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A bovine-derived anti-staphylococcus aureus eukaryotic expression single chain antibody, a preparing method thereof and uses of the antibody

A staphylococcus, single-chain antibody technology, applied in the field of genetic engineering, can solve the problems of difficult to cure, easy to produce resistance to antibiotics, unsatisfactory effect, etc., to achieve the effect of long duration, reduced content, and reduced infiltration

Inactive Publication Date: 2017-02-15
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 Staphylococcus aureus is contagious and the antibiotics used for treatment are easy to produce drug resistance, so it is difficult to cure completely; currently, vaccines against the whole strain of Staphylococcus aureus and various virulence factors are also used for the prevention of cow mastitis , but the effect is not ideal

Method used

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  • A bovine-derived anti-staphylococcus aureus eukaryotic expression single chain antibody, a preparing method thereof and uses of the antibody
  • A bovine-derived anti-staphylococcus aureus eukaryotic expression single chain antibody, a preparing method thereof and uses of the antibody
  • A bovine-derived anti-staphylococcus aureus eukaryotic expression single chain antibody, a preparing method thereof and uses of the antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of bovine phage single-chain antibody library

[0029] 1. Collect the blood of dairy cows suffering from mastitis. When the serum antibody titer is greater than 1:20000 by ELISA, continue the follow-up experiment. Bovine peripheral blood leukocytes were extracted from 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).

[0030] 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 according to the FR region (Table 1), wherein VH F and VH R are used to amplify the VH region; VL F and VL R are used to amplify the VL reg...

Embodiment 2

[0035] Example 2 Screening of bovine-derived anti-Staphylococcus aureus single-chain antibody

[0036] 1. Prepare the whole bacterial antigen of Staphylococcus aureus (ATCC25923) by enrichment and panning, and coat overnight at 4°C; seal the 96-well plate with PBS containing 4% skimmed milk powder and incubate at 37°C for 2h; 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: wash with PBS 20 times after elution with PBST f...

Embodiment 3

[0038] Example 3 Recombinant scFv sequence analysis

[0039] The obtained single-chain antibody coding gene was sequenced to prove that it was inserted into the phagemid vector pCANTAB5E according to the correct reading frame sequence. The amino acid sequence is shown in SEQ ID No.3, and its sequence sequence is VL-Linker-VH ( image 3 ).

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Abstract

A bovine-derived anti-staphylococcus aureus eukaryotic expression single chain antibody, a preparing method thereof and uses of the antibody are disclosed. The eukaryotic expression single chain antibody at least comprises a bovine-derived single chain antibody segment VL-Linker-VH formed by connecting a bovine antibody light chain variable region VL having an amino acid sequence shown as SEQ ID No.1, a bovine antibody heavy chain variable region VH having an amino acid sequence shown as SEQ ID No.2, and a middle connecting peptide Linker according to a VL-Linker-VH order. After a coding gene of the segment is inserted into an eukaryotic expression vector to construct a single chain antibody eukaryotic expression plasmid pcDNA3.1-scFv, the pcDNA3.1-scFv is directly injected to a mouse mammary tissue, and therefore contents (P<0.05) of cell factors IFN-gamma and IL-4 can be significantly increased, the content of an inflammatory factor TNF-alpha (P<0.05) can be reduced, relative integrity of the mammary structure can be maintained, infiltration of inflammatory cells is reduced and a mouse is provided with certain protection.

Description

technical field [0001] The invention relates to a bovine-derived eukaryotic expression single-chain antibody against Staphylococcus aureus, a preparation method and an application thereof, belonging to the technical field of genetic engineering. 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 to enter ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/70A61K39/40A61P31/04A61P15/14
CPCC07K16/1271A61K2039/507Y02A50/30
Inventor 朱建国王曼王婷婷
Owner SHANGHAI JIAO TONG UNIV
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