Antigen mimotope ph3 of bisphenol A and its application

A technology that mimics epitopes and antigens. It is applied in the direction of recombinant DNA technology, DNA/RNA fragments, and material inspection products. It can solve the problems of increasing detection costs, restricting the application and promotion of immunological methods, and easily causing harm to testing personnel and the environment. , to reduce the harm to human health, save costs and achieve good results

Inactive Publication Date: 2016-01-27
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method must use standard products, which not only increases the cost of detection, but also easily causes harm to the detection personnel and the environment, which restricts the application and promotion of immunological methods to a certain extent.

Method used

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  • Antigen mimotope ph3 of bisphenol A and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Affinity panning and identification of mimic epitope of bisphenol A antigen

[0018] Affinity panning of bisphenol A antigen mimotope: the specific method is: dilute the anti-bisphenol A monoclonal antibody with 10mM PBS (pH7.4), and coat 96-well microtiter plate at a final concentration of 60μg / mL, 4℃ Incubate overnight. The next day, it was washed 10 times with TBST (50mM NaCl, pH7.5 containing 0.1% Tween-20 (v / v)), and 300μL blocking solution (3%BSA-PBS) was added to incubate at 4°C for 2 hours. Discard the blocking solution after two hours, and dilute the phage stock solution with TBS 10-fold, about 1.0×10 11 pfu). The reaction was shaken at 22°C for 0.5 hours. The unbound phage was discarded, washed with TBST 10 times, the bound phage was eluted with 0.2MGlycine-HCl (pH 2.2), and immediately neutralized with 15 μL 1MTris-HCl (pH 9.1). Take 10 μL of the eluted phage to measure the titer, and the rest is used to infect 20 mL of E. coliER2738 strain that gr...

Embodiment example 2

[0022] Implementation case 2. Sequencing of bisphenol A antigen mimotope coding gene and determination of its amino acid sequence

[0023] The phage displaying mimic epitope of bisphenol A antigen was identified in Example 1 through indirect competitive ELISA, and then the DNA sequencing template of the phage was extracted. The brief process is as follows: Perform phage amplification, transfer 800 μL of phage-containing supernatant into a new centrifuge tube after the first centrifugation. Add 200 μL PEG / NaCl to precipitate the phage. After centrifugation, the pellet was resuspended in 100μL of iodide buffer (10mMTris-HCl (pH8.0), 1mM EDTA, 4MNaCl), 250μL of absolute ethanol was added to precipitate the DNA, and the pellet was washed with 70% ethanol (DNA sequencing template) after centrifugation. The pellet was finally resuspended in 20μL sterile water, and 2μL was taken for agarose gel electrophoresis analysis; 5μL of phage template was taken for DNA sequencing, and its -96gⅢ ...

Embodiment example 3

[0024] Implementation case 3 Application of bisphenol A antigen mimotope as a competitive antigen in ELISA

[0025] (1) Sample extraction

[0026] Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200rpm for 5min; filter the extract with Whatman No. 1 filter paper, take 1ml of filtrate and add 4ml of PBS (phosphate buffer Solution, pH=7.2) After mixing, it is the sample extraction solution, ready for use.

[0027] (2) Coating and sealing

[0028] Dilute the anti-bisphenol A monoclonal antibody with 10mM PBS (pH7.4), coat the plate with 10μg / mL, and incubate overnight at 4℃. After washing with PBST (10mMPBS, 0.05%Tween-20(v / v)) for 3 times the next day, blocking with PBS containing 3% skimmed milk powder, incubating at 37°C for 1 hour, washing the plate with PBST 6 times for later use .

[0029] (3) Establishment of standard curve

[0030] Take out the slats processed in step (2), and put 50μL of phage displaying mimotopes of bisphenol ...

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Abstract

The invention belongs to the field of biotechnologies and relates to an antigenic mimic epitope of bisphenol A. The amino acid sequence of the antigenic mimic epitope is YPAYTYNNLMHN. The antigenic mimic epitope of bisphenol A, disclosed by the invention, can replace a bisphenol A standard which is expensive in price and strong in toxicity, is applied to the immunological detection of bisphenol A as a competitive antigen or solid-phase coating antigen, has the immunoreaction characteristics similar to those of natural bisphenol A molecules and has a very good effect, so that the injury to the health of a human body caused by bisphenol A is reduced, the cost is reduced, and the application value is very high.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to bisphenol A antigen mimotopes and applications thereof. Background technique [0002] Bisphenol A (Bisphenol A, BPA) is a chemical material used to produce shatterproof plastics. Data show that bisphenol A is a toxic chemical. Animal experiments have found that bisphenol A has the effect of mimicking estrogen. Even a very low dose can make animals produce precocious females, decreased sperm count, and increased prostate. In addition, according to the literature, bisphenol A has certain embryo toxicity and teratogenicity. BPA can cause endocrine disorders and threaten the health of fetuses and children. Obesity caused by cancer and metabolic disorders is also considered to be related to this. In the human living environment, BPA is everywhere. From beverage water bottles, medical equipment to food packaging, many plastic products contain BPA. Even detection of bisphenol A is...

Claims

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Application Information

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