Antigenic mimic epitope of aflatoxin (AF) B1 and application thereof

An aflatoxin and mimetic epitope technology, applied in the biological field, can solve problems such as restricting the application and promotion of immunological detection methods, detecting personnel health and environmental threats, strong carcinogenicity, etc., achieving cost saving, good effect, and reducing human body Effects of health hazards

Inactive Publication Date: 2014-12-03
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, AFB must be used 1 Standards are used as raw materials to prepare competing antigens or solid-phase coated antigens, AFB 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 detection methods to a certain extent

Method used

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  • Antigenic mimic epitope of aflatoxin (AF) B1 and application thereof
  • Antigenic mimic epitope of aflatoxin (AF) B1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. AFB 1 Affinity panning and identification of antigen mimotopes

[0020] 1) AFB 1 Affinity 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 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, discard the blocking solution, wash 5 times with TBST, add 100 μl phage peptide library (phage display ring heptapeptide library or dodecapeptide library, purchased from NEB Company, dilute the phage stock solution 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 with 15 μl 1 M...

Embodiment 2

[0024] Example 2. AFB 1 Sequencing of genes encoding antigen mimotopes and determination of their amino acid sequences

[0025] The phages displaying the AFB1 antigen mimic epitope identified by indirect competition ELISA 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'-...

Embodiment 3

[0026] Example 3. AFB 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-AFB 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 AFB 1 Antigen mimotope phage (1.0×10 11 pfu) and a...

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Abstract

The invention belongs to the field of biotechnology and relates to an antigenic mimic epitope of aflatoxin B1. The amino acid sequence of the antigenic mimic epitope is CDPRKHIHC or VPYSPHYFERMI. The antigenic mimic epitope of aflatoxin B1 can replace an expensive AFB1 standard substance with strong toxicity and be applied to an immunological detection of AFB1 as a competitive antigen or a solid phase envelope antigen. The antigenic mimic epitope has immunoreaction characteristics similar to natural AFB1 molecules, and has good effects. Harm to human health caused by AFB1 is reduced, the cost is saved, and the antigenic mimic epitope of AFB1 has great application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to aflatoxin B 1 Antigen mimotope 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, conv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K7/08C12N15/31C12N15/70G01N33/68G01N33/577G01N33/543G01N33/558
Inventor 许杨何庆华贺贞云
Owner NANCHANG UNIV
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