Bungatotoxin antigen epitope gene and application of bungatotoxin antigen epitope gene to preparation of gene vaccine and antigen
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A technology of antigen and vaccine, applied in the field of biomedicine
Inactive Publication Date: 2014-06-18
中国人民解放军成都军区疾病预防控制中心
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Embodiment 1
[0068] (1) Acquisition of SEQ ID NO: 1
[0069] (1) Antigenic sites obtained by bioinformatics analysis
[0070] Two representative sequences extracted from the long chain of α-bungarotoxin (BGT), the A and B chains of β-BGT, and the κ-BGT class: AF056407, AF056407, AJ242012, AJ242011, Y12100, Y12101, Y12265, Y12266 (The above is the ID number in NCBI Genbank).
[0071] Using SignalP software ( http: / / www.cbs.dtu.dk / services / SignalP-2.0 / ) to analyze the signal peptide and propeptide sequences of the above 8 sequences, remove the signal peptide and propeptide sequences and retain the mature peptide sequence for analysis. The secondary structure of the amino acid sequence is predicted by the Jameson-Wolf method combined with the Gamier-Robson and Chou-Fasman method, the hydrophobicity is analyzed by the Kyte-Doolittle method, the flexibility is analyzed by the Karplus-Schulz method, and the site surface possibility is analyzed by the Emini method, Finally, the combination o...
Embodiment 2
[0098] (3) Application of SEQ ID NO: 3 in preparation of antigen by prokaryotic expression
[0099] Design of SEQ ID NO: 3
[0100] On the basis of SEQ ID NO: 1, the Not I (gtg cgg ccg c) and BamH I (gga tcc gc) restriction site sequences on SEQ ID NO: 1 were replaced with BamH I (gta gga tcc) and SalI ( taa gtc gac gta) restriction site sequence and then handed over to a professional company for whole gene synthesis.
[0101] (1) Submit SEQ ID NO: 3 to Beijing Liuhe Huada Gene Technology Co., Ltd. for synthesis. The pUC57 plasmid containing the synthetic gene and the prokaryotic expression plasmid pET-28a(+) were subjected to Sal I / BamH I double enzyme digestion, respectively, and the digestion conditions were carried out according to the instructions of the corresponding enzymes.
[0102](2) The digested product was confirmed to be about 350 bp in size by agarose electrophoresis, and the target fragment was excised, and then recovered according to the instructions of the G...
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Abstract
The invention relates to a bungatotoxin antigen epitope gene and application of the bungatotoxin antigen epitope gene to the preparation of a gene vaccine and an antigen. Two representative sequences are extracted from each of a long chain of alpha-BGT, chains A and B of beta-BGT and kappa-BGT of a cDNA sequence of bungatotoxin, the signal peptide and propeptide parts of the sequences are removed, mature peptide parts are collected, 10 antigen sites are obtained through analysis by a bioinformatics method, and a gene sequence containing the 10 sites is manually synthesized, and is connected to an eukaryotic expression vector pIRESneo to immunize an animal as the gene vaccine to obtain an anti-bungatotoxin neutralizing antibody; the sequence can also be connected to a prokaryotic expression vector PET-28a and then transformed to expression bacterium BL21 to induce the expression of the protein, and the target protein is separated to immunize the animal as an immunogen to obtain the anti-bungatotoxin neutralizing antibody.
Description
technical field [0001] The invention relates to the preparation of a gene and its vaccine, in particular to an artificially synthesized polypeptide sequence from a plurality of antigenic epitopes in α, β and κ silvercycline toxins, a gene and its application, belonging to the field of biomedicine. technical background [0002] Venomous snakebites have long been a serious public health problem in rural areas of Asian and African countries. Snake venom toxins can be divided into three categories according to their properties: nerve poisons, blood circulation poisons, and mixed poisons. Antivenom is the most effective treatment for poisonous snake bites. However, due to the complex composition of snake venom itself, which contains dozens or even hundreds of proteins with different functions, the antivenom produced by horses immune to venom contains not only Antibodies that neutralize toxin components also contain a large number of antibodies against other non-toxin components ...
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