Antigenic mimic epitope of fumonisin B1 and application thereof

A mimetic epitope and fumonisin technology, applied in the fields of application, biochemical equipment and methods, material inspection products, etc., can solve the constraints on the application and promotion of immunological detection methods, threats to the health of testing personnel and the environment, and high prices problem, to achieve cost savings, good effect, and reduce the effect of human health hazards

Inactive Publication Date: 2013-10-09
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of establishing an immunological detection method, it is necessary to use FB 1 Standards are used as raw materials to prepare competing antigens or solid-phase coated antigens, FB 1 It is not only expensive but also highly carcinogenic, which poses a great threat to the health of testing personnel and the environment, thus restricting the application and promotion of immunological testing methods to a certain extent

Method used

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  • Antigenic mimic epitope of fumonisin B1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. FB 1 Affinity panning and identification of antigen mimotopes

[0019] 1) Facebook 1 Affinity panning of antigen mimotope: The specific method is: dilute anti-FB with 10 mM PBS (pH 7.4) 1 Monoclonal antibody was used to coat a 96-well microtiter plate at a final concentration of 100 μg / mL and incubated overnight at 4°C. The next day, after washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 4°C for 2 hours. After 2 hours, the blocking solution was discarded, washed 5 times with TBST, and 100 μl of phage peptide library (phage display dodecapeptide library, purchased from NEB Company) was added to each well, and the phage stock solution was diluted 10 times with TBS, about 1.0×10 11 pfu), shake and react for 1 hour at 22-26°C. Unbound phages were discarded, washed 10 times with TBST, bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2), and immediately neutralize...

Embodiment 2

[0023] Example 2. FB 1 Sequencing of genes encoding antigen mimotopes and determination of their amino acid sequences

[0024] The phages identified by indirect competition ELISA displaying FB1 antigen mimic epitopes were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: carry out phage amplification, after the first step of centrifugation, transfer 800 μl of phage-containing supernatant into a new centrifuge tube. Add 200 μl PEG / NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100 μl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 μl absolute ethanol to precipitate DNA, centrifuge and wash with 70% ethanol Precipitation (DNA sequencing template). The pellet was finally resuspended in 20 μl of sterilized water, and 2 μl was taken for agarose gel electrophoresis analysis; 5 μl of phage template was used for DNA sequencing, and its -96 gIII sequencing primer was: 5'- HO ...

Embodiment 3

[0026] Example 3. FB 1 Application of Antigen Mimotope as Competing Antigen in ELISA

[0027] (1) Sample extraction

[0028] Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH = 7.2) After mixing, it is the sample extract, ready for use.

[0029] (2) Coating and sealing

[0030] Dilute anti-FB with 10 mM PBS (pH 7.4) 1 Monoclonal antibody, 10 μg / mL coated microtiter plate, incubated overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v / v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, then wash the plate 6 times with PBST stand-by.

[0031] (3) Establishment of standard curve

[0032] Take out the strips treated in step (2), and put 50 μl into each well showing FB 1 Antigen mimotope phage (1.0×10 11 pfu) and a se...

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Abstract

The invention belongs to the technical field of biology, and relates to an antigenic mimic epitope of fumonisin B1 (FB1). The antigenic mimic epitope has the amino acid sequence as follows: NNAAMYSEMATD, FYTSPGRTSHYM, IHQELRYTKDSP, GDGVHKSHDIRG, TTLQMRSEMADD, SMLNDYRDYTTH or TRDKSSMLERWP. The FB1 antigenic mimic epitope can be used for replacing a standard FB1 with expensive price and strong toxicity, and can be used as a competitive antigen or a solid-phase enveloped antigen applied to the immunological detection of the FB1. The immunoreaction characteristic of the antigenic mimic epitope is similar to that of a natural FB1 molecule, i.e., the effect of the antigenic mimic epitope is very good. In addition, the antigenic mimic epitope also has advantages that the harm of the FB1 to human health is reduced and the cost is saved, so that the application value is relatively high.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to fumonisin B 1 Antigen mimotope and its application. Background technique [0002] Fumonisin B 1 (Fumonisins B 1 , FB 1 ) is a common mycotoxin mainly produced by Fusarium moniliforme. Studies have shown that FB 1 It has strong toxic effects on humans and animals, including neurotoxicity, reproductive toxicity, embryotoxicity and teratogenic carcinogenicity, etc. The International Cancer Research Center has put FB 1 Divided into Group 2B, which is a possible carcinogen to humans. At present, most countries have already restricted the use of FB in grains, grains and foodstuffs. 1 The residual content of the food has set the limit standard, such as the United Nations Food and Agriculture Organization (FAO) stipulates the daily maximum allowable intake of FB 1 , FB 2 , FB 3 The total amount is 2 μg / kg body weight; Switzerland regulates FB in food 1 and FB 2 The total residue...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/31G01N33/68
Inventor 何庆华许杨陈波
Owner NANCHANG UNIV
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