Monoclonal antibody against Eastern Equine Encephalitis Virus e2 protein (eeev-5e4) and its recognized B-cell epitope polypeptide and application
An EEEV-5E4, monoclonal antibody technology, applied in antiviral immunoglobulins, antiviral agents, viral antigen components, etc., can solve the problems of poor prognosis of patients and late start of research
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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 EEEVE2 protein and prokaryotic expression and purification of EEEVE2 protei...
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] Follow 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, add 75...
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