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Anti-Bluetongue Virus Serum Type 16 VP2 Protein Monoclonal Antibody btv16-2b4 and Its B-cell Epitope Recognized and Its Application

A bluetongue virus and monoclonal antibody technology, applied in antiviral immunoglobulin, antiviral agents, antibodies, etc., can solve problems such as poor protection, complicated vaccine immunity, and no vaccine

Inactive Publication Date: 2016-07-06
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

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

Method used

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  • Anti-Bluetongue Virus Serum Type 16 VP2 Protein Monoclonal Antibody btv16-2b4 and Its B-cell Epitope Recognized and Its Application
  • Anti-Bluetongue Virus Serum Type 16 VP2 Protein Monoclonal Antibody btv16-2b4 and Its B-cell Epitope Recognized and Its Application
  • Anti-Bluetongue Virus Serum Type 16 VP2 Protein Monoclonal Antibody btv16-2b4 and Its B-cell Epitope Recognized and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0036] Example 1 Eukaryotic expression and purification of BTV-16VP2 protein

[0037] 1. Primer design

[0038] Bac-to-Bac baculovirus expression system was used for eukaryotic expression, and PCR amplification primers were designed according to the L2 gene sequence (JN671907) of BTV16 registered in GenBank, BTV16L2-22F: 5'-ACGAATTCATGGAGGAGCTAGTTATACC-3'(EcoRI); BTV16L2 -2901R: 5'-CCGTCGACTTAAATATTTAGAAGCTTCGTG-3' (SalI).

[0039] 2. Construction of eukaryotic expression vector of BTV-16VP2 protein and eukaryotic expression and purification of VP2 protein

[0040] First, use primers BTV16L2-22F and BTV16L2-2901R to amplify the BTV-16VP2 gene (the size of the amplified fragment is 2896bp), and perform gel recovery. Then utilize restriction endonuclease EcoRI and SalI to reclaim the VP2 gene that obtains and the shuttle vector pFastBac of baculovirus TM HTA was double digested separately. Digested VP2 gene and pFastBac using T4 DNA fast ligase TM HTA was connected, and con...

Embodiment 2

[0054] The preparation of embodiment 2 monoclonal antibody

[0055] 1. Mice Immunization

[0056] Four 6-week-old female BALB / c mice were immunized with the BTV16VP2 recombinant protein expressed and purified by the Bac-to-Bac baculovirus expression system, and immunized three times with two weeks between each immunization. Mix and emulsify with an equal volume of Freund's complete adjuvant, and mix and emulsify the recombinant VP2 protein with an equal volume of Freund's incomplete adjuvant for the second and third immunizations.

[0057] One week after the second and third immunizations, blood was collected from the tail of the mice, the serum was separated (4°C, 10,000 rpm, 20 min), and the antibody level was detected by indirect ELISA. Three days before cell fusion, booster immunization was performed on BALB / c mice with good immune effect, and each mouse was intraperitoneally injected with 50 μg of immune antigen (without adjuvant).

[0058] 2. Cell Fusion

[0059] Feed...

Embodiment 3

[0064] Identification of embodiment 3 monoclonal antibody

[0065] 1. Subclass identification of monoclonal antibodies

[0066] Subclass identification of the monoclonal antibody obtained in Example 1 was carried out according to the operating instructions of the SBAC Clonotyping™ System / HRP Antibody Subclass Identification Kit.

[0067] The result shows that the heavy chain of the monoclonal antibody BTV16-2B4 of the present invention is IgG1, and the light chain is κ chain.

[0068] 2. Western blot test

[0069]The insect cell lysate inoculated with wild-type baculovirus was used to express and purify the BTV16VP2 recombinant protein through the Bac-to-Bac baculovirus expression system, and the cell pellet and BHK-21 cell pellet after the BTV16 virus supernatant was harvested by centrifugation Afterwards, SDS-PAGE electrophoresis was carried out, and then the protein was transferred to the nitrocellulose membrane by electrotransfer, and the electrotransfer condition was 18...

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Abstract

The invention discloses monoclonal antibody BTV16-2B4 resistant to bluetongue virus serum 16 type VP2 protein, B-cell epitope identified by the monoclonal antibody BTV16-2B4, and applications of the monoclonal antibody BTV16-2B4, and belongs to the field of bluetongue prevention. The invention also relates to a hybridoma cell strain which is capable of secreting the monoclonal antibody BTV16-2B4, and monoclonal antibody secreted by the hybridoma cell strain. It is shown by indirect immunofluorescence assay that the specific reaction is observed between the monoclonal antibody and BTV16, and no cross reaction is observed between the monoclonal antibody and other serum types of bluetongue virus, Ibaraki virus or Chuzan disease virus. Further, BTV16-VP2 protein B-cell epitope polypeptide which can be identified by the monoclonal antibody is determined via cutting expression antigen short peptide and peptide scanning technology. The monoclonal antibody, and the BTV16-VP2 protein B-cell epitope polypeptide identified by the monoclonal antibody can be used for preparation of BTV16 specific differential diagnosis reagents, and foundation is provided for development of BTV epitope labeled vaccines.

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-BTV16VP2 protein monoclonal antibody and the secreted monoclonal antibody thereof; the present invention also relates to a B cell expression The present invention also relates to the above-mentioned hybridoma cell line, monoclonal antibody and B-cell epitope polypeptide in the preparation of drugs for diagnosing or preventing BTV16 infection The application belongs to the field of prevention and control of bluetongue. Background technique [0002] Bluetongue (Bluetongue, BT) is an insect-borne disease of ruminants caused by Bluetongue virus (BTV), which belongs to the genus Orbivirus of the family Reoviridae. BTV can infect most domesticated and wild ruminants, showing different clinical symptoms depending on the susceptibility of the animals. The mortality rate of susceptible animals in differ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10C07K14/14G01N33/577G01N33/569A61K39/42A61P31/14G01N33/68
Inventor 吴东来徐青元孙恩成杨涛
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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