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a fumonisin b 1 Dodecapeptide Antigen Mimotope and Its Application

A mimetic epitope and fumonisin technology, applied in the direction of peptides, DNA/RNA fragments, material inspection products, etc., can solve problems such as restrictions on the application and promotion of immunological detection methods, threats to the health of testing personnel and the environment, and high prices , to achieve cost saving, good effect, and reduce the harm to human health

Inactive Publication Date: 2017-04-05
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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  • a fumonisin b <sub>1</sub> Dodecapeptide Antigen Mimotope and Its Application

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 neutralized...

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: For phage amplification, after the first step of centrifugation, transfer 800 µl of the phage-containing supernatant to 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 the phage template was used for DNA sequencing, and its -96 gIII sequencing primer was: 5'- HO C...

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 seri...

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Abstract

The invention belongs to the field of biotechnologies and relates to a dodecapeptide antigen mimic epitope for FB1 (fumonisins B1). The amino acid sequence of the dodecapeptide antigen mimic epitope is SMLNDYRDYTTH. The FB1 antigen mimic epitope can substitute for an FB1 standard substance which is expensive and high in toxicity, can serve as a competitive antigen or a solid-phase coating antigen to be applied to immunological detection of the FB1, has immunoreaction characteristics similar to those of natural FB1 molecules and is very good in effect. The harm caused by the FB1 to human health is reduced, the cost is saved, and the dodecapeptide antigen mimic epitope has the very high application value.

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 grain 1 and FB 2 The total residue ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/11G01N33/68G01N33/577
Inventor 何庆华许杨
Owner NANCHANG UNIV
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