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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 many gaps in the research on the antigenic sites produced by FMDV broad-spectrum neutralizing antibodies

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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