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Monoclonal antibody EEEV-5E4 resisting EEEV E2 protein, B-cell epitope peptide recognized by EEEV-5E4 as well as application of EEEV-5E4 and B-cell epitope peptide

A technology of monoclonal antibody and epitope polypeptide, which is applied in antiviral immunoglobulin, antiviral agent, viral antigen components, etc., can solve the problems of poor prognosis of patients and late start of research

Inactive Publication Date: 2013-12-04
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 2003, people infected with EEEV were found in clinical cases in Fujian Province. Although there has not been an epidemic so far, there are a large number of "viral encephalitis" with unknown etiology in the southeast coast every summer. Clinical analysis shows that most cases are caused by multiple diseases. Polysyndromic syndrome of viral infection and poor patient prognosis
Furthermore, the research on EEEV started relatively late in my country and there are no effective vaccines and drugs for the prevention and treatment of EEE. Therefore, my country urgently needs to strengthen research on the disease in order to prepare corresponding technical reserves.

Method used

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  • Monoclonal antibody EEEV-5E4 resisting EEEV E2 protein, B-cell epitope peptide recognized by EEEV-5E4 as well as application of EEEV-5E4 and B-cell epitope peptide
  • Monoclonal antibody EEEV-5E4 resisting EEEV E2 protein, B-cell epitope peptide recognized by EEEV-5E4 as well as application of EEEV-5E4 and B-cell epitope peptide
  • Monoclonal antibody EEEV-5E4 resisting EEEV E2 protein, B-cell epitope peptide recognized by EEEV-5E4 as well as application of EEEV-5E4 and B-cell epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Prokaryotic and eukaryotic expression and purification of embodiment 1EEEV-E2 protein

[0032] 1. Primer Design

[0033] The prokaryotic expression vector uses pET-30a, and PCR amplification primers are designed according to the known E2 gene sequence of EEEV North American variant strain (GenBank accession number: X63135.1):

[0034] pE-E2-25F:

[0035] 5'-CTggatccGATTTGGACACTCATTTCACCCAGT-3' (BamHI);

[0036] pE-E2-1260-1241R:

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

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

[0039] pF-E2-25F:

[0040] 5'-CTggatccGGATTTGGACACTCATTTCACCCAGT-3' (BamHI);

[0041] pF-E2-1260-1241R:

[0042] 5'-GCCaagcttTTATGCCCTCGTCGGCTTAATGC-3' (Hind III).

[0043] 2. Construction of prokaryotic expression vector of EEEV E2 protein and prokaryotic expression and purification of E2 protein ...

Embodiment 2

[0064] The preparation of embodiment 2 monoclonal antibody

[0065] 1. Mice Immunization

[0066] The recombinant E2 protein expressed and purified by Bac-to-Bac eukaryotic expression system was used as the immunogen to immunize three 6-week-old female BALB / c mice intraperitoneally, 100 μg / mouse, and immunized three times in total. Mix Freund's complete adjuvant with purified recombinant E2 protein in the first dose; mix Freund's incomplete adjuvant with purified E2 protein in the second and third doses, and mix E2 protein and adjuvant in equal volumes; One week after immunization, blood was collected from the tail vein, and the serum antibody titer was detected by indirect ELISA. Three days before the fusion, the BALB / c mice with higher antibody levels were boosted, and each mouse was directly intraperitoneally injected with 100 μg of purified E2 protein.

[0067] 2. Cell Fusion

[0068] Feeder cells were prepared 1 day before fusion, and BALB / c mouse peritoneal macrophage...

Embodiment 3

[0073] Identification of embodiment 3 monoclonal antibody

[0074] 1. Subclass identification of monoclonal antibodies

[0075] According to SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions The monoclonal antibody obtained in Example 1 was used for subclass identification.

[0076] The results show that the heavy chain of the monoclonal antibody EEEV-5E4 of the present invention is IgG 1 , the light chain is a κ chain.

[0077] 2. IFA test (IFA identification of Sf9 cells infected with recombinant baculovirus)

[0078] (1) Use Sf9 insect cell plating (96 cell culture plate), when the cells grow to 80-90% of the bottom area of ​​the plate, inoculate the second-generation recombinant baculovirus BACV-E2 / P2; at the same time, inoculate the wild type under the same conditions Sf9 insect cells with baculovirus (BACV-W) served as a negative control.

[0079] (2) After 48 hours, the cells have obvious lesions, discard the culture medium,...

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Abstract

The invention discloses a monoclonal antibody EEEV-5E4 resisting an EEEV (Eastern Equine Encephalitis Virus) E2 protein, a B-cell epitope peptide recognized by the EEEV-5E4 as well as the application of the EEEV-5E4 and the B-cell epitope peptide. The invention further discloses a hybridoma cell strain which can stably secrete the EEEV-5E4 resisting the EEEV E2 protein and is named as EEEV-5E4. The microbial preservation number of the hybridoma cell strain is CGMCC No. 7008. An idiosyncratic reaction can occur between the EEEV-5E4 secreted by the hybridoma cell strain and the EEEV E2 protein while the EEEV-5E4 does not react with corresponding negative control. The EEEV-5E4 and the specific B-cell epitope peptide, recognized by the EEEV-5E4, of the EEEV E2 protein can be used for preparing reagents for identifying and diagnosing EEEVes / VEEVes (Western Equine Encephalitis Viruses / Venezuelan Equine Encephalitis Viruseses) and other alphaviruses and lay a foundation for creating serologic identifying and diagnosing methods of the EEEVes / VEEVes and other alphaviruses as well as further studying prevention and treatment measures of viruses relevant to alphaviruses.

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 a monoclonal antibody against Eastern Equine Encephalitis Virus E2 protein and the monoclonal antibody secreted thereto; It relates to a B cell epitope, especially relates to the Eastern Equine Encephalitis Virus or the Venezuelan Equine Encephalitis Virus E2 protein B cell epitope polypeptide 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 the B cell epitope polypeptide in the preparation of relevant reagents and medicines for diagnosing, preventing or treating Eastern equine encephalitis virus or Venezuelan equine encephalitis virus infection belongs to the field of prevention and treatment of equine encephalitis. Background technique [0002] Eastern equine encephalitis (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10C07K7/08G01N33/577G01N33/68A61K39/42A61K39/12A61P31/14C12R1/91
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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