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Rabies vaccines and applications based on self-assembled ferritin nano-antigen particles and prepared therefrom

An antigen and fusion protein technology, applied in the field of preparation and application of rabies vaccine, can solve the problem of restoring virulence of attenuated strains, and achieve the effects of high anti-rabies antibody level, increased immune efficacy, and increased immune range

Active Publication Date: 2022-06-21
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • 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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  • Rabies vaccines and applications based on self-assembled ferritin nano-antigen particles and prepared therefrom
  • Rabies vaccines and applications based on self-assembled ferritin nano-antigen particles and prepared therefrom
  • Rabies vaccines and applications based on self-assembled ferritin nano-antigen particles and prepared therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation and efficacy detection of RV G-Ferritin original sequence nanoparticle vaccine

[0068] 1 Configuration of solutions and media

[0069] For the configuration method of solution and medium, refer to relevant reference books (Joseph et al., Molecular Cloning Experiment Guide, 3rd Edition, 2002; Osber, et al., Refined Molecular Biology Guide, 1998).

[0070] 2. Synthesis of rabies virus G antigen protein gene sequence and ferritin gene sequence.

[0071] In order to make the fusion expression of rabies virus G antigen protein and ferritin better, the amino acid sequence of RV G rabies virus G antigen protein was analyzed by signal peptide analysis software (SignalP) and transmembrane domain analysis software (TMHMM). The signal peptide of the rabies virus G antigen protein is the first 19 amino acids, and the 1-19 amino acids are removed, and the extracellular domain is the first 458 amino acids.

[0072] In order to promote the expression efficienc...

Embodiment 2

[0158] Example 2 The original sequence of RV G-Ferritin was used for the design of the sympathetic sequence and the preparation and efficacy detection of the optimized nanoparticle vaccine

[0159] 1 Configuration of solutions and media

[0160] See Example 1 for the configuration method of the specific solution and culture medium.

[0161] 2 Gene acquisition of the conserved sequence of rabies virus G protein

[0162] The present invention compares the original amino acid sequence of rabies virus G protein in Example 1 with other 20 RV G protein amino acid sequences obtained from NCBI, and obtains a consensus sequence. This sympathetic sequence was optimized to further utilize OptimumGene TM The technology optimizes the amino acid sequence of the RV G protein, and the optimized amino acid sequence of the RV G protein and the amino acid sequence of the ferritin monomer subunit are modified according to the codon preference of the silkworm. The folded GC content, CpG dinucle...

Embodiment 3

[0184] Example 3 Nanoparticle vaccine preparation and efficacy detection of RV G-Ferritin-C-O mutants subjected to amino acid single-site mutation, double-site mutation and multi-site mutation

[0185] 1 Experimental method

[0186] 1.1 Construction of single-site, double-site and multi-site mutant genes of RV G-Ferritin-C-O amino acid sequence

[0187] Based on the results of Example 2, the present invention obtained an RV G-Ferritin-C mutant. Using the codon-optimized gene sequence of the RV G-Ferritin-C mutant as a template, multiple pairs of primers were designed to perform site-directed mutation on the conserved sequence, Site-directed mutagenesis was carried out by fusion PCR method, see Example 1 for fusion PCR method.

[0188] The mutation sites are RV G: W33N, L57M, V75H, G82S, T99P, R126M, Y135Q, R142H, L168T, G175D, N201P, V229E, V260K, P273F, E294K, E300L, K313F, I358V, I392S, A419T. The resulting mutant was named RV G-Ferritin-C-O-M (W33N, L57M, V75H, G82S, T99P...

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Abstract

The invention discloses a rabies vaccine and application based on self-assembled ferritin nanometer antigen particles and prepared therefrom. In the invention, the rabies virus G antigen protein is fused with the self-assembled ferritin nanoparticle subunit to obtain a fusion protein. In the present invention, the rabies virus G antigen protein is subjected to site mutation, and the soluble expression amount and expression efficiency of the obtained mutant are significantly improved. The present invention utilizes prokaryotic expression system, silkworm and AcMNPV-insect cell eukaryotic expression system to express recombinant protein, or performs gene presentation in vertebrate body by recombinant baculovirus to produce antigen and induce antibody production. The rabies vaccine provided by the present invention displays antigenic proteins on the surface of the ferritin cage structure of Helicobacter pylori, causing broadly neutralizing anti-rabies virus antibodies, improving immune efficacy and expanding the scope of immunity, and has the potential of a universal vaccine with cross-immunity efficacy . The vaccine preparation method of the present invention is safe and convenient, and is suitable for rapid large-scale production.

Description

technical field [0001] The present invention relates to ferritin nano-antigen particles based on self-assembly, in particular to nano-antigen particles comprising rabies virus G antigen protein and monomeric ferritin subunit fused together and a rabies vaccine prepared from the nano-particle antigen, belonging to the rabies vaccine category. Field of preparation and application. Background technique [0002] Rabies (rabies) is an acute infectious disease caused by rabies virus, which is common to both humans and animals. It is more common in dogs, wolves, cats and other carnivorous animals. The clinical manifestations are unique hydrophobia, wind phobia, pharyngeal muscle spasm, progressive paralysis, etc. Because of the prominent symptoms of hydrophobia, the disease is also known as hydrophobia. The rabies virus belongs to the Rhabdoviridae family, the genus Rabies. It is a single-stranded RNA virus that animals transmit by biting each other. Rabies in China is mainly tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/866C12N15/62B82Y40/00A61K39/205A61P31/14
CPCC07K14/195C07K14/415C07K14/435C12N15/86B82Y40/00A61K39/12A61P31/14C12N2710/14043C12N2760/20134A61K2039/5258C07K2319/00
Inventor 张志芳易咏竹李轶女刘兴健胡小元魏珍珍
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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