A mimetic fumonisin b 1 Antigen mimotope and its use

A mimetic epitope and fumonisin technology, which is applied to DNA/RNA fragments, material inspection products, instruments, 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: 2017-05-17
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 mimetic fumonisin b <sub>1</sub> Antigen mimotope and its use
  • A mimetic fumonisin b <sub>1</sub> Antigen mimotope and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1.FB 1 Affinity panning and identification of antigenic mimotopes

[0018] 1) FB 1 Affinity panning of antigenic mimotopes: the specific method is: dilute anti-FB with 10 mM PBS (pH 7.4) 1 Monoclonal antibody was coated on a 96-well microtiter plate at a final concentration of 100 μg / mL, and incubated at 4°C overnight. The next day, after washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v)), 300 μl of blocking solution (3% BSA-PBS) was added and incubated 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), 22-26 ℃ shaking reaction for 1 hour. Unbound phage was discarded, washed 10 times with TBST, and bound phage was eluted with 0.2M Glycine-HCl (pH 2.2) and immediately neutraliz...

Embodiment 2

[0022] Example 2.FB 1 Sequencing of genes encoding antigenic mimotopes and determination of their amino acid sequences

[0023] The phages identified to display the FB1 antigenic mimotopes by indirect competitive ELISA were amplified, and the DNA sequencing templates of the phages were extracted. The brief procedure is as follows: phage amplification is performed, and after the first step of centrifugation, 800 μl of the phage-containing supernatant is transferred to a new centrifuge tube. Phage was precipitated by adding 200 μl PEG / NaCl. After centrifugation, the pellet was resuspended in 100 μl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), and 250 μl absolute ethanol was added to precipitate the DNA, and the pellet was washed with 70% ethanol after centrifugation (DNA sequencing template). ). The pellet was finally resuspended in 20 μl of sterilized water, and 2 μl was used for agarose gel electrophoresis analysis; 5 μL of phage template was used for DNA se...

Embodiment 3

[0025] Example 3.FB 1 Application of Antigen Mimotopes as Competing Antigens in ELISA

[0026] (1) Sample extraction

[0027] Weigh 5g of samples (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200rpm for 5 minutes; filter the extract with whatman No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS (phosphoric acid). Salt buffer, pH=7.2) after mixing, it is the sample extraction solution, which is ready for use.

[0028] (2) Coating and sealing

[0029] Anti-FB was diluted in 10 mM PBS (pH 7.4) 1 Monoclonal antibody, 10μg / 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)), the plate was blocked with PBS containing 3% nonfat dry milk, incubated at 37°C for 1 hour, and washed 6 times with PBST for 6 times. use.

[0030] (3) Establishment of standard curve

[0031] Take out the strips processed in step (2), and put 50 μl of FB into each well. 1 Ant...

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Abstract

The invention belongs to the field of biotechnologies, and relates to an antigen mimic epitope capable of mimicking fumonisins B1. An amino acid sequence of the antigen mimic epitope is GDGVHKSHDIRG. The antigen mimic epitope of FB1 is capable of replacing an FB1 standard substance which is high in price and strong in toxicity, and is applied to immunological detection of the FB1 as a competition antigen or solid-phase envelope antigen. The antigen mimic epitope has an immunoreaction characteristic similar to a natural FB1 molecule, and a good effect. The damage of the FB1 to the health of a human body is reduced, the cost is saved, and high application value is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to fumonisin B 1 Antigen mimotopes and their applications. 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, teratogenicity and carcinogenicity. 1 Chemicals are classified into Group 2B, ie probable carcinogens to humans. At present, most countries have already restricted FB in cereals, grains and food. 1 The residual content of FB has set a limit standard, such as the United Nations Food and Agriculture Organization (FAO) stipulates the maximum allowable daily intake of FB 1 , FB 2 , FB 3 The total amount is 2μg / kg body weight; Switzerland regulates FB in food 1 and FB 2 The total residue limit is 1000 μg / L. [0003] At present, the detection of FB in...

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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