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Novel coronavirus S protein two-region subunit nano vaccine based on pyrococcus furiosus ferritin

A coronavirus and protein technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., to overcome the lack of immunogenicity of monomers, improve the level of neutralizing antibodies, and strengthen the immune response

Active Publication Date: 2020-06-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, the virus receptor binding domain (Receptorbinding domain, RBD) and fusion peptide (Fusion peptide, FP) are used together as a double antigen fragment, and a fusion protein is formed with Pyrococcus furiosus_Ferritin, Ferritin (PF) RBD-FP-PF_Ferritin realizes antigen multimerization, and at the same time adds signal peptide and purification tag, and expresses self-assembled RBD-FP-PF_Ferritin protein through plasmid transfection eukaryotic cell expression system (such as 293F cells), which can be passed through Ferritin (PF) self-assembly assembles RBD-FP-PF_Ferritin monomers into spherical twenty-tetramer nanoparticles and displays them on the surface of nanoparticles, which overcomes the shortcomings of insufficient immunogenicity of RBD monomers and can effectively induce more Strong immune response, producing antibodies that neutralize SARS-CoV-2 pseudoviruses invading target cells

Method used

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  • Novel coronavirus S protein two-region subunit nano vaccine based on pyrococcus furiosus ferritin
  • Novel coronavirus S protein two-region subunit nano vaccine based on pyrococcus furiosus ferritin
  • Novel coronavirus S protein two-region subunit nano vaccine based on pyrococcus furiosus ferritin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 constructs novel coronavirus SARS-CoV-2 antigen (fusion protein RBD-FP-PF_Ferritin)

[0067] Schematic diagram of fusion protein RBD-FP-PF_Ferritin self-assembled nanoparticles and schematic diagram of the structure are as follows figure 1 and figure 2 shown.

[0068] Specifically, the construction and preparation method of the fusion protein RBD-FP-PF_Ferritin is as follows:

[0069] 1. Preparation of vector expressing RBD-FP-PF_Ferritin fusion protein

[0070] After the 3' end of the RBD-FP-PF_Ferritin nucleotide sequence is added with a translation stop codon, it is cloned into the Xho I and Xba I restriction sites of the expression vector (pcDNA3.1-Intron-WPRE) that adds Intron and WPRE to enhance expression Between, construct the expression vector pcDNA3.1-Intron-WPRE-RBD-FP-Ferritin(PF)-IRE S-GFP (such as image 3 shown).

[0071] DH5αcompetent cells were transformed with the recombinant plasmid, cultured overnight at 37°C, and positive clones w...

Embodiment 2

[0076] Embodiment 2 mouse immunization experiment

[0077] The RBD-FP-PF_Ferritin 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.

[0078] Table 1

[0079] Antigen / Control Antigen content Adjuvant Number of animals (on...

Embodiment 3

[0080] Embodiment 3 Pseudovirus neutralization test

[0081] 1. Preparation of pseudovirus:

[0082] 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.

[0083] 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 invention discloses a novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus furiosus ferritin. The virus receptor binding domain (RBD) and fusion peptide (FP) are usedtogether as a double antigen, and are connected with pyrococcus furiosus ferritin (PF_Ferritin) to from a fusion protein RBD-FP-PF_Ferritin to realize antigen multimerization; and then an eukaryoticcell expression system is used for expressing, and a 24-mer nano antigen can be formed through self-assembly of PF_Ferritin. The scheme can overcome the shortcoming of insufficient immunogenicity of RBD monomers, the obtained vaccine can significantly increase the level of a neutralizing antibody against the virus of a host, and the produced antibody has the ability to strongly block the virus from invading target cells. In addition, the vaccine of the invention is simple in preparation method, is easy to purify, and is high in safety, and the vaccine can be relatively 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 nano-vaccine based on pyrococcal ferritin. 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 could cause human di...

Claims

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Application Information

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