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Antigenic epitope peptide imitating enrofloxacin, its preparation method and application

An antigenic epitope, enrofloxacin technology, applied in its preparation, in the field of enrofloxacin-simulating antigenic epitope peptide, can solve the problem of adaptive matching of antigenic epitope peptide immunological properties, difficulty in obtaining detection effect, and preparation method Unclear problems, to achieve high sensitivity, reduce harm to human health and ecological environment, and highlight the effect of application value

Active Publication Date: 2020-04-24
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 present invention aims at the technical defects of the prior art, and provides an antigenic epitope peptide simulating enrofloxacin, so as to solve the lack of an immunoreactive peptide similar to that of enrofloxacin 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 imitating enrofloxacin to solve the technical problem that its preparation method is not clear
[0006] The present invention also provides the application of the above-mentioned antigenic epitope peptide imitating enrofloxacin in a specific immunological detection 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 antigenic epitope peptide , so it is difficult to obtain the technical problem of the best detection effect

Method used

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  • Antigenic epitope peptide imitating enrofloxacin, its preparation method and application
  • Antigenic epitope peptide imitating enrofloxacin, its preparation method and application
  • Antigenic epitope peptide imitating enrofloxacin, its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1, Panning and Identification of ENR Antigen Mimic Epitopes

[0039] (1) Affinity panning of ENR antigen mimotope: the specific method is: use 0.1M NaHCO 3 (pH 8.6) Dilute the anti-ENR 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 6 quick washes 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, and add 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 of 1M Tris-HCl (pH 9.1). Take 5 μl of the eluted phage to measure the titer,...

Embodiment 2

[0042] Example 2, Sequencing of ENR Antigen Mimotope Encoding Gene and Determination of its Amino Acid Sequence

[0043] The phage identified as ENR 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 (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), add 250μl absolute ethanol to precipitate, let stand for 15min, and centrifuge at 14000rpm for 10min. 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 th...

Embodiment 3

[0044] Example 3, Application of ENR Antigen Mimotope as Competing Antigen in Enzyme-Linked Immunoassay Method

[0045] (1) Sample extraction

[0046] Remove the fat and connective tissue of the pork, crush it with a mixer, weigh 2 g in a 15 ml centrifuge tube, add 4 ml of 50% methanol-PBS solution, shake on a shaker for 3 min; centrifuge at 4000 rpm for 10 min. Repeat the above shaking and centrifugation steps for the precipitate, combine the supernatant twice, centrifuge again at 10,000 rpm at 4°C for 10 minutes to collect the supernatant, filter it with a microporous membrane (pore size 0.22 μm), dilute the filtrate with PBS to an equal volume, and mix well spare. Take 50 μl of the filtrate and add the sample directly into the microwell for enzyme-linked immunoassay assay.

[0047] (2) Antibody coating

[0048] with 0.1M NaHCO 3 (pH 8.6) Dilute the anti-ENR monoclonal antibody, 1μg / ml coat the microtiter plate, and incubate overnight at 4°C. The next day, wash with PBS...

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Abstract

The invention provides an enrofloxacin-mimic antigen epitope peptide, and preparation methods and an application thereof. The ENR-mimic antigen epitope peptide having a sequence represented by GKPYITWis panned from a phage random display heptapeptide library through a phage display peptide library technology with an anti-ENR monoclonal antibody as a target. The invention respectively provides a method for preparing the antigen-mimic epitope by a phage amplification technology and a method for preparing the antigen-mimic epitope by a gene recombination technology. A concrete detection method is designed based on enzyme linked immunosorbent assay, colloidal gold immunochromatography and fluorescence microsphere immunochromatography by using the immunological binding property of the antigen-mimic epitope. The antigen-mimic epitope has similar immunoreactivity characteristics to natural ENR molecules, can be used in the immunological detection of ENR to replace an ENR standard substance having strong drug resistance and high price, and the detection result is accurate and highly-sensitive.

Description

technical field [0001] The invention relates to the technical field of phage display peptide library, in particular to an antigenic epitope peptide imitating enrofloxacin, its preparation method and application. Background technique [0002] Enrofloxacin (ENR), a representative drug belonging to fluoroquinolones, is widely used in the treatment and control of bacterial and viral infectious animal diseases in the field of animal breeding due to its convenient use, low cost and remarkable curative effect. In the farming industry, some farmers do not follow the animal drug withdrawal period and abuse ENR to obtain greater economic benefits, so that ENR has become a common phenomenon in animal-derived foods. A large number of studies have shown that long-term consumption of foods containing ENR residues can cause drug resistance in humans, and cause adverse reactions and allergic reactions in the digestive system, nervous system, and genitourinary system. In recent years, my co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06G01N33/68
CPCG01N21/78G01N33/68
Inventor 邓省亮李平贺伟华赖卫华
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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