Neutralizing monoclonal antibody for resisting porcine pseudorabies virus infection and application of neutralizing monoclonal antibody
A porcine pseudorabies virus, monoclonal antibody technology, applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problem of PRV infection, and achieve low production cost, good stability, and high neutralization activity. Effect
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Embodiment 1
[0032] Example 1 BALB / c mouse immunization
[0033] Immunization of mice: Take 3 female BALB / c mice aged 4-6 weeks, and after a week in the laboratory, take the gD protein of porcine pseudorabies virus recombinantly expressed by baculovirus (provided by the Key Laboratory of Animal Immunology of Henan Academy of Agricultural Sciences) Prepared, preserved, and provided by the laboratory) emulsified with Freund's complete adjuvant (volume ratio 1:1), subcutaneously injected at multiple points, 100ug / monkey, and porcine pseudorabies virus gD emulsified with Freund's incomplete adjuvant two weeks later Protein (volume ratio 1:1) was boosted in the same way, and boosted again two weeks later.
[0034] Super-immunization: After the serum IPMA titer of the mice after booster immunization increased to above 20,000, super-immunization was performed, and the expressed and purified porcine pseudorabies virus gD protein was diluted to 0.2 μg / μL with sterile PBS buffer, and then directly ...
Embodiment 2
[0038] Example 2 Cell fusion and subcloning of monoclonal antibodies
[0039] The fusion process of monoclonal cell lines is as follows:
[0040] (1) On the third day after the mouse was hyperimmunized in Example 1, the serum titer was measured again with IPMA. On the 4th or 5th day, the eyeball of the mouse with the highest titer was removed with sterile tweezers, fully bled, and collected After the blood, the serum was separated and preserved, and then the mice were killed by decapitation and the corpse was completely immersed in 75% ethanol for disinfection;
[0041] (2) Put the sterilized mouse corpse in an ultra-clean bench, cut the mouse open with high-pressure surgical scissors and tweezers, remove the spleen and put it on a clean 200-mesh filter, and use another pair of sterilized scissors to cut the mouse open. The spleen is fully cut into small pieces, washed with serum-free 1640 medium, so that the single scattered spleen cells can penetrate the filter and flow int...
Embodiment 3
[0052] Example 3 Screening of PRV-gD monoclonal antibody
[0053] After subcloning and IPMA re-identification, a total of 13 PRV-gD positive monoclonal antibody cells were obtained, which were named according to the order and position of subcloning: 1C8, 2B9, 3D3, 4D6, 5B2, 6H7, 7D8, 8B9, 9C11, 7B6, 11C9, 12F4 and 13F8. IPMA results show that these monoclonal antibodies can specifically stain the virus on the cells at the lesion site of the cells, and after AEC color development, it turns red ( figure 1 Reaction of 9 monoclonal antibodies among them).
[0054] Expansion of PRV-HeNLH / 2017: The strain used in the present invention is PRV-HeNLH / 2017 (GenbankID: MT775883). Inoculate PK-15 cells into 5 T75 flasks. When the cells grow to 70%, pour out the medium under aseptic conditions, wash with sterile PBS buffer three times, and add 12mL of maintenance solution (the volume content of serum is 2% DMEM medium), and then inoculated according to one-thousandth, cultured in a 37°C...
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