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

A technology of single-chain antibody and staphylococcus, applied in application, antibody, antibacterial drug, etc., can solve problems such as difficult to cure, unsatisfactory effect, easy to produce drug resistance of antibiotics, etc., achieve good application prospects, inhibit adhesion and damage, inhibit the pathogenic effect of Staphylococcus aureus

Active Publication Date: 2020-10-30
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because Staphylococcus aureus is contagious and resistant to the antibiotics used to treat it, it is difficult to completely cure it
Vaccines against Staphylococcus aureus whole bacteria and various virulence factors are also used to prevent mastitis in dairy cows, but the effect is not satisfactory

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of Bovine Phage Single-Chain Antibody Primary Library

[0044] The construction of the bovine phage single-chain antibody primary library includes the following five steps, respectively:

[0045] 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. 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).

[0046] 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 o...

Embodiment 2

[0055] Example 2 Screening of bovine-derived anti-Staphylococcus aureus virulence factor LukE single-chain antibody

[0056] The screening of bovine-derived anti-Staphylococcus aureus virulence factor LukE single-chain antibody includes the following two steps, respectively:

[0057] 6) Enrichment panning

[0058] Prepare the S component (LukE) of the two-component leukocidin LukED from Staphylococcus aureus (ATCC25923), use it as an antigen, and coat it overnight at 4°C; seal the 96-well plate with PBST containing 4% skimmed milk powder, and incubate at 37°C 2h; add the single-chain antibody phage antibody primary 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; add 100ul 0.2mol per well / 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 Escherichia col...

Embodiment 3

[0061] Example 3 Prokaryotic expression and purification of single-chain antibody pGEX-4T-1-scFv

[0062] The prokaryotic expression and purification of the single-chain antibody pGEX-4T-1-scfv include the following two steps, respectively:

[0063] 8) Construction of recombinant plasmid pGEX-4T-1-scFv

[0064] With No. 8 positive clone strain as a template, as shown in Table 2, use specific primers as the second primer VL-F and the second primer VH-R to amplify the scFv target gene, wherein the nucleoside of the second primer VL-F The acid sequence is as shown in SEQ ID No.12, and the nucleotide sequence of the second primer VH-R is as shown in SEQ ID No.13; the second primer VL-F and the second primer VH-R respectively comprise enzyme cutting sites BamH I and enzyme cutting site Xho I, wherein the nucleotide sequence of enzyme cutting site BamH I is: GGATCC, the nucleotide sequence of enzyme cutting site Xho I is: CTCGAG; select restriction endonuclease BamH I and Xho I do...

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Abstract

The invention discloses a bovine-derived anti-staphylococcus aureus single-chain antibody and preparation and application thereof, and relates to the field of single-chain antibody genetic engineering. The single-chain antibody at least comprises a bovine antibody light chain variable region (VL), a bovine antibody heavy chain variable region (VH) and an intermediate linker peptide (Linker), and the connection sequence is VL-Linker-VH. The preparation method comprises the following steps of: obtaining a bovine-derived single-chain antibody gene, constructing a bovine-derived single-chain antibody primary library, enriching and elutriating, and constructing a single-chain antibody prokaryotic expression plasmid pGEX-4T-1-scFv to obtain a single-chain antibody prokaryotic expression protein.The composition can be used for treating bovine mastitis caused by staphylococcus aureus. The single-chain antibody disclosed by the invention can be specifically combined with staphylococcus aureusLukE protein to inhibit the membrane lysis effect of LukED on bovine mammary epithelial cells, so that the adhesion and damage of staphylococcus aureus to bovine mammary epithelial cells are weakened,and the single-chain antibody has a certain function of inhibiting the damage of staphylococcus aureus to mammary glands.

Description

technical field [0001] The invention relates to the field of bovine-derived antibody drugs, in particular to a bovine-derived single-chain antibody against Staphylococcus aureus (abbreviated as Staphylococcus aureus) and its preparation and application. 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 antibodies mainly include fusion expression, intracellular expression and secretory expression. Compared with intact antibodies, single-chain antibodies have the following advantages: 1) Small molecular weight, only one-sixth of the size of intact antibodies, and low immunogenicity; 2) Strong tissue penetration, easy t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/70A61K39/40A61P31/04A61P15/00
CPCC07K16/1271C07K16/005C12N15/70A61P31/04A61P15/00C07K2317/20C07K2317/56C07K2317/622C07K2317/76A61K2039/505
Inventor 朱建国张蕾王凤青闫洁新程曼玲张凡庆
Owner SHANGHAI JIAO TONG UNIV
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