Helicobacter pylori ferritin-based novel coronavirus S protein double-region subunit nanovaccine

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

Active Publication Date: 2020-09-01
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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  • Helicobacter pylori ferritin-based novel coronavirus S protein double-region subunit nanovaccine
  • Helicobacter pylori ferritin-based novel coronavirus S protein double-region subunit nanovaccine
  • Helicobacter pylori ferritin-based novel coronavirus S protein double-region subunit nanovaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction of a novel coronavirus SARS-CoV-2 antigen (fusion protein RBD-FP-HP_Ferritin)

[0069] The schematic diagram of the fusion protein RBD-FP-HP_Ferritin self-assembled nanoparticles and the schematic diagram of the structure are as follows: figure 1 with 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] The RBD-FP-HP_Ferritin nucleotide sequence (shown in SEQ ID NO: 4) was cloned into an expression vector (pcDNA3.1-Intron-WPRE) with Intron and WPRE enhanced expression after adding a translation stop codon to the 3'end Between Xho I and Xba I restriction sites, construct an expression vector pcDNA3.1-Intron-WPRE-RBD-FP-Ferritin(HP)-IRES-GFP (such as image 3 Shown).

[0073] The recombinant plasmid was transformed into DH5α competent cells, cultured overnight at 37°C, and positive clones were identifie...

Embodiment 2

[0078] Example 2 Mouse Immunization Experiment

[0079] The RBD-FP-HP_Ferritin antigen obtained in Example 1 was diluted with normal saline to 100 μg / ml according to Table 1, and was emulsified in groups with the equal volume of adjuvant SAS. Then Balb / C mice aged 6-8 weeks were immunized in groups. Immunization strategies such as Figure 8 As shown, by intraperitoneal injection, each mouse received 3 immunizations on day 0, week 3 (day 21), and week 14 (day 108), each with an inoculation volume of 200 μl (10 μg) . On days 10, 31, and 108, blood was taken from the orbit of the mice. The mouse serum was obtained by centrifugation at 4°C and 2800 rpm for 15 minutes after standing for a period of time until the serum was separated, and it was immediately used in the SARS-CoV-2 pseudovirus neutralization detection experiment.

[0080] Table 1

[0081] Antigen / Control Antigen content Adjuvant Number of animals (only) RBD-FP-HP_Ferritin 10μg SAS4 PBS 0 SAS4

Embodiment 3

[0082] Example 3 Pseudovirus neutralization test

[0083] 1. Preparation of fake virus:

[0084] According to the NCBI published sequence, the Spike protein of SARS-CoV-2 was synthesized and inserted into the pcDNA3.1 expression vector. The SARS-CoV-2Spike protein expression vector was co-transfected with pHIV-luciferase and psPAX2 plasmids into 293T cells. After 5 hours of transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium to continue the culture. After 48 hours, the supernatant was collected and centrifuged to remove cell debris. Then dissolve with a small volume of serum-free DMEM to obtain HIV-luc / SARS-CoV-2-S pseudovirus.

[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 the virus infection, so that the results of ...

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Abstract

The invention discloses a helicobacter pylori ferritin-based novel coronavirus S protein double-region subunit nanovaccine. According to the invention, a receptor binding domain (RBD) of a virus and afusion peptide (FP) are jointly used as double antigens, and the double antigens are connected with a helicobacter pylori polymer protein (HP _ Ferritin) to form a fusion protein RBD-FP-HP _ Ferritin, so that antigen multimerization is realized; and then an eukaryotic cell expression system is utilized for expression, so as to form a 24-polymer nano antigen through the self-assembly action of HP_ Ferritin. According to the scheme, the defect that RBD monomers are insufficient in immunogenicity can be overcome, the obtained vaccine can remarkably improve the level of neutralizing antibodies of a host to viruses, and the generated antibodies have the capacity of strongly preventing the viruses from invading target cells. Moreover, the vaccine is simple in preparation method, easy to purifyand high in safety, and can be quickly applied to clinical tests.

Description

Technical field [0001] The invention belongs to the field of biomedicine technology. 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 iron protein. 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 viral pneumonia; the main clinical manifestations are fever, fatigue, dry cough, etc. In severe cases, shock, Death from sepsis, respiratory failure. As the source and pathogenesis of the novel coronavirus pneumonia are still unclear, and there is a lack of specific antiviral drugs, it is extremely difficult for clinical diagnosis and treatment and control of the epidemic. [0003] At present, humans still lack effective vaccines against SARS-CoV-2. In this severe situation, a safe and effective vaccine against SARS-CoV-2 should be ...

Claims

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Application Information

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Patent Type & Authority Applications(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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