Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine
A technology of genetic engineering and inactivated vaccines, which is applied in the field of fusion protein and the preparation of the fusion protein, can solve the problems of incomplete inactivation in mass production, loose virus, and limited promotion
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Embodiment 1
[0021] Example 1 The source of the fusion protein gene
[0022] The present invention comprehensively analyzes the gene sequence, antigen structure and epidemiological research progress of Burmese 98 and pan-sub-lineage O-type foot-and-mouth disease virus, according to the amino acid sequence of its main structural proteins VP1, VP2, VP3, and uses relevant bioinformatics software to analyze its relative Analysis of water content, antigenicity, plasticity, surface accessibility and secondary structure to predict possible B cell epitopes and T cell epitopes, and comprehensively related reports to determine the 4 segments of VP1 epitopes of different strains , 2 segments of VP2 antigenic epitope, 1 segment of VP3 antigenic epitope, and at the same time introduce the dominant antigenic epitope of T cell helper epitope and the dominant antigenic epitope of killer T cell. All epitopes are connected with a flexible Linker and then connected in series with the molecular adjuvant IL-2....
Embodiment 2
[0024] Example 2 Construction of Escherichia coli expression vector and expression strain
[0025] The polypeptide coding nucleotides designed in Example 1 were sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis, and EcoR I (5' end) and HindIII (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment , The synthesized fragment was cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the related sequence (see the sequence list). The recombinant plasmid was named pMD18T-FMDV-IL-2, and the corresponding restriction endonuclease was used to digest the plasmid. The Escherichia coli expression vector was pRSETA plasmid from Invitrogen Company, and the same restriction endonuclease was also used. Digestion conditions: 10 μL reaction system, 2 μL plasmid, 5 activity units of restriction endonuclease (New England biolabs), 1 μL 10× buffer, supplemented with deionized water, di...
Embodiment 3
[0029] Example 3 Fermentation, purification and emulsification of engineering bacteria
[0030] Fermentation Inoculate the production strains into 2 mL of LB liquid medium containing 100 μL / mL ampicillin, and culture at 37°C with shaking at 180 rpm for 12 hours to activate the strains. Put the activated strains into shake flasks at an inoculum size of 1:100, shake and culture at 37°C until OD600=3, and then inoculate them into fermenters at a ratio of 10%. The medium used for fermentation is a semi-synthetic medium prepared with distilled water and does not contain any antibiotics. Calibrate dissolved oxygen and pH electrodes, turn on the tank to stir at 300 rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature is 37.0°C±0.1°C, the dissolved oxygen is controlled at about 20%, and the pH is controlled at 7.0. After inoculation, when the OD6...
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