Based on self-assembled ferritin nanoantigen particles and influenza vaccine and preparation method

An antigen and nanotechnology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of restoring virulence of attenuated strains, and achieve the effect of safe operation

Active Publication Date: 2022-07-26
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biggest drawback of using attenuated strain vaccines is the risk of the attenuated strain reverting to virulence in susceptible populations

Method used

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  • Based on self-assembled ferritin nanoantigen particles and influenza vaccine and preparation method
  • Based on self-assembled ferritin nanoantigen particles and influenza vaccine and preparation method
  • Based on self-assembled ferritin nanoantigen particles and influenza vaccine and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Preparation and efficacy detection of H9 HA-Ferritin and H5 HA-Ferritin original sequence nanoparticle vaccines

[0074] 1 Configuration of solutions and media

[0075] For the configuration method of solution and medium, refer to relevant reference books (Joseph et al., Molecular Cloning Experiment Guide, Third Edition, 2002; Osber, et al., Refined Molecular Biology Guide, 1998).

[0076] 2 Synthesis of H9, H5 hemagglutinin protein gene sequences and ferritin gene sequences.

[0077] In order to make the fusion expression of influenza virus hemagglutinin and ferritin better, the signal peptide analysis software (SignalP) and the transmembrane domain analysis software (TMHMM) were used to analyze H9 (SEQ ID NO. 4) The amino acid sequence of the influenza virus hemagglutinin protein was analyzed, and it was obtained that the signal peptide of the H9 hemagglutinin protein was the first 18 amino acids, and the extracellular domain was the first 524 amino acids. ...

Embodiment 2

[0178] Example 2 Preparation and efficacy testing of nanoparticle vaccines after H9 HA-Ferritin and H5 HA-Ferritin original sequences were designed and optimized by sympathetic sequence

[0179] 1 Configuration of solutions and media

[0180] See Example 1 for the configuration method of the specific solution and culture medium.

[0181] 2. Gene acquisition of the conserved sequence of influenza virus hemagglutinin protein

[0182] The present invention compares the original amino acid sequence of the influenza virus hemagglutinin protein in Example 1 with other 20 hemagglutinin amino acid sequences obtained from NCBI to obtain a consensus sequence. This sympathetic sequence was optimized to further utilize OptimumGene TMThe technology optimizes the amino acid sequences of H5 and H9 hemagglutinin proteins, and modifies the amino acid sequences of the optimized hemagglutinin protein amino acid sequences and ferritin monomer subunit amino acid sequences according to the codon ...

Embodiment 3

[0236] Example 3 Nanoparticle vaccine preparation and efficacy detection of H9 HA-Ferritin-C-O and H5 HA-Ferritin-C-O mutants subjected to single amino acid site mutation

[0237] 1 Experimental method

[0238] 1.1 Construction of H9 HA-Ferritin-C-O, H5 HA-Ferritin-C-O amino acid sequence single point mutant genes Based on the results of Example 2, the present invention obtained H9 HA-Ferritin-C, H5 HA-Ferritin-C mutants, Using the codon-optimized gene sequences of H9HA-Ferritin-C and H5 HA-Ferritin-C mutants as templates, multiple pairs of primers were designed to carry out site-directed mutagenesis of conserved sequences. Site-directed mutagenesis was performed by fusion PCR. The fusion PCR method See Example 1.

[0239] The mutation sites are H9: N40S, N41D, A47T, L51I, M58I, G63N, P83L, P101S, T138K, E181G, N191H, H192Q, D196E, T197A, G236R, I267V, Q304H, D367N, N419S, V469M; H5: V , K51R, T52A, K56R, S72R, N100S, H126R, G155E, E186K, D187N, V190I, L191M, G193W, I242M, N...

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Abstract

The invention discloses an influenza vaccine and a preparation method based on self-assembled ferritin nanometer antigen particles. In the present invention, the extracellular domain of the influenza virus hemagglutinin protein is fused and expressed with the N-terminal of the self-assembled ferritin nanoparticle subunit, and the influenza virus hemagglutinin protein is displayed on the surface of the self-assembled ferritin cage structure. The invention carries out mutation optimization of the influenza virus hemagglutinin protein, improves its soluble expression amount and expression efficiency, improves the immune efficacy and breadth of the vaccine, and improves the immunogenicity of the influenza virus hemagglutinin protein. The present invention utilizes E. coli prokaryotic expression system, silkworm and AcMNPV-insect cell eukaryotic expression system to express and prepare recombinant protein vaccine respectively, or generate antigen and induce antibody production by gene presentation of recombinant baculovirus in vertebrate in vivo tissue. The influenza vaccine of the invention can induce broadly neutralizing anti-influenza antibodies, can not only improve the immune efficacy but also expand the immune range, and has the potential of a universal vaccine with cross-immunity efficacy.

Description

technical field [0001] The invention relates to ferritin nano-antigen particles based on self-assembly, in particular to nano-antigen particles comprising influenza virus hemagglutinin protein and monomeric ferritin subunits fused together and influenza vaccine prepared from the nano-particle antigen, belonging to influenza vaccines field. Background technique [0002] Influenza (flu) is an acute respiratory infectious disease caused by influenza virus (influenza virus). influences. Influenza viruses are divided into three types: A (A), B (B), and C (C). The bovine influenza virus discovered in recent years will be classified as type D (D). Among them, influenza A virus frequently undergoes antigenic mutation, causing several influenza pandemics around the world. The best tool to prevent influenza virus is mainly vaccine. The main mechanism of the currently used influenza vaccine is to induce the body to produce protective antibodies against the surface antigens of influe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/866C12N15/62B82Y40/00A61K39/145A61P31/16
CPCC07K14/195C07K14/415C07K14/435C12N15/86B82Y40/00A61K39/12A61P31/16C12N2710/14043
Inventor 张志芳李轶女胡小元宋浩志刘兴健
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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