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S69 Mutation Sequence of Influenza Virus Nucleoprotein and Its Mutant and Application

A technology of influenza virus and nucleoprotein, applied in the direction of virus/phage, antisense single-stranded RNA virus, virus, etc., can solve the problem that influenza virus and influenza vaccine cannot effectively achieve preventive and protective effects

Active Publication Date: 2022-03-15
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high variability of influenza virus makes the current influenza vaccine unable to effectively prevent and protect, so it is very important to study a more suitable universal vaccine

Method used

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  • S69 Mutation Sequence of Influenza Virus Nucleoprotein and Its Mutant and Application
  • S69 Mutation Sequence of Influenza Virus Nucleoprotein and Its Mutant and Application
  • S69 Mutation Sequence of Influenza Virus Nucleoprotein and Its Mutant and Application

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preparation example Construction

[0073] The preparation method of mouse anti-M1 monoclonal antibody is described in the following documents: Koestler, T.P., Rieman, D., Muirhead, K., Greig, R.G., Poste, G., Identification and characterization of amonoclonal antibody to an antigen expressed on activated macrophages. Proceedings of the National Academy of Sciences of the United States of America, 1984, 81:4505-4509.;

[0074] Rabbit anti-NP polyclonal antibody (preparation method: use 250 mg of purified His-pET30a-NP protein to immunize 2-month-old female rabbits, with an interval of 2 weeks, boost immunization with 150 mg protein for 3 times, and take serum), the preparation method of the polyclonal antibody is recorded in In the following literatures: Liu, X., Sun, L., Yu, M., Wang, Z., Xu, C., Xue, Q., Zhang, K., Ye, X., Kitamura, Y., Liu , W., Cyclophilin A interacts with influenza A virus M1protein and impairs the early stage of the viral replication. Cellular microbiology, 2009, 11:730-741.;

[0075] Pro...

Embodiment 1

[0098] Embodiment 1, determine the NP phosphorylation site of influenza virus WSN strain

[0099] 1. Determination of NP phosphorylation site of influenza virus WSN strain

[0100] 293T cells were infected with influenza virus A / WSN / 1933 (H1N1) (the strain was rescued by a 12-plasmid reverse genetic system and propagated in MDCK cells; MOI=1). (Roche, 50ml lysate plus 1 tablet) and phosphatase inhibitor (PhosSTOP) (Sigma-Aldrich, 50ml lysate plus 1 tablet) lysate (150mM NaCl, 20mM HEPES [pH 7.4], 1mM EDTA, 10% glycerol , 1% TritonX-100, pH adjusted to 7.4) Cells were lysed at 4°C for 30 minutes, and centrifuged at 12000rpm for 10 minutes; the supernatant of the cell lysate was collected. Add rabbit anti-NP polyclonal antibody to the above supernatant, incubate at 4°C for 4 hours, enrich with protein Gagarose beads (Sigma-Aldrich), and collect protein.

[0101] Alkaline phosphatase (ALP) treatment group: 293T cells were infected with influenza virus A / WSN / 1933 (H1N1) strain (...

Embodiment 2

[0150] The impact of embodiment 2, NP S69 phosphorylation and dephosphorylation on virus pathogenicity

[0151] 1. Effect of phosphorylation or dephosphorylation of NP S69 on viral replication at the cellular level

[0152] 1. Rescue of WSN WT and NP S69 mutant viruses

[0153] Spread the 293T cells in a 60mm dish, and transfect them when they reached 90% confluence within 24 hours; the cells were replaced with Opti-MEM medium before transfection, and the reverse genetic system 12 plasmid (pH21-WSN -PB2, pHH21-WSN-PB1, pHH21-WSN-PA, pHH21-WSN-HA, pHH21-WSN-NP or pHH21-WSN-NP-S69A or pHH21-WSN-NP-S69E, pHH21-WSN-NA, pHH21 -WSN-M, pHH21-WSN-NS, pcDNA4 / TO-PB2, pcDNA4 / TO-PB1, pcDNA4 / TO-PA, pcDNA4 / TO-NP or pcDNA4 / TO-NP-S69A or pcDNA4 / TO-NP-S69E , each 1 μg); operate according to the instructions of Lipofectamine 2000; change the medium after 6 hours of transfection, and replace it with DMEM containing TPCK-treated trypsin (final concentration 2 μg / mL), and cultivate for 72-96 hou...

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Abstract

The invention discloses the S69 mutant sequence of the nucleoprotein of type A influenza virus, its mutant and its application. The invention provides a mutant of the influenza virus nucleoprotein, which is a protein obtained by mutating the 69th serine S in the amino acid sequence of the influenza virus nucleoprotein to glutamic acid E or alanine A, while keeping other amino acid residues unchanged. The present invention relates to NP S69E or S69A pathogenicity of viruses on cells and mice, viral polymerase activity, transcription and replication of viral RNA, NP self-polymerization, NP and viral polymerase protein interaction, NP and RNA binding , NP intracellular localization and so on. The present invention relates to providing influenza vaccine candidate strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutation sequence at the S69 position of nucleoprotein of type A influenza virus, a mutant and an application thereof. Background technique [0002] Influenza A virus (IAV) is the main pathogen that causes influenza epidemics, sometimes causing severe respiratory diseases. Since the host has not yet developed immunity to the newly mutated virus, virus epidemics are often accompanied by significant morbidity and morbidity in humans or animals. mortality rate. Influenza A virus is an enveloped virus belonging to the Orthomyxoviridae family, and its genome consists of 8 negative-strand RNA segments. The viral ribonucleoprotein (vRNP) complex is the core component of the virus, mainly composed of three polymerase proteins (PB1, PB2, PA), nucleoprotein (NP) and viral RNA (vRNA). play an important role in. The high variability of influenza virus makes the current influenza...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/11C12N15/44C12N7/00A61K39/145A61P31/16
CPCC07K14/005C12N7/00A61K39/12A61P31/16C12N2760/16122C12N2760/16121C12N2760/16162C12N2760/16134
Inventor 李芸刘文军郑伟楠
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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