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A 2,4-dioxotetrahydropteridine synthase-based dual-region subunit nanovaccine of the novel coronavirus S protein

A technology of dioxeteridine and coronavirus, applied in the direction of viruses, viral peptides, antiviral agents, etc., to increase the level of neutralizing antibodies, overcome the lack of immunogenicity of RBD monomers, and increase the titer of neutralizing antibodies Effect

Active Publication Date: 2020-11-10
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, the dual antigens derived from the Lumazine Synthase (LS) polymer protein (Lumazine Synthase, LS) of the Aquifexaeolicus strain and the receptor binding domain (RBD) and fusion peptide (FP) of the virus The fragments together form the fusion protein LS-RBD-FP to realize antigen multimerization, and at the same time add signal peptide and purification tag, and express self-assembled LS-RBD-FP through plasmid transfection eukaryotic cell expression system (such as 293F cells) Proteins can assemble LS-RBD-FP monomers into spherical sixtymeric nanoparticles through LS self-assembly, and display them on the surface of nanoparticles, which overcomes the shortcomings of insufficient immunogenicity of RBD monomers and can effectively induce Stronger immune response, producing antibodies that neutralize the SARS-CoV-2 pseudovirus invading target cells

Method used

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  • A 2,4-dioxotetrahydropteridine synthase-based dual-region subunit nanovaccine of the novel coronavirus S protein
  • A 2,4-dioxotetrahydropteridine synthase-based dual-region subunit nanovaccine of the novel coronavirus S protein
  • A 2,4-dioxotetrahydropteridine synthase-based dual-region subunit nanovaccine of the novel coronavirus S protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Construction of novel coronavirus SARS-CoV-2 antigen (fusion protein LS-RBD-FP)

[0065] Schematic diagram of fusion protein LS-RBD-FP self-assembled nanoparticles and schematic diagram of the structure are as follows figure 1 and figure 2 shown.

[0066] Specifically, the construction and preparation method of the fusion protein LS-RBD-FP is as follows:

[0067] 1. Preparation of vector expressing LS-RBD-FP fusion protein

[0068] The LS-RBD-FP nucleotide sequence was cloned into the Xho I and Xba I restriction sites of the expression vector (pcDNA3.1-Intron-WPRE) that added Intron and WPRE to enhance expression after adding a translation stop codon at the 3' end of the LS-RBD-FP nucleotide sequence Between, construct the expression vector pcDNA3.1-Intron-WPRE-LS-RBD-FP-IRES-GFP (such as image 3 shown).

[0069] DH5αcompetent cells were transformed with the recombinant plasmid, cultured overnight at 37°C, and positive clones were identified by screening ...

Embodiment 2

[0074] Example 2 Mouse Immunization Experiment

[0075] The LS-RBD-FP fusion protein obtained in Example 1 was diluted to 100 μg / ml with physiological saline according to Table 1, and emulsified in groups with an equal volume of adjuvant SAS. Then 6-8 weeks old Balb / C mice were immunized in groups. immunization strategies such as Figure 8 As shown, that is, by intraperitoneal injection, each mouse received 3 vaccine immunizations on the 0th day, the 3rd week (21 days), and the 14th week (108 days), each inoculation volume of 200 μl (10 μg) . On the 10th, 31st, and 108th days, blood was collected from the mice's orbits. The mouse serum was obtained by centrifuging at 2800rpm for 15 minutes at 4°C after standing for a period of time to allow the serum to separate out, and was immediately used in the SARS-CoV-2 pseudovirus neutralization detection experiment.

[0076] Table 1

[0077] Antigen / Control Antigen content Adjuvant Number of animals (only) LS-...

Embodiment 3

[0078] Embodiment 3 Pseudovirus neutralization test

[0079] 1. Preparation of pseudovirus:

[0080] According to the sequence published by NCBI, the Spike protein of SARS-CoV-2 was synthesized and inserted into the pcDNA3.1 expression vector. The expression vector of SARS-CoV-2 Spike protein was co-transfected into 293T cells with pHIV-luciferase and psPAX2 plasmids. After 5 hours of transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium to continue culturing. After 48 hours the supernatant was harvested and centrifuged to remove cell debris. Afterwards, the HIV-luc / SARS-CoV-2-S pseudovirus was obtained by dissolving with a small volume of serum-free DMEM.

[0081] The pseudovirus can effectively simulate the process of wild-type SARS-CoV-2 invading cells. When it infects production cells or target cells, the expression of the luciferase reporter gene carried by the SARS-CoV-2 pseudovirus can accurately reflect the results of virus infe...

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Abstract

The present invention discloses a novel coronavirus S protein two-region subunit nano vaccine based on lumazine synthase. A receptor binding domain (RBD) and a fusion peptide (FP) of viruses are usedas double antigens together to connect lumazine synthase (LS) derived from Aquifex aeolicus strain (lumazine synthase, LS) to form a fusion protein LS-RBD-FP to achieve antigen multimerization; and then an eukaryotic cell expression system is used for expression, and a LS self-assembly function can be used to form sixty-mer nano-antigen. The scheme can overcome shortcomings of insufficient immunogenicity of RBD monomers. The obtained vaccine can significantly increase levels of neutralizing antibodies of hosts against the viruses, the produced antibodies have ability to strongly block the viruses from invading target cells, besides, a preparation method of the vaccine is simple, easy to purify and high in safety, and the vaccine can be quickly applied to clinical trials.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to a novel coronavirus (tentative name SARS-CoV-2, also known as 2019-nCoV) S protein dual-region subunit nanovaccine based on 2,4-dioxotetrahydropteridine synthase. Background technique [0002] At present, humans still lack effective vaccines against SARS-CoV-2. Under this severe situation, developing safe and effective vaccines against SARS-CoV-2 as soon as possible to protect susceptible populations is of great importance to the health of our people. important to national security. [0003] For the development of vaccines, it is necessary to understand the structure of viruses. Coronaviruses are a class of enveloped single-positive-sense RNA viruses that are widely distributed in humans, other mammals, and birds, and cause respiratory, digestive, liver, and nervous system diseases. Before this outbreak, there were currently six known coronaviruses that cou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N9/10C12N15/62C12N15/85C12N5/10A61K39/385A61K39/215A61P31/14
CPCA61K39/12A61K39/385A61K2039/6068A61P31/14C07K14/005C07K2319/00C07K2319/735C12N9/1085C12N15/85C12N2770/20022C12N2770/20034C12N2800/107C12Y205/01078
Inventor 张辉马显才邹帆袁耀昌李镕张旭
Owner SUN YAT SEN UNIV
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