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Bovine-derived anti-staphylococcus aureus single-chain antibody, preparation method and application thereof

A single-chain antibody and Staphylococcus technology, applied in the field of genetic engineering, can solve problems such as difficult to cure and easy to produce resistance to antibiotics, and achieve good application prospects

Inactive Publication Date: 2013-01-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that on the one hand, Staphylococcus aureus is contagious, and on the other hand, Staphylococcus aureus is prone to resistance to antibiotics used for treatment, so it is difficult to completely cure

Method used

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  • Bovine-derived anti-staphylococcus aureus single-chain antibody, preparation method and application thereof
  • Bovine-derived anti-staphylococcus aureus single-chain antibody, preparation method and application thereof
  • Bovine-derived anti-staphylococcus aureus single-chain antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of Bovine Anti-Mastitis Single Chain Antibody

[0029] 1: Collect the blood of dairy cows suffering from mastitis, and when the serum antibody titer detected by ELISA is greater than 1:20000, continue the follow-up experiment. The anticoagulated blood was extracted from the peripheral nuclear monocytic cells, and the total RNA was extracted by the Trizol method (TRIZOL Reagent was purchased from TaKaRa Company). Using the extracted total RNA as a template, Oligo primer was used to synthesize the first-strand cDNA according to the product instructions of the reverse transcription kit (cDNA first-strand synthesis kit was purchased from TaKaRa Company).

[0030] 2: Design primers for amplifying the light and heavy chains of the antibody (Table 1) according to the FR region of the variable region sequence of the bovine antibody coding gene (Madhuri Kotia, 2010 Molecular Immunology; 2011, vaccine) published in the literature (Table 1), where VH1F and VH1R are use...

Embodiment 2

[0042] Indirect ELISA Screening of Antigen-specific ScFv

[0043] Prepare the whole bacterial antigen of Staphylococcus aureus (ATCC-25923) and coat it with 50mmol / L sodium bicarbonate solution (pH9.6) at 4°C overnight, block with 5% skimmed milk powder solution for 1h, and then use PBST (containing 0.1% Tween20, The same below) and wash 3 times; take 50 μL of the supernatant of the TES-treated bacteria liquid, mix it with 50 μL of 4% skimmed milk powder solution, add the above-mentioned coated Staphylococcus aureus antigen, react at 37 ° C for 2 hours, and wash with PBST; add Myc -Tag Mouse mAb (Myc-tag mouse monoclonal antibody purchased from RayBioTech Company) 100 μL (1:2000) reacted at 37°C for 2 h, washed with PBST; added Peroxidase-conjugated Affinipure GoagtAnti-Mouse IgG (purchased from Abmart Company) 100 μL (1:2000) 4000), react at 37°C for 1 h, wash with PBST; develop color with TMB, stop the reaction with 2mol / L sulfuric acid, and read the OD450 value with a micro...

Embodiment 3

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[0047]

[0048]

[0049]

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Abstract

The invention relates to a bovine-derived anti-staphylococcus aureus single-chain antibody. The antibody at least comprises a light chain variable region of an amino acid sequence which is represented in SEQ ID No.1, a heavy chain variable region of an amino acid sequence which is represented in SEQ ID No.2 and an intermediate connecting peptide which is located between the light chain variable region and the heavy chain variable region. The single-chain antibody can be specifically bound with staphylococcus aureus, has certain external neutralization activity and has good application prospects when used in related reagents or drugs for diagnosing, preventing and treating staphylococcus aureus bovine mastitis.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered single-chain antibody against Staphylococcus aureus dairy cow mastitis, an expression vector and host cell expressing the single-chain antibody, a preparation method of the single-chain antibody and its use. The single-chain antibody can specifically bind to Staphylococcus aureus and has a certain neutralizing activity in vitro. Background technique [0002] A single-chain antibody is a genetically engineered antibody that uses recombinant technology to convert the light chain variable region V of the antibody into a single-chain antibody. L and heavy chain variable region V H It is formed by connecting the end to the end of a short peptide linker, which is the smallest functional fragment that retains the complete antigen binding site. There are three forms of single-chain antibody: 1) direct expression in the cytoplasm; 2) fusion with ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/63C12N15/70C12N1/21C12P21/02A61K39/085A61P31/04G01N33/569
Inventor 朱建国王曼李本强张艳玲
Owner SHANGHAI JIAO TONG UNIV
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