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O-type foot-and-mouth disease virus structural protein VP1 broad-spectrum neutralizing antibody as well as preparation method and application thereof

A foot-and-mouth disease virus and structural protein technology, which is applied in the field of O-type foot-and-mouth disease virus structural protein VP1 broad-spectrum neutralizing antibody preparation, can solve the problems of poor specificity, few types, and weak inhibitory effect of O-type foot-and-mouth disease virus, etc., to prevent invasion, The effect of inhibiting infection

Active Publication Date: 2022-04-12
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

[0004] Neutralizing antibodies, especially broad-spectrum neutralizing antibodies, are an important part of FMD protective immunity, but the types of O-type foot-and-mouth disease virus neutralizing antibodies commonly used Less, and poor specificity, weak inhibitory effect on O-type foot-and-mouth disease virus

Method used

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  • O-type foot-and-mouth disease virus structural protein VP1 broad-spectrum neutralizing antibody as well as preparation method and application thereof
  • O-type foot-and-mouth disease virus structural protein VP1 broad-spectrum neutralizing antibody as well as preparation method and application thereof
  • O-type foot-and-mouth disease virus structural protein VP1 broad-spectrum neutralizing antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of neutralizing antibody 9H6-VP1

[0028] 1. Preparation of hybridoma cells

[0029] 1.1 Immunization of mice

[0030] The structural protein VP1 of O-type foot-and-mouth disease virus (O / Mya98) was used as an antigen to immunize 5-6 week-old Balb / c mice. The immunization method was multi-point subcutaneous injection on the back, and the injection volume of each mouse was 100 μg. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. The interval between each immunization was two weeks. The mouse serum was collected one week after the third immunization to detect the antibody titer. When the antibody titer is not low At 1:1440, impulse immunization was performed, that is, the antigen was injected intraperitoneally, and three days after the impulse immunization, the spleen of the mouse was taken for fusion with myeloma cells.

[0031] 1.2 Cell Fusion

[0032]...

Embodiment 2

[0051] Example 2 Determination of broad-spectrum reactivity of neutralizing antibody 9H6-VP1

[0052] 1. Western blot test

[0053] A-type FMD virus strains A / AF72, A / GDMM / 2013, A / WH / CHA / 09, O-type FMD virus strains O / ZK / 93, O / Mya / 98 and O / HK / 93 strains ( The virus strains were all preserved by the National Reference Laboratory of Foot-and-Mouth Disease) to infect BHK-21 cells, and the cells were collected 6 hours after infection, and the samples were prepared for WB test, and the prepared antibody 9H6-VP1 was used as the primary antibody for Western blot test.

[0054] The specific experimental process is as follows:

[0055] (1) Electrophoresis: Add the denatured O-type and A-type FMDV antigens to 12% prefabricated gel holes;

[0056] (2) Load the sample at 10 μL / well, conduct electrophoresis at a constant voltage of 90V for the stacking gel, and perform electrophoresis at a constant voltage of 120V for the separating gel;

[0057] (3) Membrane transfer: Assemble the memb...

Embodiment 3

[0075] Example 3 Determination of broad-spectrum neutralizing activity of neutralizing antibody 9H6-VP1

[0076] In order to determine the neutralizing activity of the antibody 9H6-VP1, neutralization experiments were carried out against O / ZK / 93 strains, O / Mya / 98 strains and O / HK / 93 strains, which are representative strains of different lineages of O-type foot-and-mouth disease virus. The specific operation steps are as follows:

[0077] (1) In a 96-well cell culture plate, dilute the neutralizing antibody 9H6-VP1 with cell maintenance solution at 50 μL / well from 1:4 to 1:512. Then, add 100 μL containing 100TCID to each well 50 FMDV at 37°C for 1h. Also set with 0.1, 1, 10, and 100 TCID 50 Control wells (no antibody added);

[0078] (2) Add 100 μL containing 5×10 4 The complete medium of BHK-21 cells was placed at 37°C with 5% CO 2 48 hours in the incubator;

[0079](3) Discard the supernatant cell solution, add pre-cooled fixative solution (methanol:acetone=1:1), and f...

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Abstract

The invention relates to the technical field of biology, in particular to an O-type foot-and-mouth disease virus broad-spectrum neutralizing antibody, a preparation method and application. O-type foot and mouth disease virus structural protein VP1 is used as an immunogen to immunize Balb / c mice, a positive hybridoma cell line capable of efficiently secreting a monoclonal antibody is obtained through cell fusion and screening, a mouse monoclonal antibody 9H6-VP1 is obtained, and the monoclonal antibody 9H6-VP1 is an IgG1 type monoclonal antibody. An immunoblotting test shows that the monoclonal antibody 9H6-VP1 has broad-spectrum reactivity to O-type viruses of different pedigree (O / ZK / 93, O / Mya / 98 and O / HK / 93), and does not react with a type A strain; a virus neutralization experiment shows that the monoclonal antibody has neutralization activity on O / ZK / 93, O / Mya / 98 and O / HK / 93, and can prevent virus invasion and protect an organism from infection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an O-type foot-and-mouth disease virus structural protein VP1 broad-spectrum neutralizing antibody, a preparation method and an application. Background technique [0002] Foot-and-mouth disease is an infectious disease common to cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV) infection. FMDV currently has seven serotypes: O, A, C, SAT1, SAT2, SAT3 (namely, South African foot-and-mouth disease virus types 1, 2, and 3) and Asia1 (Asian type 1). There is almost no immune protection between the various types, and animals infected with one type of FMD virus can still be infected with another type of FMD virus. FMDV is a member of the genus aphthavirus of the family picornaviridae. Type O foot-and-mouth disease is the most prevalent serotype in the world, including a variety of strains: O / GX / 09-7, O / Mya98 / XJ / 2010, O / Mya98 / BY / 2010, O / JMS / 2000, OZK / 93 etc. [0003] Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569G01N33/577A61K39/42A61P31/14
CPCY02A40/70
Inventor 郑海学杨洋茹毅秦晓东张伟杨帆曹伟军朱紫祥卢炳州赵东梅任蕊芳吴秀萍郝荣增刘华南李亚军吴国华李丹田宏张克山毛箬青
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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