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Monoclonal antibody of anti-Bluetongue virus serum 4-type VP2 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain

A hybridoma cell line, monoclonal antibody technology, applied in antiviral immunoglobulins, analytical materials, biochemical equipment and methods, etc., can solve problems such as poor protection, complicated vaccine immunity, and inability to play a protective role

Active Publication Date: 2016-12-07
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the poor cross-immune protection between each serotype, the vaccine immunity is complicated and cannot play a good protective effect. So far, there is no effective vaccine for preventing and treating bluetongue disease.

Method used

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  • Monoclonal antibody of anti-Bluetongue virus serum 4-type VP2 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain
  • Monoclonal antibody of anti-Bluetongue virus serum 4-type VP2 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain
  • Monoclonal antibody of anti-Bluetongue virus serum 4-type VP2 protein, hybridoma cell strain capable of secreting monoclonal antibody and application of hybridoma cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 14

[0032] Example 1 Prokaryotic expression and purification of 4BTV-VP2-A, 4BTV-VP2-B and 4BTV-VP2-C proteins

[0033] 1. Primer design

[0034] According to the L2 gene sequence of type 4 BTV registered in Genbank (accession number is 132566356). Using protein analysis software to analyze and predict the L2 gene, the type 4 L2 gene is divided into three segments: A, B, and C: 4A: 1-1203bp; 4B: 976-1980bp; 4C: 1861-2871bp; design PCR amplification primers, the PCR amplification primer sequences of 4A (P3-P6), 4B (P7-P10), 4C (P11-P14) are shown in Table 1 and Table 2 below, and Table 1 is for pET-28a-c ( +) The primer sequence of the vector, table 2 is the primer sequence for the pMAL-c5x vector:

[0035] Table 1 is aimed at the primer sequence of pET-28a-c (+) carrier

[0036]

[0037] Table 2 is aimed at the primer sequence of pMAL-c5x vector

[0038]

[0039]

[0040] 2. Gene amplification

[0041] Using the synthesized BTV4L2 gene as a template, the 4B segment was...

Embodiment 2

[0060] The preparation of embodiment 2 monoclonal antibody

[0061] 1. Immunization of mice

[0062] Immunization of mice Four 4-6-week-old female BALB / C mice were immunized with pET-28a(+)-VP2-4B / BL21 prokaryotically expressed recombinant BTV4-VP2-4B protein purified from pET-28a(+)-VP2-4B / BL21, and immunized three times in total. The interval between each immunization is two weeks, and the immunization dose is 50 μg per mouse. For the first time, an equal amount of Freund's complete adjuvant is used to emulsify with protein, and for the second and third times, an equal amount of Freund's incomplete adjuvant is used for emulsification. For intraperitoneal immunity. Immunization was boosted before fusion.

[0063] 2. Cell Fusion

[0064] The feeder layer cells were prepared 1 day before fusion, and BALB / C mouse peritoneal macrophages were plated in 96-well cell culture plates according to conventional methods. Blood was collected from eyeballs, serum was separated and stor...

Embodiment 3

[0067] Identification of embodiment 3 monoclonal antibody

[0068] 1. Subclass identification of monoclonal antibodies

[0069] According to the antibody subclass identification kit, among the monoclonal antibodies against BTV4VP2 protein, the subclass of 1B6 is IgG1.

[0070] 2. Western blot test

[0071] The reaction specificity of the monoclonal antibody was identified by Western blot test. The recombinant 4VP2-B protein expressed by pMAL-c5x-VP2-4B / BL21 was transferred to the NC membrane, the positive hybridoma cell line 1B6 culture supernatant was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. ECL color development.

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Abstract

The invention discloses a monoclonal antibody of anti-Bluetongue virus serum 4-type VP2 protein, a hybridoma cell strain capable of secreting the monoclonal antibody and an application of the hybridoma cell strain. According to the invention, the hybridoma cell strain which is capable of stably secreting the anti-BTV4-VP2 protein monoclonal antibody is obtained by screening positive hybridoma cells by taking VP2-4B protein, which is constructed and expressed by taking pET-28a as a vector, as an immunogen to immunize a mouse and by taking VP2-4B protein, which is constructed and expressed by taking pMAL-c5x as a vector, as an antigen, and conducting final screening. Experiments prove that the monoclonal antibody BTV4-VP2-1B6, which is secreted by the hybridoma cell strain, can participate in a specific reaction with the BTV4-VP2 protein while cannot react with other serum-type VP2 protein. The monoclonal antibody BTV4-VP2-1B6 disclosed by the invention lays a foundation for serology differential diagnosis of the BTV4 and other serum types.

Description

technical field [0001] The present invention relates to a hybridoma cell strain and the monoclonal antibody secreted thereto, in particular to a hybridoma cell strain secreting anti-BTV4VP2 protein monoclonal antibody and the secreted monoclonal antibody thereof; the present invention also relates to the above-mentioned hybridoma cell The application of the strain and the monoclonal antibody in the preparation, diagnosis or detection of BTV4 infection belongs to the field of prevention and treatment of bluetongue. Background technique [0002] Bluetongue (Bluetongue, BT) is an anti-animal vector-borne disease caused by bluetongue virus (Bluetonguevirus, BTV) of the genus Orbivirus in the Reoviridae family. Bluetongue is an acute non-contact infectious disease characterized by fever, leukopenia, and severe catarrhal inflammation of the buccal and gastrointestinal mucosa. BTV can infect most domesticated and wild animals, and the clinical symptoms vary according to the suscep...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/569
CPCC07K16/10
Inventor 唐丽杰李一经徐义刚王丽乔薪瑗刘敏刘琦李佳璇臧明鑫
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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