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Cyclic polypeptide simulating epitope of aflatoxin b1 and detection kit thereof

An aflatoxin and cyclic polypeptide technology, applied in the field of immunoanalytical chemistry, can solve the problems of large batch impact and high cost of competitive antigen preparation, and achieve the effect of improving sensitivity

Active Publication Date: 2019-12-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the preparation and application of the coated antigen, the operator will be exposed to the AFB1 environment, and the preparation of aflatoxin B1 oxime requires professional equipment such as high temperature and high pressure, which has certain potential hazards, and the competitive antigen High preparation cost and large batch impact

Method used

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  • Cyclic polypeptide simulating epitope of aflatoxin b1 and detection kit thereof
  • Cyclic polypeptide simulating epitope of aflatoxin b1 and detection kit thereof
  • Cyclic polypeptide simulating epitope of aflatoxin b1 and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Specific panning and amplification of phage-displayed polypeptides

[0048] Panning of Phage Displayed Specific Peptides

[0049] Coat 5 wells on a 96-well ELISA plate, the first well is coated with 100 μg / mL antibody, the remaining 4 wells are coated with 3% BSA, and coated overnight at 4°C, the next day, add 200 μL 0.05% PBST to wash the plate 3 times; Block the ELISA plate with 1% BSA, incubate at 25°C for 2 hours, wash the plate 3 times; add 1 μL of phage display cyclic heptapeptide library to well 1, incubate for 1 hour, add 500ppb AFB1 for competitive elution after washing the plate, incubate at 25°C for 1 hour, and Transfer the eluate to wells 2, 3, 4, and 5 at 25 μL / well. After incubation for 1 hour, collect the supernatant, which is the eluate after panning. Use the upper plate method to determine the concentration of phage in the eluate. Titer. The antibody concentrations of the second round of panning and the third round of panning were halved seq...

Embodiment 2

[0052] Example 2 Identification and sequencing of specific polypeptides

[0053] Pick a single clone from the plate of the eluted phage in the third round of the above-mentioned determination, and use the Phage ELISA method to measure the ability of the phage-displayed polypeptide clone to mimic the AFB1 antigen epitope and bind to the antibody. The specific operation is: inoculate the picked phage plaque In 2 mL of LB medium (containing 1:100 log prophase E. coli ER2738), shake culture at 37°C for 6 h, centrifuge at 10,000 rpm for 10 min, and collect the supernatant. Each clone was measured in 3 wells. Well 1 and well 2 were coated with 100 μL (10 μg / mL) of the antibody used for screening, and well 3 was coated with 1% BSA as a blank control. After washing with PBST, 3% skimmed milk was added to block for 2 h ( 300 μL / well), add 50 μL 500ppb AFB1 and 50 μL plaque culture supernatant mixture to well 1, add 50 μL 10% methanol PBS and 50 μL plaque culture supernatant mixture to ...

Embodiment 3

[0055] Example 3 Synthesis of biotin-labeled cyclic polypeptides

[0056] Biotin reacts with -NH2 in the cyclic polypeptide. There are two free -NH2 in the cyclic polypeptide sequence, one is at the N-terminal and the other is at the R group of lysine, so the cyclic polypeptide and biological Elements are connected at a ratio of 1:2. Weigh 2mg of cyclic polypeptide, add 50μL DMF and 2mL PBS buffer to dissolve the polypeptide;

[0057] Weigh 4.6 mg of biotin, add 50 μL LDMF to dissolve, take 22 μL of dissolved biotin and add dropwise to the dissolved polypeptide, ice bath for 2 hours and then dialyze for 24 hours to obtain biotin-labeled cyclic polypeptide.

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Abstract

The invention discloses a ring type polypeptide simulating antigenic epitope of aflatoxin B1 (AFB1), and a detection kit of the AFB1. The amino acid sequence of the ring type polypeptide is CVPSKPGLC,which is shown as SEQ ID No.1. The kit comprises an AFB1 competitive antigen, an elisa plate embedded with an anti-AFB1 monoclonal antibody, a standard solution of the aflatoxin B1, horseradish peroxidase labeled streptavidin and the like. The polypeptide sequence can simulate the antigenic epitope of the AFB1, can be subjected to specific binding with an antibody for screening, and is high in sensitivity and high in specificity, exposure of the AFB1 to operators is reduced, and the safer and the more efficient detection method is achieved. The ring type polypeptide simulating the antigenic epitope of the AFB1, and the detection kit of the AFB1 have the significant practical significance for achieving pollution monitoring of the AFB1 in agricultural products.

Description

technical field [0001] The invention relates to the technical field of immunoanalysis chemistry, in particular to a cyclic polypeptide simulating the epitope of aflatoxin B1 antigen and a detection kit thereof. Background technique [0002] Aflatoxins (AFTs) are mainly a class of highly toxic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. AFTs can be mainly divided into B family, G family and M family. Group B AFTs can emit blue fluorescence under the excitation of 365nm ultraviolet light, and can be divided into Aflatoxin B1 (AFB1) and AFB2. Among them, AFB1 is the most toxic, and is defined as a class I carcinogen by the World Health Organization Cancer Research Institute, that is, a substance that is confirmed to be carcinogenic to humans. Humid and hot environment, improper harvesting and storage methods will lead to the production of AFB1, which mainly pollutes rice, peanuts, corn, grain and oil products, etc. [0003] Most of the c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/64G01N33/577
CPCC07K7/64G01N33/577G01N2333/38
Inventor 王佳王小红马兰庞倩穆赫塔尔·海纳
Owner HUAZHONG AGRI UNIV
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