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Single-chain antibody and application thereof in detecting beta-stimulant

A technology of single-chain antibody and heavy chain, which is applied in application, measurement device, and introduction of foreign genetic material by carrier, etc. It can solve the problems that there is no β-stimulant multi-residue immunoassay technology, and achieve good cross-reaction rate and high Sensitivity, high-affinity effects

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

Among them, the immunoassay method is the fastest and most economical, but the antibodies currently used in immunoassays are mainly polyclonal antibodies and monoclonal antibodies, most of which are antibodies against clenbuterol, while antibodies against albuterol and other β-agonists The research on the preparation of β-agonists is still in its infancy, so there is no mature multi-residue immunoassay technology for β-agonists

Method used

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  • Single-chain antibody and application thereof in detecting beta-stimulant
  • Single-chain antibody and application thereof in detecting beta-stimulant
  • Single-chain antibody and application thereof in detecting beta-stimulant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, discovery of single-chain antibody

[0028] Using mouse splenocytes as the gene source, a natural phage single-chain antibody display library was constructed, and clenbuterol was used as the antigen to screen and obtain immunoaffinity single-chain antibody from the library. Based on this single-chain antibody, the 3D structure of the protein was simulated through homology modeling, and the computer simulation docking technology was used to analyze the recognition site and binding ability of the single-chain antibody to different β-agonists. Under the guidance of this antigen-antibody interaction analysis, site-directed mutagenesis of key sites was carried out and a ribosome display library was constructed, from which single-chain antibodies with broad-spectrum recognition ability and high binding ability to β-agonists were screened.

[0029] 1. Construction and screening of natural phage single-chain antibody display library

[0030] (1) Library construct...

Embodiment 2

[0047] Embodiment 2, the preparation of single-chain antibody

[0048] 1. Construction of recombinant plasmids

[0049] 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 374th nucleotides are the heavy chain variable region Coding sequence, the 375th to 434th nucleotides are the coding sequence of the connecting peptide, the 435th to 761st nucleotides are the coding sequence of the light chain variable region, and the 771st to 800th nucleotides are the C-myc tag tag The coding sequence, the 801st to 806th nucleotides are the XhoI restriction recognition sequence).

[0050] 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.

[0051] F1: 5'- CCATGG AAGAAGTTCAGC-3';

[0052] R1: 5'- CTCGAG CAGGTCTT...

Embodiment 3

[0067] Embodiment 3, the application of single-chain antibody

[0068] 1. Preparation of CLE-OVA

[0069] Clenbuterol hydrochloride (CLE): purchased from Sigma-Aldrich, USA, with catalog number C275.

[0070] 1. Coating agent and its preparation

[0071] The hapten CLE is coupled to the carrier protein OVA by the diazotization method. Since CLE has a hapten with a primary aryl amino group, the primary aryl amino group generates an electrophilic diazonium salt under strong acid and cooling conditions, and the strong electron-donating group in the carrier protein A reaction occurs to generate diazonium products, forming the coating antigen CLE-OVA. The preparation process is as follows:

[0072] (1) Dissolve 5 mg of clenbuterol hydrochloride in 3 ml of 0.1M hydrochloric acid aqueous solution to obtain an aqueous solution of clenbuterol hydrochloride, and pre-cool to 0°C; add 10 mg of NaNO to the aqueous solution of clenbuterol hydrochloride 2, stirred at 4°C for 5 h to form ...

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Abstract

The invention discloses a single-chain antibody and application thereof in detecting a beta-stimulant. 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 122nd 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 138th to 246th 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 beta-stimulants, is suitable for quick immune detection for various types of multi-residue beta-stimulants and has great significance for detecting the beta-stimulant.

Description

technical field [0001] The invention relates to a single-chain antibody and its application in detecting beta-agonists. Background technique [0002] β-agonist (β-agonist) full name β-adrenoceptor agonist (β-adrenoceptor agonist), is a class of phenethylamines (phenethylamines, PEAs). Common β-stimulants are clenbuterol, ractopamine, albuterol, etc. [0003] According to the substituents of the benzene ring, PEAs can be divided into aniline-type and phenol-type: aniline-type PEAs have primary aryl amine groups in their structure and are moderately polar; phenol-type PEAs generally have an ortho (resorcinol) or phenol structure. β-agonists can selectively bind to β-adrenergic receptors on the cell membrane to regulate sympathetic nerve excitation and relax tracheal smooth muscle. Therefore, they are often used clinically in the treatment of asthma-like diseases in humans and animals. When the dosage of β-agonist is 5-10 times of the clinical dosage, it can affect the flow ...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N15/13C12N15/63C12N1/21C12N5/10C12P21/08G01N33/577
Inventor 沈建忠温凯王战辉张素霞史为民丁双阳江海洋吴聪明孟辉
Owner CHINA AGRI UNIV
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