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A novel coronavirus S protein single-region subunit nanovaccine based on Helicobacter pylori ferritin

A technology of Helicobacter pylori and coronavirus, applied in the directions of viruses, viral peptides, antiviral agents, etc., to achieve the effects of easy purification, simple preparation method, and overcoming insufficient immunogenicity of monomers

Active Publication Date: 2021-07-09
GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, the receptor binding domain (RBD) of the virus is used as the antigen fragment, and the fusion protein RBD-HP_Ferritin is formed with the Helicobacter pylori polymer protein (Helicobacter pylori_Ferritin, Ferritin (HP)) to realize antigen multimerization , and add signal peptide and purification tag at the same time, express self-assembled RBD-HP_Ferritin protein through plasmid transfection eukaryotic cell expression system (such as 293F cells), and assemble RBD-HP_Ferritin monomer through Ferritin (HP) self-assembly Spherical twenty-tetramer nanoparticles, which are displayed on the surface of nanoparticles, overcome the shortcomings of insufficient immunogenicity of RBD monomers, can effectively induce stronger immune responses, and produce neutralized SARS-CoV-2 pseudoviruses Antibodies that invade target cells

Method used

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  • A novel coronavirus S protein single-region subunit nanovaccine based on Helicobacter pylori ferritin
  • A novel coronavirus S protein single-region subunit nanovaccine based on Helicobacter pylori ferritin
  • A novel coronavirus S protein single-region subunit nanovaccine based on Helicobacter pylori ferritin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1 constructs novel coronavirus SARS-CoV-2 antigen (fusion protein RBD-HP_Ferritin)

[0075] The schematic diagram and structural diagram of fusion protein RBD-HP_Ferritin self-assembled nanoparticles are as follows figure 1 and figure 2 shown.

[0076] Specifically, the construction and preparation method of the fusion protein RBD-HP_Ferritin is as follows:

[0077] 1. Preparation of vector expressing RBD-HP_Ferritin antigen

[0078] After the 3' end of the RBD-HP_Ferritin nucleotide sequence is added with a translation stop codon, it is cloned between the Xho I and Xba I restriction sites of the expression vector (pcDNA3.1-Intron-WPRE) that adds Intron and WPRE to enhance expression , construct the expression vector pcDNA3.1-Intron-WPRE-RBD-Ferritin (HP)-IRES-GFP (such as image 3 shown).

[0079] DH5αcompetent cells were transformed with the recombinant plasmid, cultured overnight at 37°C, and positive clones were identified by screening and PCR. Ext...

Embodiment 2

[0084] Embodiment 2 mouse immunization experiment

[0085] The RBD-HP_Ferritin antigen 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.

[0086] Table 1

[0087] Antigen / Control Antigen content Adjuvant Number of animals (only) R...

Embodiment 3

[0088] Embodiment 3 Pseudovirus neutralization test

[0089] 1. Preparation of pseudovirus:

[0090] 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.

[0091] 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 single region region subunit nano vaccine based on Helicobacter pylori ferritin. The present invention uses virus receptor binding domain (Receptor binding domain, RBD) as antigen, and connects with Helicobacter pylori polymer protein (HP_Ferritin) to form fusion protein RBD-HP_Ferritin, realizes antigen multimerization; reuses eukaryotic cells Expressed by the expression system, it can form tetratetramer nanoantigens through the self-assembly of HP_Ferritin. This solution can overcome the disadvantage of insufficient immunogenicity of RBD monomer, and the resulting vaccine can significantly increase the level of neutralizing antibodies of the host against the virus, and the antibodies produced have the ability to strongly block the virus from invading target cells. Moreover, the preparation method of the vaccine of the present invention is simple, easy to purify, and has high 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 (2019-nCoV) S protein single-region subunit nanovaccine based on Helicobacter pylori ferritin. Background technique [0002] The clinical manifestations of pneumonia caused by the new coronavirus (tentatively named SARS-CoV-2, also known as 2019-nCoV) are very similar to those of viral pneumonia; the main clinical manifestations are fever, fatigue, dry cough, etc., and severe cases may cause shock, Sepsis, respiratory failure and death. As the virus source and pathogenesis of the novel coronavirus pneumonia are not yet clear, and there is a lack of specific antiviral drugs, it has brought great difficulties to clinical diagnosis, treatment and control of the epidemic, causing serious social burden and crisis. [0003] At present, humans still lack effective vaccines against SARS-CoV-2. Under this severe situation, developing safe and effe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61P31/14A61P11/00
CPCC07K14/005C07K14/20C12N15/85A61K39/12A61K39/39A61P31/14A61P11/00C07K2319/00C12N2770/20022C12N2770/20034A61K2039/55511C12N15/62A61K39/385A61K2039/6068A61K2039/55566A61K2039/55555A61K39/215C07K2319/02C07K2319/21C12N15/86C12N2770/20043
Inventor 张辉马显才邹帆袁耀昌李镕张旭
Owner GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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