Foot-and-mouth disease virus non-structural protein 3B epitope peptide and application thereof

A foot-and-mouth disease virus and non-structural protein technology, which is applied in the non-structural protein antigen epitope of foot-and-mouth disease virus and its application field, can solve the problems of undetectable serum, inability to ensure the broad-spectrum of foot-and-mouth disease strains, cumbersome differential diagnosis methods for foot-and-mouth disease, etc.

Active Publication Date: 2019-02-01
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) The epitope targeted by the monoclonal antibody has not been identified, and the extent of the broad spectrum of the monoclonal antibody cannot be determined: the epitope sequence and its key amino acids or important amino acids targeted by the existing monoclonal antibody are not clear, if If the key amino acids and important amino acids vary between strains, the degree of broad-spectrum will change, which cannot ensure the broad-spectrum of foot-and-mouth disease strains, and correspondingly lacks real application value;
[0006] (2) Poor broad-spectrum of monoclonal antibodies: The antigenic epitopes targeted by monoclonal antibodies are poorly conserved, resulting in no reaction with some strains of foot-and-mouth disease;
[0007] (3) Poor competitiveness of monoclonal antibodies: Some monoclonal antibodies have poor competitive ability with FMD infection serum, which makes it impossible to establish an effective blocking ELISA detection kit
Monoclonal antibodies with no competition or poor competition are only suitable for coating ELISA pl

Method used

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  • Foot-and-mouth disease virus non-structural protein 3B epitope peptide and application thereof
  • Foot-and-mouth disease virus non-structural protein 3B epitope peptide and application thereof
  • Foot-and-mouth disease virus non-structural protein 3B epitope peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Screening and Identification of Foot-and-Mouth Disease Virus Nonstructural Protein Monoclonal Antibody 2H1

[0062] 1 test material

[0063] 1.1 Viruses, cells, strains and experimental animals

[0064] The strains used for specific analysis of monoclonal antibodies by IFA include: A FMDV A / KT / 58 (GenBankaccession number: AJ131665), A / XJBC / CHA / 2010, Asia1FMDV Asia1 / YS / CHA / 05 (GU931682), O FMDV O / Tibet / CHA / 99 (AJ539138), O / GD / 86 (AJ131468) (Wang et al., 2011); BHK-21 and SP2 / 0 myeloma cells of suckling hamster kidney cells were preserved in our laboratory; clean Grade female BALB / c mice were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Type A, O and Asia1 FMDV infection and immune sera were donated by Xinjiang Tiankang Biological Co., Ltd. The baculovirus expresses FMDV 3ABC protein, and the prokaryotic system expresses FMDV 3AB protein is preserved by our laboratory. The mo...

Embodiment 2

[0089] The assembly of embodiment 2 foot-and-mouth disease virus antibody blocking ELISA detection kit

[0090] Foot-and-mouth disease virus antibody detection blocking ELISA kit, including: horseradish peroxidase-labeled monoclonal detection antibody, ELISA plate coated with foot-and-mouth disease virus non-structural protein antibody, coated antibody, foot-and-mouth disease virus antigen, foot-and-mouth disease virus positive control serum , FMD virus negative control serum, serum diluent, washing solution, substrate solution and stop solution.

[0091] The horseradish peroxidase-labeled foot-and-mouth disease virus detection monoclonal antibody: secreted by a cell line with a deposit number of CCTCCNO: 16212, and then purified;

[0092] The coating antibody is secreted by a cell line with the deposit number CCTCC NO: 16213, and then purified;

[0093] Positive control serum: pig and bovine serum immunized with foot-and-mouth disease virus inactivated vaccine;

[0094] Neg...

Embodiment 3

[0099] The assembly of embodiment 3 foot-and-mouth disease antibody detection ELISA kit

[0100] FMD antibody detection ELISA kit, including: ELISA detection plate, FMD virus coated antigen, enzyme-labeled antibody, FMD virus infected serum, immune serum, FMD virus negative serum, diluent, washing solution, substrate solution and color developing solution ;

[0101] Wherein, the coating antigen of the foot-and-mouth disease virus is a linear epitope peptide (KPLKVK);

[0102] The enzyme-labeled antibody is an HRP-labeled goat anti-bovine IgG antibody;

[0103] ELISA plate coated with inactivated antigen of foot-and-mouth disease virus: Dilute the concentration of antigen coated with foot-and-mouth disease virus to 1 ng / μL, coat the ELISA plate with 100 μL / well, incubate at 37°C for 2 hours or overnight at 4°C;

[0104] Diluent: PBS (pH7.4) solution containing 0.05% (V / V) Tween and 0.5% BSA (mg / ml);

[0105] The substrate liquid is OPD;

[0106] Washing solution: PBST solut...

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Abstract

The invention discloses a foot-and-mouth disease virus non-structural protein 3B epitope peptide and an application thereof. A hybridoma cell line 2H1 is obtained through screening using a cell fusiontechnology, and a monoclonal antibody secreted by the hybridoma cell line 2H1 has a broad spectrum and a high affinity, and is highly competitive with serum. The antigen epitope recognized by the monoclonal antibody 2H1 is further identified accurately, and the amino acid sequence of the monoclonal antibody 2H1 is represented by SEQ ID No.2; and the antigen epitope peptide is mutated to obtain aseries of mutants. The antigen epitope peptide and the mutants thereof have good reaction ability with FMDV antibody positive serums of foot-and-mouth disease infected pigs, cattle and sheep, and an antibody ELISA detection kit established by using the antigen epitope peptide as envelope antigens can specifically distinguish FMDV infected serum and antigen immunized serum, can be adopted to detectthe serum of any animal species, spans the inter-species barrier, and has the advantages of high reliability, wide application range, good broad spectrum, strong competitiveness, and quickness and convenience in detection.

Description

technical field [0001] The present invention relates to antigenic epitopes of foot-and-mouth disease virus, especially the non-structural protein 3B antigenic epitopes of foot-and-mouth disease virus and their applications. Protein antigenic epitopes and their fields of application. Background technique [0002] Foot-and-mouth disease (FMD) is caused by foot-and-mouth disease virus (FMDV). It is a highly contagious infectious disease that mainly affects livestock such as cattle, pigs, and sheep, as well as a variety of wild cloven-hoofed animals. Humans can also be infected. The disease causes the death of young animals, decreased milk production, decreased meat consumption, decreased meat production, and decreased animal production performance. It is highly contagious and can form a large-scale epidemic, causing huge economic losses. Foot-and-mouth disease virus is a member of the foot-and-mouth disease virus genus of the Picornaviridae family. It is a single-stranded posi...

Claims

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Application Information

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IPC IPC(8): C07K14/09C12N15/42G01N33/569
CPCC07K14/005C12N2770/32122G01N33/56983G01N2333/09
Inventor 周国辉于力杨德成王海伟孙超
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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