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A kind of epitope peptide simulating dienestrol, its preparation method and application

An epitope, diethylstilbestrol technology, applied in the direction of peptides containing affinity tags, peptides containing MBP tags, peptides, etc., can solve the adaptive matching of antigenic epitope peptide immunological properties, difficult to obtain detection effect, lack etc.

Active Publication Date: 2021-04-20
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to aim at the technical defects of the prior art, and to provide an antigenic epitope peptide simulating dienestrol, so as to solve the lack of an immunoreactive peptide similar to dienestrol in the prior art and capable of replacing it. Technical Problems of Sensitive Immunoassay Detection Components
[0005] The present invention also provides a method for preparing the above-mentioned antigenic epitope peptide simulating dienestrol, 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 dienestrol 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 , so it is difficult to obtain the technical problem of the best detection effect

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  • A kind of epitope peptide simulating dienestrol, its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1, Panning and Identification of DIS Antigen Mimic Epitopes

[0036] (1) Affinity panning of DIS antigen mimotope: the specific method is: use 0.1M NaHCO 3 (pH 8.6) dilute the anti-DIS 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

[0039] Example 2, Sequencing of DIS Antigen Mimotope Encoding Gene and Determination of its Amino Acid Sequence

[0040] The phage identified as the mimotope of the DIS antigen by indirect competitive ELISA 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. Resuspend the pellet in 30μl...

Embodiment 3

[0041] Example 3, Application of DIS Antigen Mimotope as Competing Antigen in Enzyme-Linked Immunoassay Method

[0042] (1) Sample extraction

[0043] Remove the fat and connective tissue of the pork, break it with a mixer, weigh 2g into a 50ml centrifuge tube, add 6ml of 10% methanol-PBS solution, shake on the shaker for 3min; add 6ml of 0.05M carbonate Buffer solution (pH9.6), then add 10ml of ethyl acetate, shake at 150rpm for 30min, and centrifuge at 4000rpm for 10min. Pipette the upper organic layer into another test tube, add 1.25ml 0.1M NaOH solution and 0.625ml 1.0M MgCl to it 2 solution in a water bath at 60°C for 30 minutes. Take out the test tube and add 1g of anhydrous Na after cooling for a while 2 SO 4 , Shake on a shaker for 5 minutes and then centrifuge for 5 minutes. Transfer the upper organic layer to a new test tube, and dry it in a nitrogen blower at about 55°C. Add 100 μl of methanol solution to dissolve, then add 900 μl of PBS to resuspend, and filt...

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Abstract

The invention provides an epitope peptide simulating dienestrol, its preparation method and application. The technical solution is firstly based on the phage display peptide library technology, with the anti-DIS monoclonal antibody as the target, and panning from the phage random display heptapeptide library to obtain a DIS antigen mimic epitope whose sequence is CTLNWAC. The present invention respectively provides methods for preparing the antigen mimetic epitope by means of phage amplification and gene recombination. The present invention designs specific detection methods based on enzyme-linked immunosorbent assay, colloidal gold immunochromatography and fluorescent microsphere immunochromatography around the immunological binding characteristics of the antigen mimic epitope. The antigen mimic epitope has similar immune response characteristics to the natural DIS molecule, and can replace the expensive DIS standard substance with teratogenic and carcinogenic effects for the immunological detection of DIS, with accurate detection results and high sensitivity.

Description

technical field [0001] The invention relates to the technical field of phage display peptide library, in particular to an epitope peptide simulating dienestrol, its preparation method and application. Background technique [0002] Dienestrol (Dienestrol, DIS) is a synthetic non-steroidal estrogen, and it is also one of the main metabolites of the typical banned veterinary drug Dienestrol in animals. It has obvious effect in one aspect and has been widely used in animal husbandry as a growth-promoting agent. Studies have shown that excessive use of DIS will lead to high residues in animal tissues, and directly cause teratogenic and carcinogenic hazards to humans through the food chain. Although our country has stipulated that the use of DIS in animal husbandry is prohibited, the illegal use still exists due to the drive of interests. Therefore, timely detection of DIS residues in food is not only an effective means to prevent and control the hazards of DIS, but also an effe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C12N15/11C12N15/62C12N15/70G01N33/558
CPCC07K7/06C07K2319/24C12N15/70G01N33/558
Inventor 邓省亮李平贺伟华涂祖新张莉莉
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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