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, monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, antiviral agents, antibodies, etc., can solve the problems of no vaccine, poor protection, and inability to play a protective role

Inactive Publication Date: 2013-09-18
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
View PDF2 Cites 5 Cited by
  • 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

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

[0040] 1. Primer design

[0041] According to the registered BTV8VP2 gene sequence 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 BamH I and Hind III restriction sites, and the length of the amplified product is expected to be 2886bp.

[0045] 2. BTV8 virus RNA extraction and reverse transcription

[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] Steps of RNA extraction by Trizol method: harvest BHK-21 cells infected with BTV8 1-5×10 7 Add 1mL Trizol and mix well, let stand for 5min at room temperature, add 0....

Embodiment 2

[0075] Example 2 Preparation of monoclonal antibodies

[0076] 1. Mouse Immunization

[0077] Five 6-week-old female BALB / c mice were immunized with the purified prokaryotic expression recombinant VP2 protein. A total of three immunizations were carried out with an interval of two weeks between each immunization. The immunization dose was 50 μg / mouse. The immunization route was intraperitoneal immunization.

[0078] One week after the second and third immunizations, the mice were tail-cleaved to collect blood, the serum was separated (4℃, 10000rpm, 20min), and the antibody level was detected by indirect ELISA. Three days before the cell fusion, BALB / c mice with good immune effect were boosted again, and each mouse was injected with 50μg immune antigen.

[0079] 2. Cell Fusion

[0080] One day before fusion, feeder cells were prepared, and BALB / c mouse peritoneal macrophages were plated in 96-well cell culture plates according to conventional methods for use. Mice with spleen to be tak...

Embodiment 3

[0085] Example 3 Identification of monoclonal antibodies

[0086] 1. Subclass identification of monoclonal antibodies

[0087] According to the SBA ClonotypingTM System / HRP antibody subclass identification kit operating instructions, the monoclonal antibody obtained in Example 2 was subclassed.

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

[0089] 2. Western blot test

[0090] After centrifugation to harvest the BTV8 virus supernatant, the cell pellet and BHK-21 cell pellet were processed for SDS-PAGE electrophoresis, and then the protein was transferred to a nitrocellulose membrane by electrotransfer. The electroporation condition was 18V for 30 min; degreasing with 5% The milk powder blocking solution will block the transferred nitrocellulose membrane overnight at 4°C; add the monoclonal antibody supernatant and incubate at room temperature for 1 hour, and wash with PBST (pH7.4 PBS buffer...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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 line and its secreted monoclonal antibodies, in particular to a hybridoma cell line that secretes anti-BTV8-VP2 protein monoclonal antibodies and its secreted monoclonal antibodies; the present invention also relates to a B Cell epitope polypeptides, especially related to the B-cell epitope polypeptides of BTV8-VP2 protein recognized by the aforementioned monoclonal antibodies; the present invention also relates to the aforementioned hybridoma cell lines, monoclonal antibodies, and B-cell epitope polypeptides used in the preparation of diagnostics, detection or The application of drugs to prevent BTV8 belongs to the field of prevention and treatment of BTV8. Background technique [0002] Bluetongue (Bluetongue, BT) is a ruminant-borne infectious disease caused by the Bluetongue virus (BTV) of the circovirus family Reoviridae. BTV can infect most domestic and wild ruminants, and show different clinical sympt...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap