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Monoclonal antibody BTV12-NS1-1F8 of anti-bluetongue-virus 12-type NS1 protein, B cell epitope recognized by monoclonal antibody and application of monoclonal antibody

A bluetongue virus and monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, antiviral agent, antibody, etc., can solve the problems of lack of cross immune protection, detection and prevention and control difficulties

Inactive Publication Date: 2016-01-06
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

The disease was first discovered in sheep in South Africa in the 19th century. It was named "Bluetongue Disease" in 1905. Due to the large number of genetic and antigenic variations in BTV, 26 BTV serotypes have been found in the world so far, and The lack of effective cross-immune protection among various serotypes has brought great difficulties to the detection and prevention of BT

Method used

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  • Monoclonal antibody BTV12-NS1-1F8 of anti-bluetongue-virus 12-type NS1 protein, B cell epitope recognized by monoclonal antibody and application of monoclonal antibody
  • Monoclonal antibody BTV12-NS1-1F8 of anti-bluetongue-virus 12-type NS1 protein, B cell epitope recognized by monoclonal antibody and application of monoclonal antibody
  • Monoclonal antibody BTV12-NS1-1F8 of anti-bluetongue-virus 12-type NS1 protein, B cell epitope recognized by monoclonal antibody and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0033] Example 1 Prokaryotic and eukaryotic expression and purification of BTV12-NS1 protein

[0034] 1. Primer Design

[0035] Prokaryotic expression uses pET-30a prokaryotic expression system, and PCR amplification primers are designed according to NCBI's BTV12-NS1 gene sequence (GenBank accession number: X63135.1): pET-BTV12-NS1-1-22F:

[0036] 5'-GCGGATCCATGGAGCGCTTTTTGAGAAAAT-3'(BamHI); pET-BTV12-NS1-1659-1640R:

[0037] 5'-ACGAAGCTTGATACTCCATCCACATCTGCGAC-3' (Hind III).

[0038] For eukaryotic expression, the Bac-to-Bac insect baculovirus expression system was used, and PCR amplification primers were designed according to the above sequences:

[0039] pF-BTV12-NS1-1-22F:

[0040] 5'-GCGGATCCGATGGAGCGCTTTTTGAGAAAAT-3'(BamHI); pF-BTV12-NS1-1659-1635R:

[0041] 5'-ACGAAGCTTCTAATACTCCATCCACATCTGCGAC-3' (Hind III).

[0042] 2. Construction of prokaryotic expression vector of BTV12-NS1 protein and prokaryotic expression and purification of NS1 protein

[0043] First, the...

Embodiment 2

[0050] The preparation of embodiment 2 monoclonal antibody

[0051] 1. Immunization of Mice

[0052]The recombinant BTV12-NS1 protein expressed and purified by the Bac-to-Bac eukaryotic expression system was used as an immunogen to immunize three 8-week-old female BALB / c mice in the peritoneal cavity, 100 μg / mouse, immunized three times in total, and one mouse was not immunized BALB / c mice were bred under the same conditions as negative controls. For the first immunization, Freund's complete adjuvant was mixed with purified eukaryotic recombinant NS1 protein in equal volume ratio; for the second and third immunization, Freund's incomplete adjuvant was mixed with purified eukaryotic recombinant NS1 protein in equal volume ratio. One week after the second immunization and the third immunization, blood was collected from the tail vein, and the serum and purified prokaryotic recombinant NS1 protein were diluted in multiples, and then indirect ELISA was used to detect the serum an...

Embodiment 3

[0059] Identification of embodiment 3 monoclonal antibody

[0060] 1. Subclass identification of monoclonal antibodies

[0061] Follow SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions Subclass identification of the monoclonal antibody obtained in Example 1, the specific process is as follows: the prokaryotic expressed recombinant BTV12-NS1 protein is used as the coating antigen, and the monoclonal antibody obtained in Example 2 is used as the coating antigen. Monoclonal antibody BTV12-NS1-1F8 was used as the primary antibody, and HRP-labeled IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, κ chain, λ chain, and goat anti-mouse antibody were used as secondary antibodies for indirect ELISA identification.

[0062] The results show that the heavy chain of the monoclonal antibody BTV12-NS1-1F8 of the present invention is IgG 1 , the light chain is a λ chain.

[0063] 2.IFA test (IFA identification of BHK-21 cells infected with recombinant baculovirus...

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Abstract

The invention discloses a monoclonal antibody BTV12-NS1-1F8 of anti-bluetongue-virus 12-type NS1 protein, a B cell epitope recognized by the monoclonal antibody and application of the monoclonal antibody. The monoclonal antibody is secreted by a screened hybridoma cell strain which is preserved in microbial preservation number of CGMCC No.10207; and experiments prove that the anti-BTV12 NS1 protein monoclonal antibody secreted by the hybridoma cell strain can undergo a specific reaction with BTV12-type NS1 protein. In addition, the invention also discloses a BTV12 NS1 protein specific B cell epitope recognized by the monoclonal antibody. The monoclonal antibody and the BTV12 NS1 protein specific B cell epitope recognized by the monoclonal antibody can be used for preparing reagents for identifying, diagnosing, preventing or treating BTV12 infection; meanwhile, the invention lays foundations for the establishment of an epitope based diagnosis method and the design of an epitope-labeled vaccine, and provides material reserves for further research and treatment of BTV12 type.

Description

technical field [0001] The present invention relates to a hybridoma cell strain and the monoclonal antibody secreted thereof, in particular to a hybridoma cell strain secreting anti-bluetongue virus type 12 NS1 protein monoclonal antibody and the secreted monoclonal antibody thereof; Involving the linear B cell epitope of the BTV12 type NS1 protein recognized by the above monoclonal antibody and the above hybridoma cell line, monoclonal antibody and linear B cell epitope in the preparation of identification, diagnosis, prevention or treatment of bluetongue virus type 12 The invention relates to the application of infection-related reagents and medicines, and the invention belongs to the field of prevention and treatment of bluetongue. Background technique [0002] Bluetongue (Bluetongue, BT) is an infectious disease of ruminant insects (such as Culicoides, Aedes, etc.) caused by Bluetongue Virus (BTV), an orbivirus of the Reoviridae family. Its clinical features are fever, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10C07K7/08G01N33/577G01N33/569A61K39/42A61P31/14C12R1/91
Inventor 吴东来孙恩成徐青元杨涛
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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