Preparation and application of genetic engineering subunit vaccine for infectious bursal disease
A subunit vaccine and bursal disease technology, applied in the fields of bioengineering and immunology, can solve the problems of non-naturalness and affecting immunogenicity
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Embodiment 1
[0067] Embodiment 1, the acquisition of target gene
[0068] 1. Extraction of viral genome total RNA
[0069] The bursa tissue was obtained from SPF chickens infected with a highly virulent strain of infectious bursal disease (vvIBDV). The bursa tissue was ground and homogenized, and frozen and thawed three times. Take 0.02g of tissue into a DEPC-treated 1.5mL EP tube, add 1mL Trizol, mix well, and place at room temperature for 5min. Add 200 μL of chloroform (generally added at a ratio of 0.2 mL chloroform / mL Trizol) to the treated sample, shake gently for 15 seconds, and place in an ice bath for 3 minutes. Centrifuge at 12,000 rpm for 15 min at 4°C. Take the supernatant colorless aqueous phase into a new EP tube (DEPC pretreated), add 500 μL of pre-cooled isopropanol, mix well, and let stand at room temperature for 10 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Carefully discard the supernatant, add 1 mL of 95% (v / v) ethanol (freshly prepared with DEPC water), and mi...
Embodiment 2
[0079] Embodiment 2, construction and identification of recombinant expression plasmid
[0080] The above-mentioned purified target gene VP2 and the expression vector pPICZαA were both digested with XhoI / NotI, recovered and purified by 1% agarose gel, and ligated with T4 ligase. The recombinant expression plasmid obtained was named pPICZαA- VP2. The recombinant expression plasmid was transformed into Escherichia coli JM109 competent cells. Single colonies were isolated in low-salt LB medium containing the antibiotic Zeocin. The plasmid was extracted from the kit, and the pPICZαA-VP2 recombinant plasmid was identified by double enzyme digestion using XhoI / NotI (gene fragments of 3.6kb and 1.4kb in size respectively) (see figure 2 ), using the 5' and 3' AOX1 primers at both ends of the pPICZαA vector (to amplify about 2.0kb gene fragment) and the upstream and downstream primers of the target gene (to amplify about 1.4kb gene fragment) for PCR identification (see image 3 , 4...
Embodiment 3
[0082] Example 3, Screening of recombinant plasmid electroporation of Pichia pastoris and highly resistant recombinant yeast strains
[0083] (1) Linearization and purification of recombinant plasmids
[0084] The recombinant plasmid pPICZαA-VP2 was digested with SacI, bathed in water at 37°C for 2 hours, and then at 65°C for 20 minutes to inactivate the enzyme. Add an equal volume of phenol / chloroform and mix well, centrifuge at 12,000 rpm for two minutes, add 2.5 times the volume of absolute ethanol and 1 / 10 volume of 3M sodium acetate to the supernatant, mix well, and place at -20°C for 10 minutes to precipitate DNA. Centrifuge at 4°C and 12000rpm for 5 minutes, discard the supernatant, wash the pellet with 80% (v / v) ethanol, dry at room temperature and wash with 10 μL sterilized ddH 2 Resuspended in O, the purified linearized recombinant plasmid was stored at -20°C for later use.
[0085] (2) Preparation of Pichia Competent Cells
[0086] The competent preparation metho...
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