Hirame rhabdovirus genetic engineering subunit vaccine
A technology of genetic engineering and flounder bomb is applied in the field of flounder rhabdovirus genetic engineering subunit vaccine, which can solve problems such as economic loss
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Embodiment 1
[0014] Example 1 Obtaining of Flounder Rhabdovirus Novel Fusion Protein rG Gene
[0015] Using the biological software DNAStar to analyze the antigenic epitope of the G protein of flounder rhabdovirus (GenBank accession number: AB103462), remove the signal peptide of 20 amino acids at the N-terminus, and the strong hydrophobic region composed of 168 amino acids at the C-terminus that affects the recombinant expression of the protein , and introduce a general immune-enhancing T cell epitope (TAKSKKFPSYTATYQF) behind the 42-position amino acid at the N-terminal to obtain a novel fusion protein rG, the amino acid sequence of which encodes the protein is SEQ ID NO: 1, and uses online bio The software http: / / www.jcat.de / optimized the rare codons of Pichia pastoris to obtain the nucleotide sequence SEQ ID NO:2 of the fusion protein rG.
[0016] The nucleotide sequence of the newly obtained fusion protein rG was subjected to whole gene synthesis, and XhoI and XbaI restriction sites ...
Embodiment 2
[0017] The construction of embodiment 2 genetic engineering protein expression vector and the acquisition of engineering bacteria
[0018] 1. Both the vector containing the rG gene of the new fusion protein of the flounder rhabdovirus and the yeast expression vector were digested with XhoI and XbaI, and the digested products were recovered and ligated for PCR identification and sequencing.
[0019] 2. After the positive plasmid was linearized by SacI single enzyme digestion, it was added to the competent cell suspension of Pichia pastoris. After electroporation, spread evenly on YPDS selection plate containing 100 μg / mL Zeocin, and incubate at 30°C for 3-5 days. When the positive transformants on the YPDS plate grow larger, inoculate each transformant onto the YPDS selection plate containing Zeocin 200 μg / mL, 500 μg / mL, and 1000 μg / mL in order to eliminate the normal growth of colonies on the high-concentration Zeocin plate. possible high copy recombination strain.
[0020...
Embodiment 3
[0021] Example 3 Preparation of flounder rhabdovirus genetically engineered subunit vaccine
[0022] 1. Fermentation of recombinant bacteria
[0023] 1) Inoculate the positive recombinants obtained by screening into Erlenmeyer flasks with 1%-10% inoculum volume after activation, and inoculate 10L with 5%-20% inoculation volume at 28-30°C and 200r / min shaker for 16-24h Fermentation tank (installed culture medium 6L), temperature 28-30 ℃, rotation speed 500-1500r / min, medium pH value 5.0-6.0, ventilation volume 0.1-1.0VVM (the amount of oxygen introduced into 1L fermentation broth for 1min), dissolved Fermentation is carried out under the condition of oxygen>20%, and 50% glycerol is fed for 4 hours after culturing for 18-24 hours. When the dissolved oxygen suddenly rises to 100%, methanol is fed until the end of fermentation. The whole fermentation lasts for 48-72 hours.
[0024] 2) After the fermentation, centrifuge at 5000r / min for 10min, and collect the fermentation supernat...
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