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

A coronavirus, a new type of technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., to achieve the effect of easy purification, simple preparation method, and increase the level of neutralizing antibodies

Active Publication Date: 2021-07-09
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 (Receptor binding domain, RBD) and fusion peptide (Fusion peptide, FP) are used together as a double antigen fragment, and it is composed of Helicobacter pylori_Ferritin, Ferritin (HP) The fusion protein RBD-FP-HP_Ferritin realizes the multimerization of the antigen, and at the same time adds a signal peptide and a purification tag, and expresses the self-assembling RBD-FP-HP_Ferritin protein through a plasmid transfection eukaryotic cell expression system (such as 293F cells), which can Self-assembly of RBD-FP-HP_Ferritin by Ferritin (HP) self-assembles RBD-FP-HP_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 Elicit a stronger immune response and produce antibodies that neutralize the SARS-CoV-2 pseudovirus invading target cells

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 constructs novel coronavirus SARS-CoV-2 antigen (fusion protein RBD-FP-HP_Ferritin)

[0069] The schematic diagram and structural diagram of fusion protein RBD-FP-HP_Ferritin self-assembled nanoparticles are as follows figure 1 and figure 2 shown.

[0070] Specifically, the construction and preparation method of the fusion protein RBD-FP-HP_Ferritin is as follows:

[0071] 1. Preparation of vector expressing RBD-Ferritin antigen

[0072] After the 3' end of the RBD-FP-HP_Ferritin nucleotide sequence (shown in SEQ ID NO: 4) is added with a translation stop codon, it is cloned into the expression vector (pcDNA3.1-Intron-WPRE) that adds Intron and WPRE to enhance expression Between the Xho I and Xba I restriction sites, construct the expression vector pcDNA3.1-Intron-WPRE-RBD-FP-Ferritin (HP)-IRES-GFP (such as image 3 shown).

[0073] DH5α competent cells were transformed with the recombinant plasmid, cultured overnight at 37°C, and positive clones were...

Embodiment 2

[0078] Embodiment 2 mouse immunization experiment

[0079] The RBD-FP-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 immunize groups of Balb / C mice aged 6-8 weeks. Immunization strategies such as Figure 8 As shown, that is, by intraperitoneal injection, each mouse received 3 times of vaccine immunization on day 0, week 3 (day 21), and week 14 (day 108), with an inoculation volume of 200 μl each time (10 μg) . On the 10th, 31st, and 108th days, blood was collected from the mice's orbits. After standing for a period of time to allow the serum to separate out, the mouse serum was obtained by centrifuging at 2800rpm for 15 minutes at 4°C, and was immediately used in the neutralization detection experiment of SARS-CoV-2 pseudovirus.

[0080] Table 1

[0081] Antigen / Control Antigen content Adjuvant Number of animals (only) ...

Embodiment 3

[0082] Embodiment 3 Pseudovirus neutralization test

[0083] 1. Preparation of pseudovirus:

[0084] 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.

[0085] 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 Helicobacter pylori ferritin. The present invention uses virus receptor binding domain (Receptor binding domain, RBD) and fusion peptide (Fusion peptide, FP) as dual antigens, and connects with Helicobacter pylori polymer protein (HP_Ferritin) to form fusion protein RBD-FP- HP_Ferritin realizes the multimerization of antigens; it can be expressed by eukaryotic cell expression system to form tetratetramer nanoantigens through 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 (tentative name SARS-CoV-2, also known as 2019-nCoV) S protein double-region subunit nano-vaccine based on Helicobacter pylori ferritin. Background technique [0002] The clinical manifestations of pneumonia caused by the novel 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. In severe cases, shock, Sepsis, respiratory failure and death. As the virus source and pathogenesis of 2019-nCoV pneumonia are still unclear, and there is a lack of specific antiviral drugs, it has brought great difficulties to clinical diagnosis, treatment and control of the epidemic. [0003] At present, humans still lack effective vaccines against SARS-CoV-2. Under this severe situation, developing safe and effective vaccines agains...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/215A61P31/14
CPCC07K14/005C07K14/205C12N15/85A61K39/12A61P31/14C12N2770/20022C07K2319/00C12N2800/107C12N2770/20034C12N15/625C07K14/195C07K2319/40A61K2039/6031A61K2039/55555A61K2039/575A61K2039/55566A61K39/215A61K2039/523A61K2039/6068A61K2039/627C12N15/86
Inventor 张辉马显才邹帆袁耀昌李镕张旭
Owner SUN YAT SEN UNIV
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