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Bovine derived anti-staphylococcus aureus fusion antibody scFv-Fc and preparation method thereof

A scfv-fc, staphylococcus technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory vaccine treatment effect and health threat

Inactive Publication Date: 2018-12-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, cow mastitis caused by Staphylococcus aureus is mainly treated with antibiotics, but this treatment method leads to the emergence of more and more drug-resistant strains, and the resistant milk also poses a certain threat to human health; on the other hand On the one hand, vaccines are mainly used. At present, there are many vaccines made against virulence factors such as live or inactivated bacteria of Staphylococcus aureus, isolated adhesins, toxins and polysaccharides. It may be due to the variation of bacterial antigens that the pathogenic factors The structure has changed, and none of these vaccines are effective therapeutically

Method used

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  • Bovine derived anti-staphylococcus aureus fusion antibody scFv-Fc and preparation method thereof
  • Bovine derived anti-staphylococcus aureus fusion antibody scFv-Fc and preparation method thereof
  • Bovine derived anti-staphylococcus aureus fusion antibody scFv-Fc and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of bovine phage single-chain antibody library

[0031] 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. Anticoagulated blood was extracted from peripheral nuclear lymphocytes, 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).

[0032] 2: Design primers for amplifying the light and heavy chains of the antibody (Table 1) according to the FR regions of the variable region sequence of the bovine antibody coding gene (Madhuri Kotia, 2010 Molecular Immunology; 2011, Vaccine) published in the literature, wherein VH F and VH R Used to amplify the VH...

Embodiment 2

[0038] Example 2 Screening of anti-Staphylococcus aureus single-chain antibody

[0039] 1: Enrichment and panning to prepare the whole bacterial antigen of Staphylococcus aureus (ATCC25923), coat overnight at 4°C; seal the 96-well plate with PBS containing 4% skimmed milk powder and incubate at 37°C for 2 hours; add the above step to the 96-well plate The prepared single-chain antibody phage antibody library was incubated at 37°C for 2 hours, washed 10 times with PBST and PBS respectively, and unbound free phages were washed away; 100 μl of 0.2 mol / L Gly-Hcl buffer (pH=2.2) was added to each well The specifically bound phages were eluted, and 50 μl of 1mol / L Tris-Hcl (PH=9.1) was added to neutralize the eluate; after the remaining part of the eluate was infected with Escherichia coli TG1, the above steps were repeated. Repeat this for 3-5 rounds. After the first round, the stringency of washing should be increased. Before elution, wash with PBS 20 times after elution with PBST...

Embodiment 4

[0043] Example 4 Expression and Purification of Bovine Fusion Antibody scFv-Fc

[0044] After extracting the plasmid from the positive clones screened in Example 2, they were double-digested with NcoI and NotI endonucleases, and the bovine IgG Fc gene amplified in Example 3 was double-digested with NotI and XhoI endonucleases. The pET-28a(+) vector digested with NcoI and XhoI was ligated to construct a recombinant plasmid, and then transformed into Rosetta cells for induced expression, and then the bovine fusion antibody scFv-Fc was purified by Ni-NTA affinity chromatography.

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Abstract

The invention discloses a bovine derived anti-staphylococcus aureus fusion antibody scFv-Fc and a preparation method thereof. The fusion antibody scFv-Fc at least has a light chain variable region ofthe amino acid sequence shown as SEQ ID No:1, a heavy chain variable region of the amino acid sequence shown as SEQ ID No:2, an intermediate connecting peptide between the light chain variable regionand the heavy chain variable region, and a Fc fragment of the amino acid sequence shown as SEQ ID No:3. The ELISA result shows that the fusion antibody can be specifically bound to a staphylococcus aureus antigen; in in-vitro bacteriostatic test, the fusion antibody can completely inhibit the growth of staphylococcus aureus within 24h; moreover, the fusion antibody can mediate dairy cow peripheralblood phagocytes in terms of playing the role of conditioning phagocytosis. The fusion antibody can be used in related reagents or drugs for diagnosis, prevention and treatment of staphylococcus aureus cow mastitis.

Description

technical field [0001] The invention relates to a bovine-derived anti-staphylococcus aureus fusion antibody scFv-Fc and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Cow mastitis refers to the inflammation of cow mammary gland tissue, which is a common and frequently-occurring disease that affects the development of the dairy industry and brings huge losses to dairy production. There are many pathogenic bacteria that cause mastitis in dairy cows, among which Staphylococcus aureus is the most important pathogenic bacteria. On the one hand, cow mastitis caused by Staphylococcus aureus is mainly treated with antibiotics, but this treatment method leads to the emergence of more and more drug-resistant strains, and the resistant milk also poses a certain threat to human health; on the other hand On the one hand, vaccines are mainly used. At present, there are many vaccines made against virulence factors such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/73A61K39/40A61P31/04A61P15/14G01N33/569
CPCA61P15/14A61P31/04C07K16/1271C07K2317/31C07K2317/622C07K2317/72C07K2317/76C12N15/73G01N33/56938G01N2333/31G01N2800/365
Inventor 朱建国王曼王婷婷王凤青张波
Owner SHANGHAI JIAO TONG UNIV
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