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Canine-derived anti-canine parvovirus antibody, antibody library and construction method thereof

A canine parvovirus and a construction method technology, applied in the field of antibody engineering, can solve the problems of cumbersome process, high preparation cost, host immune rejection or allergic reaction, etc.

Active Publication Date: 2020-04-07
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) Traditional mouse-derived monoclonal antibodies have strong heterogeneity and immunogenicity to non-mouse-derived organisms, which can easily cause immune rejection or allergic reactions of the host when used in vivo, which reduces the curative effect of mouse-derived monoclonal antibodies, and even It has serious consequences in the body, which greatly limits the effect of clinical application of mouse monoclonal antibodies
[0006] (2) Traditional mouse-derived monoclonal antibodies require animals, high preparation costs, and long production cycles
[0007] (3) Traditional mouse-derived monoclonal antibody hybridomas require single-cell cloning, the process is cumbersome, and the reaction intensity of monoclonal antibodies is not high
Canine parvovirus is one of the most serious infectious diseases that harm dogs. At present, there is no anti-canine parvovirus antibody that is widely accepted by consumers in the market. An efficient and low-cost anti-canine parvovirus antibody It is not only the needs of the general market, but also plays a very important role in the research and prevention of canine parvovirus and the development of my country's dog industry

Method used

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  • Canine-derived anti-canine parvovirus antibody, antibody library and construction method thereof
  • Canine-derived anti-canine parvovirus antibody, antibody library and construction method thereof
  • Canine-derived anti-canine parvovirus antibody, antibody library and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Construction of canine-derived anti-canine parvovirus single-chain antibody library

[0081] 1. Isolation of Canine Peripheral Blood Lymphocytes

[0082] Dogs that were infected and resistant to parvo were collected from a dog farm and immunized with vaccines. The interval between each immunization was 2 weeks, and a total of 3 immunizations were performed. Two weeks after the dog group was immunized for three times, the peripheral blood of the dogs was collected. Take 5-10mL of fresh blood and mix evenly with whole blood and tissue diluent at a ratio of 1:1-1:2. Add cell separation solution to a 15mL centrifuge tube, and gently add an equal volume of diluted anticoagulant blood along the tube wall. The horizontal centrifuge is centrifuged at a speed of 400g-800g and a time of 15min-25min. After centrifugation, the liquid in the centrifuge tube should be divided into four layers, from top to bottom: the first layer: plasma layer; the second layer: lymphocyt...

Embodiment 2

[0089] Example 2 Screening of canine-derived anti-canine parvovirus single-chain antibody library

[0090] The configuration of the main solution:

[0091] Blocking solution: 0.05g BSA was dissolved in 100mL PBS, fully dissolved, and stored in a 4°C refrigerator.

[0092] Wash buffer PBST: NaCl 8.0g, KCl 0.2g, NaCl 2 HPO 4 1.15g, KH 2 PO 4 0.2g, 950mL deionized water, and dilute to 1000mL. Add 100 μL Tween 20.

[0093] Stop solution: 0.5g BSA, 0.75g glycine dissolved in 100mL ddH 2 O, adjust the pH to 2.2 with 1M hydrochloric acid, and store in a refrigerator at 4°C.

[0094] Neutralization buffer: Tris 12.2g, ddH 2 O 50mL, fully dissolved, frozen in 4 ℃ refrigerator.

[0095] 1. Preparation of Phage Antibody

[0096] Add 1 mL of phage primary antibody library to 3 mL OD 600 =0.5 XLI-Blue bacteria solution, put it at 37°C for 45 minutes, add 6 mL of LB liquid medium containing Amp+ (100 μg / mL), and add glucose (1mol / L) at a ratio of 1:1000 to continue the cultivat...

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Abstract

The invention belongs to the technical field of genetic engineering, and discloses a canine-derived anti-canine parvovirus antibody, an antibody library and a construction method thereof. The canine-derived anti-canine parvovirus antibody is a heavy chain variable region with an amino acid sequence of SEQ ID NO: 1 or a light chain variable region with an amino acid sequence of SEQ ID NO: 2. The nucleotide sequence of DNA molecule of the heavy chain variable region is SEQ ID NO: 3. The nucleotide sequence of DNA molecule of the light chain variable region is SEQ ID NO: 4. Compared with a monoclonal antibody produced by immune animals, the phage display technology of the invention has the advantages of fast production, simple operation and higher efficiency, can be adopted to produce canine-derived antibodies, and has higher utilization value. According to the invention, by constructing the phage display antibody library, the process of antibody generation can be simulated in vitro, andantibodies against any antigen can be screened.

Description

technical field [0001] The invention belongs to the technical field of antibody engineering, and in particular relates to a canine-derived anti-canine parvovirus antibody, an antibody library and a construction method. It specifically relates to a heavy chain and light chain variable region of a canine anti-canine parvovirus antibody, including its amino acid sequence and nucleotide sequence. Background technique [0002] At present, the closest prior art: canine parvovirus disease is a highly contagious severe infectious disease of dogs, clinically characterized by acute hemorrhagic enteritis and myocarditis, and is the most important severe infection that endangers dogs one of the diseases. For canine parvovirus infection, there is currently no specific treatment product in the market, and symptomatic treatment, supportive therapy, and specific therapy such as hyperimmune serum and monoclonal antibody are often used. [0003] Canine parvovirus monoclonal antibody can inh...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/11C40B40/10C40B50/06
CPCC07K16/081C40B40/10C40B50/06C07K2317/56C07K2317/622
Inventor 原昆鹏尹燕博
Owner QINGDAO AGRI UNIV
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