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Monoclonal antibody BTV (Bluetongue virus)-NS3-3D8 of anti-BTV NS3 protein, B cell epitope identified by monoclonal antibody and application

A bluetongue virus and monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, antiviral agent, antibody, etc., can solve the problems that the biological function has not yet been determined, and the structure and function of NS3 protein are not clear.

Inactive Publication Date: 2016-01-20
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

At present, the research on the structure and function of NS3 protein is still not thorough, and its biological function has not yet been determined. Relevant scholars believe that the main biological function of NS3 protein may be related to the release of viruses, and its mechanism of action is still being explored stage

Method used

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  • Monoclonal antibody BTV (Bluetongue virus)-NS3-3D8 of anti-BTV NS3 protein, B cell epitope identified by monoclonal antibody and application
  • Monoclonal antibody BTV (Bluetongue virus)-NS3-3D8 of anti-BTV NS3 protein, B cell epitope identified by monoclonal antibody and application
  • Monoclonal antibody BTV (Bluetongue virus)-NS3-3D8 of anti-BTV NS3 protein, B cell epitope identified by monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0032] Prokaryotic expression and purification of embodiment 1BTV15NS3 protein

[0033] 1. Primer Design

[0034] PCR amplification primers were designed according to the known BTV15 type S10 gene sequence (GenBank accession number: AGJ83560.1), and restriction sites EcoRI and HindIII were respectively introduced into the 5' ends of the upstream and downstream primers, and the prokaryotic expression vectors were PET -30a and pMAL-C4X:

[0035] B15-PET-NS3-F:5'-CCgaattcATGCTATCCGGGCTGATCC-3'

[0036] (EcoRI)

[0037] B15-PET-NS3-R:5'-GCaagcttTCAGGTTAATGGCATTTCGA-3'

[0038] (HindIII)

[0039] B15-pC4X-NS3-F:5'-CCgaattcATGCTATCCGGGCTGATCC-3'

[0040] (EcoRI)

[0041] B15-pC4X-NS3-R:5'-GCaagcttTCAGGTTAATGGCATTTCGA-3'

[0042] (HindIII)

[0043] 2. Construction, expression and purification of BTV15NS3 protein prokaryotic expression vector

[0044] The plasmid pEASY-NS3 already in the laboratory was amplified by PCR to obtain the S10 gene and recovered from the gel, and th...

Embodiment 2

[0046] The preparation of implementation example 2 monoclonal antibody

[0047] 1. Immunization of Mice

[0048] Five 6-week-old BALB / c female mice were immunized with the recombinant BTV15-NS3 protein expressed by the gel-cut purified prokaryotic expression vector PET-30a as an immunogen by intraperitoneal and subcutaneous multipoint injection, 100 μg / mouse. A total of two immunizations. Since no adjuvant was used in the immunization process, the handle of a 20ml syringe was used to crush the SDS-PAGE gel strip containing BTV15-NS3 protein each time the immunogen was prepared, and an appropriate amount of PBS buffer was added, and the freeze-thaw was repeated 3 times. Until the rubber strips are completely broken into a soft porridge. The immunization procedure is as follows: the interval between the first immunization and the second immunization is 14 days, the blood is collected by docking the tail one week after the second immunization, and the serum antibody titer of th...

Embodiment 3

[0077] Example 3 Identification of Monoclonal Antibody Characteristics

[0078] 1. Identification of Monoclonal Antibody Subclasses

[0079] Follow SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions The monoclonal antibody obtained in Example 2 was used for subclass identification.

[0080] Its identification result shows that monoclonal antibody BTV-NS3-3D8 of the present invention is IgG 1 type.

[0081] 2. Indirect Immunofluorescence Assay (IFA)

[0082] (1) Plate the BHK cells in good growth state on a 96-well cell plate. When the cells grow to 80-90% of the bottom area of ​​the plate, inoculate BTV1-24 viruses with DMEM basal medium; at the same time, do not inoculate under the same conditions Viral BHK cells served as a negative control.

[0083] (2) After about 24 to 36 hours, when the cells have not undergone obvious lesions, discard the culture medium, add -20°C pre-cooled 75% ethanol in an amount of 100 μL / well, and fix at ...

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Abstract

The invention discloses a monoclonal antibody of anti-BTV (Bluetongue virus) NS3 protein, a B cell epitope identified by the monoclonal antibody and an application and further discloses a hybridoma cell strain which is obtained through screening and can stably secrete the monoclonal antibody. The hybridoma cell strain is named as BTV-NS3-3D8, and the microbial preservation number of BTV-NS3-3D8 is CGMCC (China General Microbiological Culture Collection Center) No.10209. The experiment proves that the monoclonal antibody secreted by the hybridoma cell strain can have the group specificity reaction with 1-24 types of NS3 protein of the BTV. Besides, the invention further discloses the specific B cell epitope, identified by the monoclonal antibody, of BTV NS3 protein. The monoclonal antibody and the specific B cell epitope, identified by the monoclonal antibody, of the BTV NS3 protein can be used for establishing a BTV antigen group identification and diagnosis method and laying the certain foundation for fundamental research of structures and functions of the NS3 protein.

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-bluetongue virus type 1-24 NS3 protein monoclonal antibody and the secreted monoclonal antibody thereof; The present invention also relates to a B cell epitope, in particular to the B cell epitope of the bluetongue virus type 1-24 NS3 protein recognized by the above-mentioned monoclonal antibody; the present invention also relates to the above-mentioned hybridoma cell line, monoclonal antibody and The application of B cell epitopes in the study of the structure and function of bluetongue virus NS3 protein and in the preparation of reagents and medicines for identification, diagnosis, prevention or treatment of bluetongue virus 1-24 infection, the invention belongs to the prevention and treatment of bluetongue disease field. Background technique [0002] Bluetongue (Bluetongue, BT) is caused by B...

Claims

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Application Information

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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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