducing fat synthesis. Studies have shown that excessive use has also brought about the problem of high residues of DES in animal tissues. Highly residual DES will cause teratogenic and carcinogenic hazards to humans through the food chain. my country has listed DES as a banned veterinary drug. However, according to literature reports, the detection rate of DES in animal-derived foods is still high, and timely detection of DES residues in foods is an effective means to prevent and control its hazards. [0003] At present, ...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11C12N15/62C12N15/70G01N33/558
CPCC07K7/06C07K2319/24C12N15/70G01N33/558
Inventor 邓省亮李平贺伟华
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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