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Deoxynivalenol mimetic antigen based on nanobody and its application

A deoxynivalenol and nanobody technology, applied in the biological field, can solve the problems of restricting the application and promotion of immunological detection methods, threats to the health of detection personnel and the environment, high price, etc., achieving good effects, cost saving, Harm reduction effect

Active Publication Date: 2017-04-12
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 unavoidable to prepare competing antigens or solid-phase coated antigens with DON standard products as raw materials. DON standard products are not only expensive but also have strong toxicity. It poses a great threat to the health and environment of the country, which restricts the application and promotion of immunological detection methods to a certain extent.

Method used

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  • Deoxynivalenol mimetic antigen based on nanobody and its application
  • Deoxynivalenol mimetic antigen based on nanobody and its application

Examples

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Effect test

Embodiment 1

[0025] Example 1. Construction of camel-derived natural single domain heavy chain antibody library

[0026] 1) Separation of camel-derived leukocytes: add lymphocyte separation solution to the centrifuge tube, then add blood samples, centrifuge at 700g for 20min; carefully draw the leukocytes suspended in the middle layer into a new centrifuge tube, add 1 / 2 volume of PBS, Centrifuge at 800g for 15min; discard the supernatant, wash the leukocytes on the wall of the centrifuge tube with PBS, and centrifuge at 850g for 10min; discard the supernatant, resuspend the leukocytes in 300μL PBS and count them; add lysate (RNAiso) at a volume ratio of 1:10 and save for later use.

[0027]2) Extraction of total RNA: Add 1 / 5 volume of chloroform to the above lysate, shake vigorously for 15 s to fully emulsify, let stand at room temperature for 5 min; centrifuge at 12000g at 4°C for 15 min, take the supernatant and transfer to another fresh centrifuge into the tube; add an equal volume of i...

Embodiment 2

[0049] Example 2. Affinity panning and identification of DON mimic antigen

[0050] 1) Affinity panning of DON mimic antigen: First, dilute anti-DON monoclonal antibody with PBS (pH 7.4), coat the microtiter plate with a final concentration of 10 μg / mL, and incubate at 37 °C for 4 hours. The next day, after washing 3 times with PBST (10mM PBS, 0.5% Tween-20 (v / v) ), 300 μL of 7% BSA-PBS (3% OVA-PBS) was added to block for 2 hours at 37°C. After 2 hours, the blocking solution was discarded, washed 5 times with PBST, and 100 μL camel-derived natural single domain heavy chain antibody library (titer about 2.0×10 11 cfu), incubate at 37°C for 1 hour. Aspirate unbound phage, wash with PBST 10 times, add 100 μL of Glycine-HCl (0.2M, pH 2.2) to elute for 6-8min, and immediately neutralize with 15 μL of Tris-HCl (1M, pH 9.1). Take 10 μL of the eluted phage to determine the titer, and the rest is used to infect 20 mL of E. coli The TG1 strain was amplified. On the third day, the ...

Embodiment 3

[0054] Example 3. The sequencing of DON mimic antigen coding gene and the determination of its amino acid sequence

[0055] The phage clones displaying the DON mimic antigen identified by indirect competition ELISA were subjected to DNA sequencing, and the amino acid sequence of the DON mimic antigen could be obtained according to the DNA sequencing results and the codon table. The amino acid of the simulated antigen of the present invention is obtained, and the sequence is as SEQ ID NO.:1.

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Abstract

The invention belongs to the biotechnical field and relates to nano antibody-based deoxynivalenol mimic antigen and application thereof. The amino acid sequence is shown in SEQIDNO:1. The invention further relates to nucleotide which codes the amino acid. The nano antibody-based deoxynivalenol mimic antigen provided by the invention can be used for replacing a DON (deoxynivalenol) standard substance which is high in price and strong in toxicity and is used as competitive antigen or solid phase coated antigen which is applied to immunological detection of DON. The mimic antigen has an immunoreaction characteristic which is similar to that of a natural DON molecule and is good in effect. Compared with conventional polypeptide-based antigen mimic epitopes and IgG-based conventional anti-idiotype antibody, VHH has the characteristics of being more stable in structure, resistant to acid and base and high temperature, high in detection sensitivity and the like. Therefore, the immunodetection stability is extremely enhanced, and meanwhile, the endurance capacity on the environment is further enhanced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a deoxynivalenol mimetic antigen based on a nanobody (variable domain of heavy chain of heavy chain antibody, VHH) and an application thereof. Background technique [0002] Deoxynivalneol (deoxynivalneol, DON) is a trichothecene toxin, mainly produced by Fusarium, widely present in barley, wheat, corn, oats, rice and other crops and their products. DON has cytotoxicity, embryotoxicity and immunotoxicity, and can cause symptoms such as anorexia, vomiting, diarrhea, fever and slow growth, which seriously threaten the health of humans and animals. Due to the high contamination rate and high toxicity of DON, the detection of DON in food is of great significance. [0003] At present, the methods for detecting DON in food mainly include thin layer chromatography, high performance liquid chromatography, gas chromatography, surface plasmon resonance, and immunological detection. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11G01N33/68G01N33/577
CPCC07K14/00G01N33/6854
Inventor 何庆华许杨邱雨楼
Owner NANCHANG UNIV
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