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US 2014/0227310 A1Published application
Recombinant influenza virus highly expressing ha protein and preparation method and use thereof
AssigneeShanghai Veterinary Research Institute, Caas
InventorsZejun Li, Qiaoyang Teng
Life of this patent
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Abstract
A PR8 recombinant influenza virus contains an HA and/or NA gene of H1, H3, H4, H5, H6, H7, H9 or H10 subtype influenza virus, and 6 internal genes (PB1, PB2, PA, NP, M and NS genes) of PR8 virus, in which the NS and/or NP gene have the following mutation sites: the NS2 protein encoded by the NS gene has an E67S point mutation, E74S point mutation, or E67S/E74S point mutation, and the NP protein encoded by the NP gene has a G132A point mutation.
Claims19
A PR8 recombinant influenza virus comprising
- an HA and/or NA gene of an influenza virus, and
- 6 internal genes (PB1, PB2, PA, NP, M and NS genes) of PR8 virus,
- wherein the NS and/or NP gene comprises the following mutation sites:
- the NS2 protein encoded by the NS gene has an E67S point mutation, E74S point mutation or E67S/E74S point mutation, and
- the NP protein encoded by the NP gene has a G132A point mutation, and
- wherein the influenza virus is one selected from the group consisting of an H1 subtype influenza virus other than PR8 virus, an H3 subtype influenza virus, an H4 subtype influenza virus, an H5 subtype influenza virus, an 116 subtype influenza virus, an H7 subtype influenza virus, an H9 subtype influenza virus, and an H10 subtype influenza virus.
3 more elements
A method for preparing a PR8 recombinant influenza virus, comprising:
- constructing a recombinant plasmid comprising the HA and NA genes of the H1 other than PR8 virus, H3, H4, H5, H6, H7, H9 or H10 subtype influenza virus;
- constructing a recombinant plasmid comprising a PR8 virus mutant gene fragment, the PR8 virus mutant gene fragment being selected from the group consisting of the following mutant NS or NP gene fragments:
- a PR8 virus NS gene fragment encoding the NS2 protein containing the E67S point mutation, E74S point mutation or E67S/E74S point mutation, and a PR8 virus NP gene fragment encoding the NP protein containing the G132A point mutation;
- co-transfecting the recombinant plasmid of the HA and NA genes, the recombinant plasmid comprising the PR8 virus mutant gene fragment, and the plasmids respectively comprising the PA, PB1, PB2, M, NP or NS internal gene of PR8 virus, into a 293T cell, and culturing the transfected cell;
- inoculating the cultured cell supernatant into chicken embryos, and culturing the chicken embryos in an incubator for a proper time period to acquire chicken embryo allantoic fluid, detecting the hemagglutination condition of the allantoic fluid, and in case of the presence of hemagglutinin activity, determining the absence of unexpected mutations by sequence analysis to acquire the PR8 recombinant influenza virus.
2 more elements
Dependencies are inferred from the claim text.