Anti-DON single-chain antibody ScFv and preparing method and application thereof

A single-chain antibody and anti-vomiting technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of antibody hybridoma cell lines that are prone to mutation, expensive, and cell line instability

Inactive Publication Date: 2011-03-09
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the shortcomings of the above prior art, and aims at many shortcomings such as easy mutation of antibody hybridoma cell lines in large-scale production, unstable cell lines, easy loss of genes, and high cost. The present invention uses genetic engineering methods to provide a A heavy chain and light chain variable region gene and encoded polypeptide of a monoclonal anti-vomitoxin DON antibody, after recombination, an antibody active fragment that specifically recognizes and binds to vomitoxin DON is expressed, making it possible to produce antibodies on a large scale and at low cost

Method used

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  • Anti-DON single-chain antibody ScFv and preparing method and application thereof
  • Anti-DON single-chain antibody ScFv and preparing method and application thereof
  • Anti-DON single-chain antibody ScFv and preparing method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1. Establishment of anti-vomitin monoclonal antibody McGill078 hybridoma cell line and preparation of anti-vomitoxin monoclonal antibody

[0026] The antigen was synthesized by literature method (Casale WL, Journal of Agriculture and Food Chemistry, 1988, 36:663-668), and the hybridoma cell line was established according to conventional methods (Wu Jianxiang, Acta Microbiologica Sinica, 2000, 40 (6 ): 638-645) named McGill078, the McGill078 hybridoma cell line was cultured, subcultured, resuspended and inoculated in the abdominal cavity of mice, and about 15 ml of ascites was collected after the abdomen was swollen 10 days later. The anti-vomitin antibody in rat ascites was purified by rapid batch adsorption method, and concentrated by ultrafiltration membrane dialysis; the concentration of this monoclonal antibody was determined to be 9.2 mg / ml, and the antibody type was IgG1 (γ 1 , κ), with good antigen specificity and antigen affinity.

Embodiment 2

[0027] Example 2. Cloning of heavy chain and light chain variable region genes of anti-vomitin monoclonal antibody McGill078

[0028] 1. The monoclonal hybridoma cell line McGill078 used can secrete antibodies that specifically recognize vomitoxin DON, and the molecular subtype of the secreted antibodies is IgG 1 , whose heavy and light chain isotypes are γ 1 / K.

[0029] 2. Take the McGill078 hybridoma cells (2×10) in the logarithmic growth phase 6), using Trizol (Invitrogen) reagent to extract total RNA, isolate and purify mRNA (PolyATtract mRNA Isolation Systems, Promega), according to Invitrogen kit (SuperScript III First-Strand Synthesis System for RT-PCR), with Oligo(dT)20 as primer , RT-PCR reverse transcription to synthesize cDNA, then amplify the light and heavy chain variable region genes with specific primers, 1% agarose gel electrophoresis, recover the fragments with gel recovery test kit (promega company), TA clone insert pMD18- T vector, sequence determination...

Embodiment 3

[0098] Example 3. Construction and expression of single-chain antibody ScFv

[0099] ScFv was assembled by overlapping extension (SOE) with a DNA fragment encoding a hydrophilic polypeptide linker (G 4 S) 3 The cloned antibody light and heavy chain variable region genes are connected to form a 5' VH-Lingker-VL3' format. ScFv and expression vector pET26b (+) (Novagen Company) were digested with restriction endonucleases BamHI and HindIII, connected with T4 ligase, transformed into Escherichia coli TOP10F' for expression, and then targeted for expression in the form of inclusion bodies (IBS) ScFv expression system, optimization of IPTG-induced expression conditions, inclusion body isolation, denaturation, and renaturation. The results showed that ScFv was successfully expressed in Escherichia coli.

[0100] The relevant operation steps are as follows:

[0101] (1) Cloning of VH and VL genes in pMD18-T plasmid

[0102] pMD 18T-VH template (50ng)

[0103] (Or pMD18T-VL) (50n...

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Abstract

The invention relates to an anti-DON single-chain antibody ScFv and a preparing method and application thereof, belonging to biological product field. In the invention, variable region gene Fab section of heavy chain and light chain of antibody is cloned through a PCR method in a hybridoma cell strain McGill078 which secretes anti-DON monoclonal antibody; two genes are recombined through a gene engineering method; a prokaryotic expression plasmid vector is constructed and is transformed to Escherichia coli to express an ScFv antibody active fragment which can specifically recognize DON; and then the recombined single chain antibody ScFv is purified and renatured. The anti-DON recombined single-chain antibody ScFv has an activity for binding with DON and has the advantage of residual detection for DON owing to recognition and binding activity to DON and having antibody medicament prospect for treating DON poisoning after humanized modification; and the constructed transformation vector plasmid is easy to preserve and produce in a great quantity.

Description

Technical field: [0001] The invention relates to an anti-vomitin DON single-chain antibody ScFv and a preparation method thereof, a polypeptide encoded by the gene, a carrier containing the gene and a preparation method of the gene and the polypeptide, belonging to the field of biological products. Background technique: [0002] Deoxynivalenol (deoxynivalenol, DON), also known as vomitoxin, is mainly trichothecene toxins, one of the secondary metabolites produced by Fusarium fungi. The main toxin-producing bacteria are Fusarium graminearum, Snow Fusarium rot, etc. It is currently considered to be one of the major natural toxins present in scab wheat. It can cause human and animal poisoning, because of its widespread existence and a large amount of food pollution, it poses a great harm to humans and animals. At the 3rd Conference on Food Additives and Contaminants jointly held by the Food and Agriculture Organization of the United Nations and the World Health Organization i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N15/13C12N15/63C12N15/70G01N33/577C12R1/19
Inventor 张存政张晓刘贤进刘媛祭芳张强王耘
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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