Foot-and-mouth disease virus type a antigen polypeptide, fusion antigen polypeptide and vaccine
A technology of foot-and-mouth disease virus and antigenic polypeptide, applied in the directions of virus antigenic components, virus peptides, multivalent vaccines, etc., can solve the problems of inability to A-type strain antigens, fusion polypeptides and vaccine work enlightenment, inability to immunize each other, and no protective effect.
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Embodiment 1
[0158] Embodiment 1. The solid-phase synthesis of polypeptide
[0159] Equipment: PSI 300B automatic peptide synthesizer, using Rink Amide MBHA resin as a solid phase carrier; Pall Centrasette tangential flow ultrafiltration system, using Omega series membrane cassettes.
[0160] Raw materials and reagents: 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid is used as the starting material, and other reagents used in the solid-phase synthesis are detailed in the following parts of the examples. All the raw materials and reagents used in the synthesis can be purchased in the market or obtained after simple preparation by those skilled in the art according to known techniques.
[0161] The fusion antigen polypeptides represented by SEQ ID NOs: 48-51 (referred to as fusion antigen polypeptides 1, 2, 3, and 4) were synthesized by Merrifield solid-phase synthesis method.
[0162] 1. Polypeptide synthesis
[0163] The synthesis sequence of Merrifield solid-phase synthesis is f...
Embodiment 2
[0182] Example 2. Preparation of Antigen Polypeptide Vaccine
[0183] 1. Water phase preparation
[0184] The fusion antigen polypeptides 1, 2, 3, and 4 obtained in Example 1 were diluted to 50 μg / ml with sterilized water for injection, and filtered through a filter with a pore size of 0.2 μm.
[0185] 2. Preparation of oil phase
[0186] The oil phase adjuvant Montanide ISA 50V2 was sterilized at 120°C for 30 minutes before use.
[0187] 3. Emulsification
[0188] First add the oil phase into the emulsification tank, then stir at 80-100r / min, and at the same time slowly add the water phase, the ratio of the oil phase / water phase is 1 / 1, stir for 2 minutes after adding, and then stir at 8500r / min After 6 minutes, it was emulsified to form a water-in-oil emulsion to obtain the foot-and-mouth disease virus vaccine of the present invention, which was recorded as vaccine 1, 2, 3, and 4.
Embodiment 3
[0189] Example 3. Detection of antibody levels after different doses of antigen immunization
[0190] 1. Test material
[0191] Test samples: vaccines 1, 2, 3, and 4 prepared in Example 2.
[0192] Experimental animals: healthy pigs with negative foot-and-mouth disease, weighing about 40kg / head, about 4 months old.
[0193] 2. Test method
[0194] Take 25 μg, 8.33 μg and 2.78 μg of vaccines 1, 2, 3, and 4 formulated with fusion antigen polypeptides, and inoculate 20 pigs once in each dose group by intramuscular injection behind the root of the ear, respectively, on the 7th, 14th, 21st, and 28th day Blood was collected, serum was separated, and the antibody level after antigen immunization was detected by liquid phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) method for FMD A antibody. The basic principle of the liquid-phase blocking enzyme-linked immunosorbent assay is as follows: coat the ELISA plate with rabbit anti-foot-and-mouth disease virus antibody, tr...
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