Preparation method for fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses

A technology for foot-and-mouth disease virus and bovine antibody, applied in chemical instruments and methods, anti-viral immunoglobulin, immunoglobulin and other directions, to achieve the effect of good genetic diversity and high efficiency

Active Publication Date: 2018-12-14
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Neutralizing antibodies are an important part of FMD protective immunity, but there are still ma...

Method used

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  • Preparation method for fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses
  • Preparation method for fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses
  • Preparation method for fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses

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preparation example Construction

[0034] The invention provides a method for preparing a broad-spectrum neutralizing antibody of whole bovine source O-type foot-and-mouth disease virus, comprising the following steps:

[0035] 1) Use O-type foot-and-mouth disease virus to immunize cattle for the first time, within 30-60 days after the first immunization, carry out the second immunization, and within 120-150 days after the first immunization, carry out the third immunization; Described O type foot-and-mouth disease virus comprises following three lineages: Southeast Asia topological strain, Chinese topographic strain and Central and Southeast Asian topological strain; Foot-and-mouth disease viruses have different lineages;

[0036] 2) After the third immunization, separate peripheral blood mononuclear cells, and use bait antigens to screen O-type foot-and-mouth disease virus antigen-specific single B cells; the bait antigens include biotin or fluorescent protein-labeled O-type foot-and-mouth disease virus;

[...

Embodiment 1

[0049] animal immunity

[0050]A total of 3 healthy 1-year-old Qinchuan cattle were selected for the preparation of bovine-derived monoclonal antibodies, numbered #2334, #1217 and #0005, respectively. All the cattle used in the experiment were raised in the Lanzhou Veterinary Research Biosafety Level 3 (P3) laboratory of the Chinese Academy of Agricultural Sciences. For the first immunization, 2ml of O / Mya98 / JX / 2010FMDV (Southeast Asia topological type) containing 10,000 BID50 cattle-adapted O / Mya98 / JX / 2010FMDV (topological type in Southeast Asia) was inoculated by subcutaneous inoculation on the tongue surface; on the 35th day after the first immunization, 5ml was inoculated by neck muscle injection. O / HN / CHA / 93FMDV (Chinese topological type) emulsified with ISA201 adjuvant for the second immunization; then on the 132nd day after the first immunization, 5ml of O / Tibet / 99FMDV (Central Southeast Asia topological type) was immunized for the third time. Regularly collect blood...

Embodiment 2

[0054] bovine antigen-specific IgG + , IgM + , IgD + Isolation of single B cells

[0055] Bovine peripheral blood mononuclear cell isolation

[0056] Use lymphocyte separation fluid (ρ=1.083, Histopaque, Sigma-aldrich) to separate peripheral blood mononuclear cells (PBMCs) from bovine peripheral blood, and specific method is as follows:

[0057] a) Take the lymphocyte separation solution and place it at room temperature to equilibrate, and add 6 mL of the lymphocyte separation solution to each 15 mL centrifuge tube.

[0058] b) Use PBS solution to dilute bovine EDTA anticoagulant blood at a ratio of 1:1, then pipette 8 mL of diluted whole blood, and slowly add it to the upper layer of the lymphatic separation solution, taking care to avoid blood mixing into the lymphatic separation solution.

[0059] c) Put the centrifuge tube into a centrifuge equipped with a horizontal rotor, set the centrifuge speed up to 6, speed down to 1, and set the temperature to 25°C. Centrifuge ...

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Abstract

The invention relates to a preparation method for a fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses, belonging to the technical field of preparation of antibodies. The preparation method comprises the following steps: 1) performing primary, secondary and tertiary immunization on cattle by using O-type foot-and-mouth disease viruses; 2) screening antigen-specific individual B cells of the O-type foot-and-mouth disease viruses; 3) amplifying heavy-chain and light-chain variable genes of a bovine antibody; 4) acquiring the heavy-chain and light-chain constant region sequences of the bovine antibody; 5) preparing a full-length heavy-chain vector of a fully-bovine-derived monoclonal antibody and a full-length light-chain vector of the fully-bovine-derived monoclonal antibody; and 6) applying the full-length heavy-chain vector and the full-length light-chain vector of the fully-bovine-derived monoclonal antibody to cotransfection of cells, taking a cell culture supernatant, and purifying the supernatant so as to obtain the fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses. The preparation method of the invention utilizes different foot-and-mouth disease virus strains for infecting cattle and carries out screening to obtain the neutralizing antibody capable of neutralizing O-type foot-and-mouth disease viruses of three pedigrees; and the preparation method can prepare the fully-bovine-derived broad-spectrum neutralizing antibody.

Description

technical field [0001] The invention relates to the technical field of antibody preparation, in particular to a method for preparing a broad-spectrum neutralizing antibody of whole bovine source O-type foot-and-mouth disease virus. Background technique [0002] Foot-and-mouth disease (FMD) is a major animal disease that endangers the healthy development of animal husbandry in my country. It mainly affects cloven-hoofed animals such as cattle, sheep and pigs, and has a significant impact on the international trade of livestock and their products. Foot-and-mouth disease virus (FMDV) is a small RNA virus with quasi-species characteristics of RNA virus. The seven serotypes of viruses have formed many topological types with regional characteristics due to different endemic regions. A topology contains multiple lineages. There are currently three lineages of O-type FMDV in China, which are Southeast Asian topological type (O / Mya98), Central Southeast Asian topological type (O / Pan...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/85
CPCC12N15/85C07K16/1009C07K2317/76C07K2317/20C07K2317/14
Inventor 李坤卢曾军刘在新曹轶梅包慧芳陈应理王省李平花孙普白兴文李冬
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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