Avian infectious bronchitis virus (IBV)-like particle and preparation method and application thereof
A bronchitis and virus-like technology, applied in the fields of genetic engineering and medical immunology, can solve the problem of few veterinary VLPs vaccines, achieve good innate immunity and mucosal immune response, strong immune protection, and good immune effect
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Embodiment 1
[0039] Preparation and identification of embodiment 1 recombinant S, M, E protein
[0040] FastBac TM / HBM-TOPO transposable vector and DH10Bac TM Competent cells were purchased from Invitrogen; Sf9 insect cells and IBV GX-YL5 strain were preserved by the Institute of Poultry Breeding and Poultry Diseases, Guangxi University.
[0041] 1) Primer design and synthesis
[0042] Refer to the IBV GX-YL5 complete gene sequence included in NCBI GenBank (GenBank serial number: HQ848267.1), find out the corresponding S, M and E gene sequences, and use the SignalP4.1 Server online software to analyze the signal peptides of the three genes respectively. Prediction, after removing the signal peptide sequence, start codon and stop codon (there are start codon and stop codon on the carrier), design primers corresponding to the 3 genes and M13 for later PCR identification of recombinant bacmid and post-transfection PCR identification The universal primers for the vector were synthesized by...
Embodiment 2
[0149] Preparation and identification of embodiment 2 SME-VLPs
[0150] 1) Preparation of SME-VLPs
[0151] Under the optimal conditions of exploration, press 2×10 6 cells / mL inoculated Sf9 cells (vitality > 90%) in cell suspension culture flasks, and after suspension culture in a constant temperature incubator at 27°C for 12 hours, the cells grew stably. According to the same MOI (S:M:E= 3:3:3) co-infect the Sf9 insect cells in suspension culture, and harvest the cell suspension at 84h.
[0152] 2) Sucrose density gradient centrifugal purification of SME-VLPs
[0153] The above harvested cell suspension was centrifuged at 4°C and 2000r / min for 20min, the supernatant was discarded, the cell pellet was collected and resuspended in pre-cooled PBS, and then placed at -80°C for three times of repeated freezing and thawing, and then ultrasonicated on ice ( Work for 5s, rest for 5s) to fully release the virus particles, sonicate until it is no longer viscous, then the crushing is...
Embodiment 3
[0161] Immunogenicity and immune protection research of embodiment 3SME-VLPs
[0162] Preparation of oil-emulsion seedlings
[0163] (1) The inactivated GX-YL5 allantoic fluid is made into an aqueous phase by containing 2 μg S protein / feather volume ratio, virus liquid: autoclaved Tween-80=96:4, No. 10 white oil: Siben- 80=10:1 mix well, make oil phase after autoclaving. The water phase is added dropwise to the oil phase (water phase: oil phase = 2:3), fully oscillated evenly, and the inactivated oil emulsion seedlings of the water-in-oil property are made;
[0164] (2) SME-VLPs containing 2 μg of S protein / feather were completely emulsified and mixed with an equal amount of Freund's complete adjuvant to prepare an oil-free emulsion vaccine. Completely emulsify and mix with Freund's incomplete adjuvant according to the same dose, and prepare the second free oil emulsion vaccine.
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