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Canine single-chain antibody, and construction method and application thereof

A technology of single-chain antibody and construction method, which is applied in the biological field and can solve problems such as the difficulty of canine blood type monoclonal antibodies

Inactive Publication Date: 2014-09-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the information of canine blood group antigens is unknown, it is not possible to obtain a relatively large amount of canine blood group antigens through gene expression, biosynthesis and antigen purification.
It is very difficult to prepare monoclonal antibodies of canine blood type by the conventional method of preparing monoclonal antibodies by hybridoma method, because more antibodies to other red blood cell antigens will be produced due to the impurity of antigens, which poses a great challenge to the preparation and screening of specific antibodies challenge

Method used

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  • Canine single-chain antibody, and construction method and application thereof
  • Canine single-chain antibody, and construction method and application thereof
  • Canine single-chain antibody, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Amplification of scFv gene

[0055] (1) Isolation of Canine Peripheral Blood Lymphocytes

[0056] Immune DEA1.1-negative dogs with canine DEA1.1-positive antigen red blood cells, and immunize 5ml of 5% red blood cells through the abdominal cavity and vein each time. or heparin) anticoagulant. Whole blood was diluted with an equal volume of PBS or 0.9% NaCl (normal saline). Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. The volume ratio of separation medium, anticoagulated undiluted whole blood, and PBS (or normal saline) is 1:1:1. At room temperature, centrifuge with a horizontal rotor at 700-800g (2000-2500rpm) for 20-30min. After centrifugation, the bottom of the centrifuge tube is red blood cells, the middle layer is the separation liquid, the top layer is the plasma / tissue homogenate lay...

Embodiment 2

[0089] Example 2 Construction and panning of phage antibody library

[0090] (1) scFv linked to pCANTAB-5E

[0091] Proceed as follows:

[0092]

[0093] Ligate at 16°C for 12-16 hours, and extinguish T4 ligase at 70°C for 10 minutes.

[0094] (2) Preparation of electrotransformation competent bacteria TG1

[0095] Resuspend the ligated vector in 1 mL LB medium. Incubate overnight at 37°C, 250rpm. Inoculate into MMP (minimal medium plate) medium and culture overnight (12h) at 37°C. Pick a single colony and culture in 5mL LB medium, 37°C, 250rpm, 10h. Pipette 900 μL of stable growth phase Ecoli . For TG1 cells, add 250 μL sterilized 80% glycerol, mix well and store at -70°C. Specifically include the following steps:

[0096] A. Pick a single colony from the micro-medium plate, inoculate it in 10mL 2×YT medium, and cultivate it overnight at 37°C with shaking;

[0097] B. Dilute the above-mentioned overnight bacteria 1:100 and inoculate them into 1000mL fres...

Embodiment 3

[0128] Example 3 Detection of Positive Phage Single-Chain Antibody

[0129] (1) Positive phage antibody gene amplification and sequencing

[0130] A. Add 400 μL of 2×YT-AG medium to 96 tubes, randomly pick 96 single clones from the SOBAG plate after the third round of elutriation, and culture them overnight at 200 rpm at 30°C;

[0131] B. Add 400 μL of 2×YT-AG medium to a new tube, take 40 μL of overnight cultured cells into a new tube, and save the remaining bacterial solution in glycerol for later use;

[0132] C. Take a new tube and incubate at 30°C for 2 hours at 200 rpm. Centrifuge at room temperature at 1500g for 20min, discard the supernatant;

[0133] D. Centrifuge to prepare 50mL 2×YT-AI medium, transfer 400μL to each tube;

[0134] E. Incubate at 30°C for 3-4 hours, 200rpm. Centrifuge at 1500 g for 20 min at room temperature;

[0135] F. Carefully transfer 320 μL of supernatant to a new tube. Add 80 μL blocking solution to block 320 μL supernatant, incuba...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a canine single-chain antibody, and a construction method and application thereof. By designing the canine antibody variable region degenerate primers, the canine heavy chain variable region VH and light chain variable region VL genes are successfully amplified. On such basis, a bacteriophage display technique is utilized to successfully construct the canine single-chain antibody scFv library. The scFv single-chain antibody library has diversity and enough storage capacity. A Dot-ELISA method is utilized to screen 3 single chain antibodies capable of being combined with the canine DEA 1.1 blood group antigen from the scFv single-chain antibody library by using the canine DEA 1.1 blood group antigen, wherein Clone 16 has stronger combination characteristic. A pET-32a-Clone16 recombinant protein is constructed according to the Clone 16 gene to perform prokaryotic expression, and the purified antibody has combination activity. The canine single-chain antibody lays solid foundation for preparing canine DEA 1.1 blood grouping reagents.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, to a canine single-chain antibody and its construction method and application. Background technique [0002] Traditional antibody preparation, including hybridoma-monoclonal antibody technology, needs to immunize the body with antigen first, and the performance of the obtained antibody depends on the effectiveness of in vivo immunization. Therefore, it is difficult to prepare antibodies against weak antigens, self-antigens, toxic antigens, and human antibodies. The phage antibody library technology developed in the 1990s is a major advance in the field of antibody engineering, and it has become an important way to develop human monoclonal antibodies. The phage antibody library technology simulates the process of antibody production in vivo, which provides the possibility to prepare antibodies without immunization, and has gradually become one of the important means of prep...

Claims

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Application Information

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IPC IPC(8): C07K16/34C12N15/11C12N15/13C12N15/70C40B40/10C40B50/06G01N33/80
Inventor 李守军李华涛贾坤孙凌霜
Owner SOUTH CHINA AGRI UNIV
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