Hybridoma cell strain and secretion monoclonal antibody and application thereof
A technology of hybridoma cells and monoclonal antibodies, applied in the direction of antibodies, analytical materials, antiviral agents, etc., can solve the problems of high cost, re-emergence of piglets, single preventive effect of vaccines, etc., and achieve good neutralizing activity and high binding strength Effect
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[0056] Example 1 Acquisition of hybridoma cell line
[0057] 1.1 Propagation and purification of PEDV virus
[0058] 1.1.1 Preparation of antigens
[0059] The Vero cells that have grown into a good monolayer (Vero cells are from Shanghai Institute of Biochemistry) were washed 2-3 times with PBS, and then inoculated with the inoculation dose of MOI=0.001 to PEDV HN1301 strain. The total amount of virus solution and maintenance solution during inoculation Incubate at 37°C for 60 minutes, mix gently every 20 minutes, discard the liquid in the bottle after adsorption, add 5mL of fresh maintenance solution, and place at 37°C CO 2 In an incubator, the virus solution was harvested when the cytopathic effect reached 90%, and was frozen at -20°C for later use.
[0060] 1.1.2 Purification of antigen
[0061] Take 200mL of virus solution, add 20mL of TritonX-100, shake at room temperature for 10min, centrifuge at 5000rpm for 30min in a high-speed refrigerated centrifuge, take the cen...
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[0110] Example 2 Preparation and identification of monoclonal antibodies
[0111] 2.1 Preparation of monoclonal antibody ascites BALB / C female mice were first sensitized with sterilized liquid paraffin, 0.5 mL / mice was injected into the abdominal cavity, and 0.5 mL (about 2.0 × 10) was injected into the abdominal cavity after 7-10 days. 6 cells / mL) of hybridoma cells. It was observed that ascites was withdrawn when the abdomen of the mouse was significantly distended. The ascites was placed at 37°C, 1 h later at 4°C overnight. The next day, the supernatant was collected by centrifugation, aliquoted, and stored at -70°C.
[0112] 2.2 Identification of monoclonal antibody Ig types
[0113] Sigma monoclonal antibody typing reagent was used to identify the Ig type of the monoclonal antibody according to the instructions. The heavy chain of 8A3A10 monoclonal antibody was IgG2a subtype, and the light chain was Kappa chain.
[0114] 2.3 Determination of monoclonal antibody titer ...
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[0134] Example 3 Evaluation of the effect of monoclonal antibody on prevention and treatment of viral infection
[0135] 3.1 Evaluation of the efficacy of monoclonal antibodies in preventing viral infection
[0136] Screening 15 2-3 day-old piglets with double-negative PEDV, RV, TGEV antigens and antibodies, and randomly divided them into 3 groups. The first group and the second group were orally administered 2 mL of the monoclonal antibody prepared in Example 2, and the third group was orally administered 2mL of PBS buffer solution, 24 hours after oral administration, the first and third groups were orally administered with 2mL of PEDV epidemic strain HN1301 enterovirus (including 1000 minimum incidence doses), and the second group was orally administered with CV777 enterovirus (including 1000 minimum incidence doses), The clinical symptoms of piglets were observed daily for 14 consecutive days, and the efficacy of the monoclonal antibody in preventing diarrhea virus infectio...
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