Monoclonal antibody BTV8-VP2-3E11 resistant 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 agents, antibodies, etc., can solve the problems of complex immunity of vaccines, inability to play a protective role, poor protection, etc.
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Embodiment 1B
[0038] Prokaryotic expression and purification of embodiment 1BTV8-VP2 protein
[0039] 1. Primer Design
[0040] According to the BTV8VP2 gene sequence registered in Genbank (accession number: AJ585129), design PCR amplification primers, the sequence is as follows:
[0041] BTV8VP2-BamHI-atg-1-18:5'-CG GGATC CATGGAGGAGCTAGCAATTC-3'
[0042] BTV8VP2-HindⅢ-2903-2884R:5’-GTC AAGCTT CTATACATTGAGCAGRTTAG-3'
[0043] 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.
[0044] 2. Extraction and reverse transcription of BTV8 viral RNA
[0045] 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.
[0046] 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
[0074] The preparation of embodiment 2 monoclonal antibody
[0075] 1. Mice Immunization
[0076] Five 6-week-old female BALB / c mice were immunized with prokaryotic-expressed recombinant BTV8-VP2 protein purified from gel cutting, and immunized three times in total, with a two-week interval between each immunization. The immunization dose was 50 μg / mouse, and the immunization route was intraperitoneal immunization .
[0077] 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.
[0078] 2. Cell Fusion
[0079] 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 conve...
Embodiment 3
[0084] Identification of embodiment 3 monoclonal antibody
[0085] 1. Subclass identification of monoclonal antibodies
[0086] 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.
[0087] The results show that the heavy chain of the monoclonal antibody 3E11 of the present invention is IgG 1 , the light chain is a κ chain.
[0088] 2. Western blot test
[0089] SDS-PAGE electrophoresis was carried out after the cell pellet and BHK-21 cell pellet were harvested by centrifugation, and then the protein was transferred to the nitrocellulose membrane by electrotransfer. 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 wash with PBST (pH7.4 PBS b...
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