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West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application

A West Nile virus and monoclonal antibody technology, applied in the direction of antibodies, antiviral agents, anti-animal/human immunoglobulin, etc., can solve the problem that antibodies cannot be used for differential diagnosis of viruses

Inactive Publication Date: 2011-04-20
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

In addition, due to the serious serological cross-reactivity between flaviviruses, the general diagnostic method for detecting antibodies in serum cannot effectively differentiate the viruses of this genus, and some studies have shown that the NS1 protein carries more The specific site can induce multiple specific antibodies that have no cross-reaction in flaviviruses, and if a hybridoma cell line that can secrete specific antibodies is screened, and the single secreted antibody is identified Cloning the WNV-NS1 protein-specific B-cell epitope recognized by the antibody, then an ELISA assay for the differential diagnosis of WNV and other flaviviruses can be established

Method used

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  • West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application
  • West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application
  • West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of Monoclonal Antibody

[0030] 1. Mice Immunization

[0031] Immunize five 6-week-old female BALB / c mice with the purified prokaryotic recombinant NS1 protein, and immunize 3 times in total, with an interval of two weeks between each immunization. Firstly, mix the recombinant NS1 protein with an equal volume of Freund's complete adjuvant Emulsification, second immunization and third immunization The recombinant NS1 protein was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, the immunization dose was 50 μg / rat, and the immunization route was intraperitoneal immunization.

[0032] One week after the second immunization and the third immunization, blood was collected from the tail of the mice, and 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, booster immunization was performed on BALB / c mice with good immune effect, and each mouse wa...

Embodiment 2

[0039] Example 2 Identification of monoclonal antibodies

[0040] 1. Subclass identification of monoclonal antibodies

[0041] 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.

[0042] The results show that the heavy chain of the monoclonal antibody WN-3D10 of the present invention is IgG 1 , the light chain is a κ chain.

[0043] 2. Western blot test

[0044] The WNV-NS1 protein and JEV-NS1 protein were subjected to SDS-PAGE electrophoresis and then electrotransferred. The electrotransfer condition was 18V for 30min. The transferred nitrocellulose membrane was blocked overnight at 4°C in PBS blocking solution containing 50 g / L skimmed milk powder; the blocked nitrocellulose membrane was placed in the supernatant of positive hybridoma culture medium for 1 hour at room temperature, and then blocked with PBST. (PBS buffer s...

Embodiment 3

[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 the phage display random 12 peptide library of NEB Company, the prepared and purified monoclonal antibody WN-3D10 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 for 10-fold serial dilution, take 10 μL for each dilution and inoculate 200 μL logarithmic growth phase ER2738 host bacteria, and after 5 minutes of action, mix all the bacterial solution with 3 mL agar me...

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Abstract

The invention discloses a west nile virus NS1 protein monoclonal antibody, an identified B cell epitope thereof and application. A hybridoma cell strain which stably secretes anti-WNV (West Nile Virus)-NS1 protein monoclonal antibody is obtained by screening and has the microorganism preservation number of CGMCC No.4242. The monoclonal antibody WN-3D10 secreted by the hybridoma cell strain carries out specific reaction with WNV-NS1 protein but does not react with JEV (Japanese Encephalitis Virus)-NS1 protein. The monoclonal antibody and the WNV-NS1 protein virus specific B cell epitope identified by the monoclonal antibody can be used to be prepared into a reagent for identifying or diagnosing west nile virus and Japanese encephalitis virus, and lay a foundation for the serology differential diagnosis method for the WNV and Flavivirus virus.

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-West Nile virus NS1 protein monoclonal antibody and the secreted monoclonal antibody thereof; the present invention also relates to A B cell epitope, especially relates to the West Nile virus NS1 protein B cell epitope recognized by the above-mentioned monoclonal antibody; Or the application of the medicine for treating West Nile virus belongs to the field of prevention and control of West Nile virus disease. Background technique [0002] West Nile Fever (WNF) is an acute zoonotic infectious disease caused by West Nile virus (WNV) and transmitted by mosquitoes. WNV is a single-stranded positive-sense RNA virus belonging to the Flaviviridae genus, and WNV is closely related to Japanese encephalitis virus (JEV), St. Louis encephalitis virus (Saint-Louis encephalitis virus, SLEV) and Murray Valley ...

Claims

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Application Information

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IPC IPC(8): C12N5/18C07K16/18G01N33/577A61K39/395A61P31/14
CPCY02A50/30
Inventor 吴东来杨涛刘霓红王群孙恩成林燕田野
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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