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Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof

A technology of single-chain antibody and staphylococcus, applied in application, antibody, anti-bacterial immunoglobulin, etc., can solve the problem of no anti-staphylococcus aureus and achieve the effect of inhibiting the pathogenic activity of staphylococcus aureus

Pending Publication Date: 2021-10-12
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no genetically engineered single-chain antibody against Staphylococcus aureus

Method used

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  • Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof
  • Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof
  • Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of bovine phage single-chain antibody library

[0055] 1. Collect the blood of dairy cows suffering from mastitis. When the ELISA method detects that the serum antibody titer is greater than 1:20000, continue the follow-up experiment. Bovine peripheral blood leukocytes were extracted with anticoagulated blood, and total RNA was extracted by 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).

[0056] 2 Analyze the variable region sequence of the bovine antibody coding gene in the published literature, and design primers for amplifying the light and heavy chains of the antibody (Table 1) according to the FR region, wherein VH F and VH R are used to amplify the VH region; VL F and VL R are used t...

Embodiment 2

[0063] Example 2 Screening of bovine-derived anti-Staphylococcus aureus virulence factor LukD single-chain antibody

[0064] 1 Enrichment and panning to prepare the F component (LukE) of the two-component leukocidin LukED of Staphylococcus aureus (ATCC25923), use it as an antigen, and coat overnight at 4°C; block 96 wells with PBST containing 4% skimmed milk powder Plate, incubate at 37°C for 2h; add the single-chain antibody phage antibody library prepared in the above steps to the 96-well plate, incubate at 37°C for 2h, wash with PBST and PBS 10 times each, and wash off unbound free phage; each well Add 100ul 0.2mol / L Gly-Hcl buffer (PH=2.2) to elute the specifically bound phage, add 50ul 1mol / L Tris-Hcl (PH=9.1) to neutralize the eluate; infect the remaining part of the eluate After E. coli TG1, repeat the above steps. Repeat this for 3-5 rounds. After the first round, the stringency of washing should be increased: wash with PBS 20 times after elution with PBST for 20 time...

Embodiment 3

[0066] Example 3 Prokaryotic expression and purification of single chain antibody pGEX-4T-1-scFv-22

[0067] 1 Construction of the recombinant plasmid pGEX-4T-1-scFv Using the No. 22 positive clone strain as a template, use specific primers (as shown in Table 2) to amplify the scFv-22 target gene, and select restriction enzymes EcoR I and Xho I double-enzyme-digest the target gene and the prokaryotic expression vector pGEX-4T-1, connect to obtain the recombinant plasmid after digestion, transform it into DH5α competent, colony PCR and double-enzyme digestion of the plasmid verify that the correct clone is sent to Shanghai Boshang Biotechnology Limited Sequencing;

[0068] Table 2 Primers for amplifying single-chain antibodies and the size of their amplified fragments

[0069]

[0070]

[0071]The clones with correct sequencing were extracted from the plasmids, and then the recombinant plasmids were transformed into BL21 competent cells, and single clones were picked, co...

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Abstract

The invention relates to a bovine-derived single-chain antibody for resisting a staphylococcus aureus LukD virulence factor and preparation and application thereof, and specifically comprises the bovine-derived single-chain antibody for resisting the staphylococcus aureus LukD virulence factor, a carrier and a host cell for screening the single-chain antibody, a preparation method of the single-chain antibody and application of the single-chain antibody. The prokaryotic expression single-chain antibody at least comprises a light chain variable region with an amino acid sequence shown as SEQ ID No.1, a heavy chain variable region with an amino acid sequence shown as SEQ ID No.2 and an intermediate connecting peptide located between the light chain variable region and the heavy chain variable region. Compared with the prior art, the bovine-derived single-chain antibody for resisting the staphylococcus aureus LukD virulence factor can be specifically combined with staphylococcus aureus LukD protein and inhibit the membrane lysis effect of LukED on bovine mammary epithelial cells, so that the adhesion and damage of staphylococcus aureus to the bovine mammary epithelial cells are weakened, and the bovine-derived single-chain antibody has a certain function of inhibiting the damage of staphylococcus aureus to mammary gland.

Description

technical field [0001] The invention belongs to the field of bovine-derived antibody drugs, in particular to a bovine-derived single-chain antibody against Staphylococcus aureus (referred to as Staphylococcus aureus) LukD virulence factor and its preparation and application. The invention specifically relates to a bovine-derived single-chain antibody against Staphylococcus aureus LukD virulence factor, a carrier and a host cell for screening the single-chain antibody, a preparation method of the single-chain antibody and an application of the single-chain antibody. Background technique [0002] Single-chain antibody is a genetically engineered antibody, which is formed by linking the light chain variable region VL and the heavy chain variable region VH of the antibody through a short peptide linker end-to-tail through DNA recombination technology, and is the smallest functional fragment that retains the complete antigen-binding site . The expression forms of single-chain an...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N15/11C12N15/70C12N1/21A61K39/395A61P15/14C12R1/19
CPCC07K16/1271C12N15/70A61P15/14C07K2317/622C07K2317/56A61K2039/505
Inventor 朱建国张蕾王凤青程曼玲
Owner SHANGHAI JIAO TONG UNIV
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