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A novel coronavirus S-protein polymer nanovaccine based on Helicobacter pylori ferritin

A technology of Helicobacter pylori and coronavirus, applied to viruses/bacteriophages, polypeptides containing positioning/targeting motifs, viruses, etc., can solve the problems of lack of vaccines, social burden crisis, lack of specific antiviral drugs, etc., and achieve easy Simple purification and preparation method, and the effect of improving immunogenicity

Active Publication Date: 2021-10-12
GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] 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 an effective vaccine against SARS-CoV-2. Under this severe situation, developing a safe and effective vaccine against SARS-CoV-2 as soon as possible to protect susceptible populations is of great importance to the health of the people and the country. safety matters

Method used

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  • A novel coronavirus S-protein polymer nanovaccine based on Helicobacter pylori ferritin
  • A novel coronavirus S-protein polymer nanovaccine based on Helicobacter pylori ferritin
  • A novel coronavirus S-protein polymer nanovaccine based on Helicobacter pylori ferritin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Construction of novel coronavirus SARS-CoV-2 antigen (fusion protein RBD-HPF and RBD-HR-HPF)

[0068] Electron microscope counterstaining images of RBD-HPF subunit multimer protein and RBD-HR-HPF double subunit multimer protein self-assembled into nanoparticles figure 1 .

[0069] Specifically, the construction and preparation method of RBD-HPF subunit multimeric protein is as follows:

[0070] 1. Preparation of expressed His-tag-ST-RBD protein

[0071] Add a translation stop codon to the 3' end of the nucleotide sequence corresponding to SP-His-tag-ST-RBD and clone it into an expression vector (pcDNA3.1-Intron-WPRE) that adds Intron and WPRE to enhance expression to construct an expression vector .

[0072] DH5αcompetent cells were transformed with the recombinant plasmid, cultured overnight at 37°C, and positive clones were identified by screening and PCR. The endotoxin-free plasmid was extracted and used for the expression of nanoantigen protein after e...

Embodiment 2

[0082] Embodiment 2 mouse immunization experiment

[0083] The RBD-HPF protein and RBD-HR-HPF protein obtained in Example 1 were diluted to 100 μg / ml with physiological saline according to Table 1, and emulsified in groups with an equal volume of adjuvant SAS. Then BALB / c mice aged 6-8 weeks were immunized in groups. By intraperitoneal injection, each mouse was immunized with the vaccine three times on day 0, week 3 (day 21), and week 14 (day 108), with an inoculation volume of 200 μl (10 μg) each time. On the 10th, 31st, and 108th days, blood was collected from the mice's orbits. The mouse serum was obtained by centrifuging at 2800rpm at 4°C for 15 minutes after standing for a period of time to allow the serum to separate out, and was immediately used in the SARS-CoV-2 virus neutralization detection experiment.

[0084] Table 1

[0085] Antigen / Control Antigen content Adjuvant Number of animals (only) RBD-HPF protein 10μg SAS 4 RBD-HR-HPF pro...

Embodiment 3

[0086] Embodiment 3 Pseudovirus neutralization test

[0087] 1. Experimental method

[0088] Day 0:

[0089] Vero E6 cells were divided into 2 × 10 per well 4 cells at a density of 96 wells.

[0090] Day 1: (Wait for cell growth density to reach 100%.)

[0091] (1) Dilute serum: Serum is inactivated at 56°C for 30 minutes (this inactivation step is generally performed immediately after obtaining the serum), and the serum is diluted 5 times or 10 times according to the experimental requirements. Here, two dilution gradients are selected, namely 100 times and 1000 times. times. For example, to obtain a serum system with a final concentration of 10 times, 100 times and 100 times dilution, dilute as shown in Table 2 below:

[0092] Table 2

[0093]

[0094] (2) Dilute virus: 6000-8000 FFU per milliliter of DMEM culture medium, here, according to our laboratory experience, add 0.25ul of virus stock solution per well. Prepare a sufficient amount of diluted virus solution acc...

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Abstract

The invention discloses a novel coronavirus S protein subunit nano vaccine based on Helicobacter pylori ferritin. In the present invention, the protein expressed by the receptor binding domain (RBD) of the novel coronavirus and the N-terminus of the Helicobacter pylori polymer protein (HP_Ferritin, HPF) are intermolecularly differentiated through the SpyTag / SpyCatcher (ST‑SC) system. Peptide bonds connect the subunit multimer protein to realize antigen multimerization; or combine the RBD expressed protein with the heptad repeat region (Heptad Repeat, HR) expressed protein, and covalently combine with the HPF protein to form a double Subunit multimeric protein, realizing antigen multimerization. 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. At the same time, based on sequence analysis, the scheme of the present invention is expected to develop effective vaccines against a variety of coronaviruses. 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 (SARS-CoV-2, also known as 2019-nCoV) S protein dual-region subunit nano-vaccine based on Helicobacter pylori ferritin. Background technique [0002] 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 an effective vaccine against SARS-CoV-2. Under this severe situation, developing a safe and effective vaccine against SARS-CoV-2 as soon as possible to protect susceptible populations is of great importance to the health of the people and the country. Security matters. Therefore, it is imminent to develop vaccines with high immunogenicity and neutralization efficiency against coro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/215A61P31/14
CPCC07K14/005C07K14/195A61K39/12A61P31/14C12N2770/20022C12N2770/20034C07K2319/21C07K2319/02
Inventor 张辉邹帆
Owner GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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