Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof
A porcine foot-and-mouth disease virus and recombinant antigen technology, applied in the field of biological vaccines, can solve problems such as short duration, inability to meet disease prevention and control, and insufficient protection rate of virus attack, and achieve high antibody levels and good application prospects
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Embodiment 1
[0029] The preparation of embodiment 1 recombinant antigen (EoIgG)
[0030] 1. Bioinformatics analysis of O-type foot-and-mouth disease virus VP1 gene:
[0031]The structural protein VP1 of foot-and-mouth disease virus is the dominant antigen of the virus. Whether it is the isolated and purified natural VP1 protein or the recombinant expression product, it can induce the body to produce protective neutralizing antibodies, which is type-specific. The full length of the foot-and-mouth disease virus VP1 gene consists of 639 nucleotides, encoding a protein with 213 amino acids, and its main epitopes are concentrated in the 140-160 amino acid and the 200-213 amino acid segment. The present invention uses DNAStar biological software to carry out sequence analysis to three strains of porcine foot-and-mouth disease virus O type representative strains (O / China / 99, O / Miandian / 98, O / ZK / 93) that China isolates, and determined that the dominant antigenic epitope is The 135th-160th amino a...
Embodiment 2
[0039] The preparation of embodiment 2 foot-and-mouth disease virus 3D protein different fragments
[0040] 1. Bioinformatics analysis of 3D protein gene of foot-and-mouth disease virus:
[0041] The non-structural protein 3D of foot-and-mouth disease virus is a viral RNA polymerase, which is highly conserved in seven serotypes of foot-and-mouth disease virus. The full length of the 3D gene is 1410 nucleotides, encoding a protein with a length of 470 amino acids. Using DNAStar biological software to analyze the sequences of 46 representative strains of seven serotypes obtained and compare them with the GenBank database, the results show that the nucleotides are homologous The sex is higher than 90%, the amino acid homology is higher than 97%, and it is highly conserved. In this study, the 3D sequence of the Asia1 type FMD virus JS / China / 05 strain was used as a reference sequence to design primers to amplify the target gene fragment and express the protein.
[0042] 2. Gene c...
Embodiment 3
[0044] Embodiment 3, vaccine preparation and immune efficacy experiment:
[0045] 1. Vaccine preparation
[0046] The purified recombinant antigen prepared in Example 1 and the purified 3D protein full-length or its different fragments (3D1\3D2\3D3) prepared in Example 2 were quantified by the Bio-Rad quantitative kit and then diluted into appropriate Concentration, after the purified 3D protein fragments and recombinant antigens were configured in a ratio of 1:2 (V / V), an equal volume of oil adjuvant ISA206 (France) was added to emulsify into vaccine preparations, and the recombinant antigens were contained in 1ml / tube. 200 μg, 100 μg of the full-length 3D protein or its fragments.
[0047] 2. Immunity efficacy test:
[0048]Use the prepared vaccine to vaccinate pigs (200 μg antigen + 100 μg full-length 3D protein or its fragments) through the muscle route according to 1 ml per head. The results of immunization experiments show that whether the recombinant antigen alone or ...
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