Recombination gene VII type Newcastle disease multi-epitope vaccine
A Newcastle disease, multi-epitope technology, applied in the field of biotechnology genetic engineering, can solve the problems of increasing the cost of immunization and the impact of immunization effects
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Embodiment 1
[0025] Example 1 Design Idea of Gene VII Type Newcastle Disease Multi-epitope Vaccine Protein
[0026] The present invention is based on the amino acid sequences of two envelope glycoproteins related to virus infection: fusion protein (F), neuraminidase (HN) and nucleocapsid protein (NP) in the genome of the main epidemic strain of gene VII Newcastle disease in China at present, The Th epitope, CTL epitope and B cell epitope were analyzed on the F protein, HN protein and NP protein of the epidemic strains by using relevant bioinformatics software DNASTAR, Bcepred, Multipre, BIMAS and SYFPEITHI. The designed epitopes were concatenated and expressed in Escherichia coli, and after fermentation, purification, emulsification and other processes, a genotype VII Newcastle disease multi-epitope vaccine with ideal immunogenicity was obtained. The vaccine prepared by the invention can effectively prevent genotype VII Newcastle disease.
Embodiment 2
[0029] Embodiment two Escherichia coli expression vector and the construction of expression bacterial strain
[0030] 1 Send the designed polypeptide encoding nucleotides to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. BamHI (5' end) and HindIII (3' end) restriction enzyme sites are designed at both ends of the nucleotide fragment respectively. After synthesis, they were respectively cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the designed sequence (see the sequence list). The recombinant plasmids were respectively named pMD18T-NDV(VII). Digest the plasmid with the corresponding restriction endonuclease. The expression vector of E. coli is the pRSETA plasmid from Invitrogen Company, which is also treated with the same restriction endonuclease. Digestion conditions: 10 μl reaction system, add 2 μl plasmid into the system , 5 activity units of restriction endonuclease (New England Biolabs), ...
Embodiment 3
[0034] Fermentation, purification and emulsification of embodiment three engineering bacteria
[0035] 1 Fermentation Take the production strain pRSETA-NDV(VII) / BL21(DE3, Plys), inoculate it in 2 mL of LB liquid medium (containing 100 μg / mL ampicillin), and culture it at 37°C and 200 rpm for 12 hours to activate the strain. Then inoculate the shake flask with an inoculation amount of 1:100, shake and culture at 37°C until OD600=3, and then inoculate it into a fermenter at a ratio of 10%. The fermentation medium is a semi-synthetic medium prepared with distilled water. Calibrate the dissolved oxygen and pH electrodes, start the tank to stir, the rotation speed is 300rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37.0°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature was 37.0±0.1°C, the dissolved oxygen was controlled at about 40%, and the pH was controlled at 7.0. After inoculation, wh...
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