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Single-chain antibody and application thereof in detecting aflatoxin

A single-chain antibody and light chain technology, which is applied in applications, measurement devices, and the introduction of foreign genetic material using carriers, can solve problems such as limited production, limited development of immunological methods, and high technical costs, achieving high affinity and good crossover The effect of increasing the reaction rate and the cross-reaction rate

Active Publication Date: 2012-09-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high cost and limited output of these two antibody preparation techniques greatly limit the further development of immunological methods in detection

Method used

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  • Single-chain antibody and application thereof in detecting aflatoxin
  • Single-chain antibody and application thereof in detecting aflatoxin
  • Single-chain antibody and application thereof in detecting aflatoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, discovery of single-chain antibody

[0043] 1. Extraction of hybridoma mRNA and construction of full-length scFv gene

[0044] Take hybridoma cells with good homogeneity and purity (secreting AFM 1 specific monoclonal antibody) 1 x 10 6 Total RNA was extracted and purified to obtain mRNA, which was reverse transcribed to obtain cDNA. Using a full set of primers, cDNA was used as a template to amplify the heavy chain variable region (VH) and light chain variable region (VL) genes, respectively, cloned into the pCR 2.1 vector and sequenced, after computer analysis and database BLAST comparison , VH and VL genes with potential immunocompetence and correct expression cassettes were identified. Overlap extension PCR (SOE-PCR) primers were designed according to the above-identified genes, and the full-length scFv gene was constructed by splicing. The scFv gene was sequenced and analyzed, and cloned into the pCR 2.1 vector for preservation and amplification. ...

Embodiment 2

[0061] Embodiment 2, the preparation of single-chain antibody

[0062] 1. Construction of recombinant plasmids

[0063] 1. Synthesize the double-stranded DNA molecule shown in Sequence 2 of the sequence listing (the 1st to 6th nucleotides from the 5' end are the NcoI restriction recognition sequence, and the 9th to 338th nucleotides are the light chain variable region Coding sequence, the 339th to 398th nucleotides are the coding sequence of the connecting peptide, the 399th to 752nd nucleotides are the coding sequence of the heavy chain variable region, and the 762nd to 791st nucleotides are the C-myc tag tag The coding sequence, the 792nd to 797th nucleotides are the XhoI restriction recognition sequence).

[0064] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0065] F1: 5'- CCATGG CACAGACGGTTG-3';

[0066] R1: 5'- CTCGAG CAGGTCT...

Embodiment 3

[0081] Embodiment 3, the application of single-chain antibody

[0082] 1. AFB 1 - Preparation of BSA

[0083] 1. AFB 1 -Synthesis of BSA

[0084] (1) Add 5mg of aflatoxin B 1 Dissolve in 5ml of pyridine, add 25mg of carboxymethylhydroxylamine hemihydrochloride, stir overnight in the dark at room temperature, spin dry at 40°C; dissolve in a small amount of methanol, separate with a TLC scraper, the developer is methanol:chloroform=1:9, take The fluorescent band of the product with RF=0.2 was extracted with 2ml of ethyl acetate, 500μl of methanol and 200μl of DMF were added to assist the extraction, filtered, and the filtrate was rotary evaporated at 40°C to obtain 300μl of the product solution.

[0085] (2) Take 300 μl of the above product solution, add 300 μl DMF and 300 μl water, add 8 mg EDC and 8 mg NHS, activate for 1 hour, react in the dark, and obtain solution Ⅰ;

[0086] (3) Dissolve 10mg BSA in 1ml carbonate buffer (0.1M, PH=9.51), fully dissolve, and stir at room...

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Abstract

The invention discloses a single-chain antibody and application thereof in detecting aflatoxin. The single-chain antibody is a polypeptide consisting of a light chain variable region, a connection peptide and a heavy chain variable region, wherein the connection peptide is positioned between the light chain variable region and the heavy chain variable region; the light chain variable region is represented by amino acid residues from 1st to 110th site of the tail ends of sequences from 1 to N of a sequence table; and the heavy chain variable region is represented by amino acid residues from 131st to 248th site of the tail ends of the sequences from 1 to N of the sequence table. The single-chain antibody has high cross reactivity, high affinity and high sensitivity to various types of aflatoxin, is suitable for quick immune detection for various types of multi-residue aflatoxin and has great significance for detecting the aflatoxin.

Description

technical field [0001] The invention relates to a single-chain antibody and its application in detecting aflatoxins. Background technique [0002] Aflatoxin (Aflatoxin, AFT) is a mycotoxin that poses a huge threat to human health and agricultural production. Its chemical structure is similar, and it is a derivative of dihydrofuranocoumarin. The molecular weight is about 312-346. Secondary metabolites produced by Aspergillus flavus, A. parasiticus. As many as 250,000 people die of mycotoxin-induced cancers around the world every year. Mycotoxin contamination also leads to the destruction of a large number of agricultural products, causing economic losses of hundreds of millions of dollars every year. The occurrence of aflatoxins in food and feed is highest in hot and humid regions. [0003] Aflatoxin is a highly toxic and highly toxic substance that can damage the liver tissue of humans and animals, and can lead to liver cancer or even death in severe cases. In 1993, afla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N15/13C12N15/63C12N1/21C12N5/10C12P21/08G01N33/577
Inventor 沈建忠温凯王战辉张素霞史为民丁双阳江海洋吴聪明
Owner CHINA AGRI UNIV
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