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

A single-chain antibody, staphylococcus technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory effect, difficult to cure, and antibiotic resistance easily, and achieve better effect.

Active Publication Date: 2015-09-23
安替奔达(宁夏)生物科技有限责任公司
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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; cu

Method used

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

[0032] 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 ampli...

Embodiment 2

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

[0039]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 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 0.2mol / 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 PBS...

Embodiment 3

[0041] Example 3 Recombinant scFv sequence analysis

[0042] Sequencing of the obtained 8 single-chain antibody ZW.1, ZW.2, ZW.12, ZW.22, ZW.33, ZW.68, ZW.73, ZW.88 encoding genes, to prove that they read correctly The frame sequence is inserted into the phagemid vector pCANTAB5E, the amino acid sequence is shown in SEQ ID No.17-24, and the sequence sequence is VL-Linker-VH.

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Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to a bovine-derived anti-staphylococcus aureus genetic engineering single-chain antibody, a preparation method and application thereof. The single-chain antibody comprises a bovine antibody light-chain variable region VL, an intermediate junction peptide Linker and a bovine antibody heavy-chain variable regional VH, the connecting sequence thereof is VL-Linker-VH. The amino acid sequence of the bovine antibody light-chain variable region VL is shown as SEQ ID No: 1-8. The amino acid sequence of the bovine antibody heavy-chain variable region VH is shown as SEQ ID No: 9-16. The screened single-chain antibody (scFv) of positive clone is jointly applied to in-vitro and in-vivo inhibition staphylococcus aureus test and achieves ideal antibacterial effect. The single-chain antibody is used for preparing drugs for preventing and curing staphylococcus aureus bovine mastitis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a bovine-derived anti-staphylococcus aureus genetically engineered single-chain antibody, a preparation method and an application thereof. 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, e...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/70C12N1/21A61K39/40A61P31/04
Inventor 朱建国王曼张艳
Owner 安替奔达(宁夏)生物科技有限责任公司
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