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Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications

A technology of BTV-4D4 and bluetongue virus, which is applied in the direction of antiviral immunoglobulin, antibody, antiviral agent, etc., can solve the problems of no vaccine, complicated vaccine immunity, and inability to play a protective role

Inactive Publication Date: 2014-03-19
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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  • Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications
  • Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications
  • Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 monoclonal antibody

[0031] 1. Mice Immunization

[0032] Take BTV8 (TCID 50 10 8.25 5 6-week-old female BALB / c mice were immunized with the whole virus for 3 times with a two-week interval between each immunization.

[0033] 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 given booster immunization, and each mouse was intraperitoneally injected with 0.5 mL immune antigen.

[0034] 2. Cell Fusion

[0035]Feeder 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. Kill the mice to be taken by neck dislocation, take the spleen aseptically and separate the splenocytes, perform cell fusion with PEG according ...

Embodiment 2

[0040] Identification of embodiment 2 monoclonal antibody

[0041] 1. Subclass identification of monoclonal antibodies

[0042] Subclass identification of the monoclonal antibody obtained in Example 1 was carried out according to the operating instructions of the SBA ClonotypingTM System / HRP Antibody Subclass Identification Kit.

[0043] The result shows that the heavy chain of the monoclonal antibody BTV-4D4 of the present invention is IgG1, and the light chain is κ chain. The results of IFA test showed that monoclonal antibody BTV-4D4 could react with 24 serotypes of BTV.

[0044] 2. Western blot test

[0045] 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 blo...

Embodiment 3B

[0047] Example 3 Identification of B cell epitopes

[0048] 1. Biopanning of phage display random peptide library and phage genome sequence determination

[0049] Referring to the operation manual of NEB company's phage display random 12 peptide library, the prepared and purified monoclonal antibody BTV-4D4 was used to perform three rounds of panning on the random peptide library, so that the displayed peptides can be specifically combined with the monoclonal antibody of bacteriophages. The coating amount of monoclonal antibody in the three rounds of panning was 100 μg / mL, 150 μL / well, and the concentrations of Tween-20 in PBST buffer used in the three rounds of panning were 0.1%, 0.3% and 0.5%, respectively.

[0050] Take 2 μL from the phage eluted in the third round and make 10-fold serial dilutions, take 10 μL for each dilution and inoculate 200 μL logarithmic growth phase ER2738 host bacteria, after 5 minutes of action, mix all the bacterial solution with 3 mL top agar me...

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Abstract

The invention discloses a kind of bluetongue virus (BTV) NS2 protein monoclonal antibody BTV-4D4, a B cell epitope recognized thereby and applications. BALB / c mice are subjected to totivirus immunization by utilization of bluetongue virus serotype 8 type (BTV8), splenic lymphocytes of the mice after immunization and SP2 / 0 cells are fused. A hybridoma cell strain BTV-4D4 secreting anti-BTV-NS2 protein monoclonal antibodies steadily is obtained after screening with BTV 8 as a coating antigen through an indirect ELISA method. The monoclonal antibodies secreted from the hybridoma cell strain and the BTV-NS2 protein are subjected to a specific reaction. The B cell epitope recognized by BTV-4D4 is 149RSNYDV154 after identification by utilization of a phage display technology. The monoclonal antibody and the BTV-NS2 protein B cell epitope recognized by the monoclonal antibody can be used for diagnosis or prevention of bluetongue virus infection.

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-BTV-NS2 protein monoclonal antibody and the secreted monoclonal antibody thereof; the present invention also relates to a B Cell epitope, especially relate to BTV-NS2 protein B cell epitope polypeptide recognized by above-mentioned monoclonal antibody; The present invention also relates to above-mentioned hybridoma cell line, monoclonal antibody and B cell epitope polypeptide in the preparation diagnosis or prevention of BTV infection The invention belongs to the field of prevention and control of bluetongue. Background technique [0002] Bluetongue (Bluetongue, BT) is an arbovirus-borne disease of ruminants caused by Bluetongue virus (BTV) belonging to the genus Orbivirus in the Reoviridae family. BTV can infect most domesticated and wild ruminants, showing different clinical symptoms depending...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10C07K7/06G01N33/577G01N33/569A61K39/42A61P31/14G01N33/68
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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