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Antigen mimic epitope AM-1 of aflatoxin B1 and application thereof

A technology of mimetic epitope and aflatoxin, which is applied in recombinant DNA technology, DNA/RNA fragments, material inspection products, etc., can solve the problems of high price, uneven quality, pollution of the surrounding environment, etc., and reduce the risk of human health. Harm, high application value, cost saving effect

Inactive Publication Date: 2015-01-28
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical synthesis method of AFB1 whole antigen still has many disadvantages: 1) As the raw material for the synthesis of whole antigen, AFB1 standard product not only has strong carcinogenicity Toxicity and high price make the preparation of AFB1 whole antigen expensive, which limits the large-scale application of AFB1 immunological detection system; 2) AFB1 The chemical synthesis and preparation process of the whole antigen is complicated, and there are many intermediate by-products, resulting in large batch errors and uneven quality of the whole antigen, which has become an important technical bottleneck affecting the accuracy of immunological detection methods; 3) AFB1 During the chemical synthesis of the whole antigen, the organic reagents, intermediate products, waste water and waste residues used are likely to poison the operators and seriously pollute the surrounding environment

Method used

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  • Antigen mimic epitope AM-1 of aflatoxin B1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1) AFB 1 Affinity competition panning of antigen mimotope: the specific method is: dilute anti-AFB 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 3 times with PBST (10mM PBS pH 7.4 containing 0.1% Tween-20 (v / v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 37°C for 1 hour. After 1 hour, the blocking solution was discarded, washed 5 times with PBST, 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 PBS, about 1.0×10 11 pfu), shake at 37°C for 1 hour. Discard the unbound phage, wash 10 times with PBST, and dissolve the bound phage with 500ng of 10% AFB 1 Standards were subjected to competitive elution. Take 10 μl of the eluted phage to measure the titer, and the rest is used to inf...

Embodiment 2

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

[0024] Will demonstrate AFB by indirect competition ELISA 1 The phage of the antigen mimic epitope is amplified, and the DNA sequencing template of the phage is extracted. The brief process is as follows: carry out phage amplification, after the first step of centrifugation, transfer 800 μL of phage-containing supernatant to a new centrifuge tube. Add 200 μL PEG / NaCl to precipitate the phage. 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 TE buffer (10 mM Tria-HCl (pH 8.0), 1 mM EDTA), add an equal volume of absolute ethanol, freeze at -20°C for 2 hours, centrifuge and wash the precipitate (DNA sequencing template) with 70% ethanol. 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 t...

Embodiment 3

[0025] Example 3. AFB 1 The application of antigen mimotope AM-1 as a competitive antigen in ELISA

[0026] (1) Sample extraction

[0027] Weigh 5g of the sample (cereals and related foods), add 25 mL of 80% methanol-PBS solution, shake at 200 rpm for 4 minutes; filter the extract with Whatman No. 1 filter paper, take 1 mL of the filtrate and add 4 mL of PBS (phosphoric acid Salt buffer, pH=7.2)

[0028] After mixing, the sample extract is ready for use.

[0029] (2) Coating and sealing

[0030] Dilute anti-AFB with 10 mM PBS (pH 7.4) 1 Monoclonal antibody, 2 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 4% 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 AFB 1 Antigen mi...

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Abstract

The invention belongs to the biotechnology field and relates to an antigen mimic epitope of aflatoxin B1. The amino acid sequence of the antigen mimic epitope is WPLYYYPWSDGA. The antigen mimic epitope of the aflatoxin B1 can substitute standard aflatoxin B1 with high price and strong toxicity, and is applied to the immunological detection of the aflatoxin B1 as a competition antigen or a solid phase envelope antigen; the antigen mimic epitope has the immunoreaction characteristic similar to the natural aflatoxin B1 molecule, has good effect, can reduce the harm of the aflatoxin B1 to human health, saves the cost and has high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to aflatoxin B 1 Antigen mimotope AM-1 and its application. Background technique [0002] Aflatoxin B 1 (aflatoxin B 1 , AFB 1 ) is a secondary metabolite mainly produced by Aspergillus flavus and Aspergillus parasiticus, and widely exists in crops such as peanut, cottonseed, corn, wheat and rice. AFB 1 It has a destructive effect on human and animal liver tissue, and can cause liver cancer or even death in severe cases. In 1993, it was classified as a class I carcinogen by the Cancer Research Institute of the WHO. Therefore, AFB in food 1 detection is of great significance to human health. [0003] Currently, detection of AFB in food 1 The methods mainly include high performance liquid chromatography, gas chromatography, thin layer chromatography and immunological detection methods. Immunological detection methods are widely used in AFB for their advantages of high sensitivity,...

Claims

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Application Information

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