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Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application

A bluetongue virus and monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, antiviral agent, antibody, etc., can solve the problems of inability to protect, no vaccine, complicated vaccine immunity, etc.

Inactive Publication Date: 2014-11-05
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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  • Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application
  • Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application
  • Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0039] Prokaryotic expression and purification of embodiment 1BTV8-VP2 protein

[0040] 1. Primer Design

[0041] According to the BTV8VP2 gene sequence registered in Genbank (accession number: AJ585129), design PCR amplification primers, the sequence is as follows:

[0042] BTV8VP2-BamHI-atg-1-18:5'- CGGGA TCCATGGAGGAGCTAGCAATTC-3'

[0043] BTV8VP2-HindⅢ-2903-2884R:5’- GTCAA GCTTCTATACATTGAGCAGRTTAG-3'

[0044] The underlined part is the introduced restriction site of BamH I and Hind III, and the length of the amplified product is expected to be 2886bp.

[0045] 2. Extraction and reverse transcription of BTV8 virus RNA

[0046] Viral genomic RNA was extracted from BHK-21 cells infected with BTV8 by Trizol method as a template, and viral cDNA was synthesized by reverse transcription with BTV8VP2-HindⅢ-2903-2884R primers.

[0047] RNA extraction step by Trizol method: Harvest BHK-21 cells infected with BTV8 1-5×10 7 Add 1mL Trizol and mix well, let stand at room temper...

Embodiment 2

[0075] The preparation of embodiment 2 monoclonal antibody

[0076] 1. Mice Immunization

[0077] Five 6-week-old female BALB / c mice were immunized with prokaryotic-expressed recombinant VP2 protein purified from gel cutting, and immunized three times with a two-week interval between each immunization. The immunization dose was 50 μg / mouse, and the immunization route was intraperitoneal immunization.

[0078] 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, BALB / c mice with good immune effect were boosted again, and each mouse was intraperitoneally injected with 50 μg of immune antigen.

[0079] 2. Cell Fusion

[0080] 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. ...

Embodiment 3

[0085] Identification of embodiment 3 monoclonal antibody

[0086] 1. Subclass identification of monoclonal antibodies

[0087] Subclass identification of the monoclonal antibody obtained in Example 2 was carried out according to the operating instructions of the SBA ClonotypingTM System / HRP Antibody Subclass Identification Kit.

[0088] The results show that the heavy chain of the monoclonal antibody 4D9 of the present invention is IgG 1 , the light chain is a κ chain.

[0089] 2. Western blot test

[0090] SDS-PAGE electrophoresis was performed on the cell pellet after centrifugation harvesting of the BTV8 virus supernatant and the BHK-21 cell pellet, and then the protein was transferred to the nitrocellulose membrane by electroblotting. The electroporation condition was 18V for 30min; Block the transferred nitrocellulose membrane overnight at 4°C with milk powder blocking solution; add the monoclonal antibody supernatant and incubate at room temperature for 1 hour, then ...

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Abstract

The invention discloses a monoclonal antibody BTV8-VP2-4D9 resistant to bluetongue virus 8 type (BTV8) VP2 protein, B-cell epitope peptide identified thereby and application, and belongs to the field of BTV8 control. A selected hybridoma cell strain capable of stably secreting a BTV8-VP2 protein resistant monoclonal antibody is stored with a microbial preservation number of CGMCC (China General Microbiological Culture Collection Center) No.7003. The experiment results show that the monoclonal antibody BTV8-VP2-4D9 secreted by the hybridoma cell strain can perform idiosyncratic reaction with the BTV8-VP2 protein but not reacts with the VP2 proteins of other serum types. The monoclonal antibody BTV8-VP2-4D9 and the BTV8-VP2 protein virus specific conserved B cell epitope peptide identified by the a monoclonal antibody can be prepared into an agent used for diagnosing BTV8 infection, thus laying a good foundation for creating serology differential diagnosis methods for BTV8 and other types of serum.

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-BTV8-VP2 protein monoclonal antibody and the secreted monoclonal antibody thereof; the present invention also relates to a B Cell epitope polypeptides, especially BTV8-VP2 protein B-cell epitope polypeptides recognized by the above-mentioned monoclonal antibodies; The application of the medicine for preventing BTV8 belongs to the field of prevention and treatment of BTV8. Background technique [0002] Bluetongue (Bluetongue, BT) is an insect-borne infectious disease of ruminants caused by Bluetongue virus (BTV), which belongs to the genus Orbivirus in the Reoviridae family. 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 different endemic areas varies greatly, ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10C07K7/08G01N33/577G01N33/569A61K39/42A61P31/14G01N33/68
Inventor 吴东来徐青元刘霓红杨涛
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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