Newcastle disease virus chimeric virus-like particle, vaccine and preparation method
A technology for Newcastle disease virus and chimeric virus is applied in the field of Newcastle disease virus chimeric virus-like particles, preparation and prevention of Newcastle disease virulent vaccines, which can solve problems such as mismatch of vaccine strains, achieve elimination of insect cell fusion, Improves synthesis and increases the number of cells growing
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Embodiment 1
[0058] Construction and Detection of Newcastle Disease Virus (NDV) Chimeric Virus-like Particle Protein Vector
[0059] According to the epidemic trend of virulent strains of Newcastle disease virus combined with antigen analysis and codon optimization at the same time, the genes of F protein and HN protein of gene type VII of virulent Newcastle disease virus strains were artificially synthesized, and two retroviruses (chicken leukemia virus ALV, Gag precursor protein gene of human immunodeficiency virus HIV-1).
[0060] Newcastle disease virus fusion protein F and hemagglutinin-neuraminidase HN are used as the membrane glycoprotein of Newcastle disease virus (NDV) chimeric virus-like particles, and have the function of virus surface antigen of virulent strain gene type VII. The Gag precursor protein will be used as the matrix protein of the NDV chimeric virus-like particle, released from the host cell in the form of budding, and self-assembled into the chimeric virus-like par...
Embodiment 2
[0072] Expression and self-assembly of NDV chimeric virus-like particle protein
[0073] Insect cell sf9 was transfected with recombinant bacmid bacmid with F gene, HN gene and Gag gene (from chicken leukemia virus ALV, or human immunodeficiency virus HIV-1), and the cell supernatant was collected six days later to obtain The first generation of recombinant baculovirus strains is called the P1 generation. Infect insect cells with the P1 virus seed to amplify the virus seed to obtain the second-generation recombinant baculovirus seed, which is called the P2 generation. Then use the P2 generation virus seeds to amplify to obtain the P3 generation. The NDV chimeric virus-like particles were prepared by using the P3 generation as the production virus seed. Briefly, Sf-9 cells in good growth state were inoculated into shake flasks, and when the cells grew to the logarithmic growth phase, the cell concentration was diluted to 3.0×10 6 cells. Inoculate the virus into cell shake f...
Embodiment 3
[0083] Purification and Electron Microscopic Observation of NDV Chimeric Virus-like Particles
[0084] NDV chimeric virus-like particles were purified by discontinuous sucrose ultracentrifugation. The collected 20mL cell supernatant was centrifuged at 4°C and 100000xg for 1 hour in an ultracentrifuge with an angle rotor, discarded the centrifuged supernatant, and added 5ml PBS to suspend and dissolve overnight. The sucrose solutions were prepared, and the mass percentage concentrations were respectively adjusted to 20%, 45% and 60%, and carefully loaded into ultracentrifuge tubes to form a discontinuous sucrose density gradient. Add overnight 5ml PBS suspension to the top layer, and perform ultracentrifugation with a horizontal rotor at 4°C, 100000xg, for 1 hour. There is a band at the junction of 20% and 45% and at the junction of 45% and 60%, and the band at the junction of 20% and 45% is the most prominent (see Figure 5 ). The bands here were collected, which contained ...
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