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Novel coronavirus S protein and subunit vaccine thereof

A subunit vaccine, coronavirus technology, applied in the field of new coronavirus S protein and its subunit vaccine, can solve the problems of unknown integration risk, interference with S protein immunogenicity, inability to effectively deal with mutant strains, etc. cut effect

Active Publication Date: 2021-07-30
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some vaccines against SARS-CoV-2 have been put on the market urgently for emergency use, there are still many problems with the current vaccine varieties: 1. Compared with nucleic acid vaccines and subunit vaccines, inactivated vaccines mainly used in my country The originality is not strong enough, the inactivation reagent will destroy the natural epitope of the antigen, and other proteins in the virus particle will also interfere with the immunogenicity of the S protein
2. Nucleic acid vaccine products are the first application in the new crown virus. Nucleic acid vaccines have not been successfully marketed before, and their long-term safety and integration risks are unknown
3. The adenovirus vector of the adenovirus vaccine will be interfered by pre-existing immunity
4. Various existing vaccines are designed for early epidemic strains, and cannot effectively deal with the mutant strains that have been produced, and do not have a conservative protective effect

Method used

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  • Novel coronavirus S protein and subunit vaccine thereof
  • Novel coronavirus S protein and subunit vaccine thereof
  • Novel coronavirus S protein and subunit vaccine thereof

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Experimental program
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Effect test

preparation example Construction

[0069] According to another typical implementation of the embodiments of the present invention, a method for preparing the S protein of a novel coronavirus is provided, the method comprising:

[0070] obtaining the recombinant expression vector;

[0071] Transfecting the recombinant expression vector into cells, and obtaining a cell line stably expressing the recombinant S protein through glutamine resistance screening and monoclonal screening of the cell population;

[0072] The cell line was secreted, expressed and purified to obtain a purified recombinant novel coronavirus S protein.

[0073] According to another typical implementation of the embodiments of the present invention, a novel coronavirus subunit vaccine is provided, and the novel coronavirus subunit vaccine comprises the recombinant S protein and a pharmaceutically acceptable adjuvant.

[0074] The adjuvant includes at least one of aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM, IL-12, aluminum hydroxi...

Embodiment 1

[0078] Example 1 Recombinant S protein vector construction and expression optimization

[0079] 1. Construction of S protein gene expressed in mammalian cell supernatant

[0080] The schematic diagram of the construction of the S protein expression gene of the present invention is shown in figure 1 . figure 1 Sequence design diagram of mammalian cell secreted S protein: the sequence retains the original signal peptide, or is mutated to tPA signal peptide / CD5 signal peptide / IgG signal peptide; Furin cleavage site is mutated from RRAR to GSAS, GS combination, (GGGS) n or (GGGGS) n or (G) n ; The C-terminal transmembrane region and the intracellular region were replaced by the minor fibrin sequence of T4 phage.

[0081] First, in order to efficiently express in eukaryotic cells, we used the JAVA Codon Adaptation software to optimize the codons of the S expression gene with mammalian preference. The nucleotide sequence of the original signal peptide was optimized as SEQ ID N...

Embodiment 2

[0093]Embodiment 2. Recombinant S protein trimer vaccine immunization and effect identification

[0094] 1. Immunization procedure of mice

[0095] The mice used in this experiment were K18-hACE male mice, 6-8 weeks old, 19-28g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All animal experiments were performed in SPF laboratory. The experimental group was inoculated with vaccine + adjuvant, a total of 4 mice; the control group was inoculated with PBS, a total of 4 mice. The second injection was inoculated 14 days after the first injection, and 35 days after the first injection (that is, 21 days after the second injection), blood was collected from the orbit of all mice. Mouse serum was taken for testing.

[0096] 2. ELISA detection process

[0097] (1) Dilute RBD (Shenzhou Science and Technology Co., Ltd., Yiqiao Shenzhou Technology Co., Ltd.) with 0.1M carbonate buffer (pH=9.6) to make the final concentration 1ng / μL, and then add 100μL to each well of the 9...

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Abstract

The invention provides a novel coronavirus S protein and a subunit vaccine thereof. The novel coronavirus S protein is characterized in that a furin cleavage site 682-RRAR-685 between two subunits S1 and S2 of the novel coronavirus S protein is replaced by a flexible protein linker. The linker is a GSAS and GS combination composed of glycine G and serine S, (GGGS) n or (GGGGS)n or (G)n, and n is an integer larger than or equal to 1. The novel coronavirus subunit vaccine comprises the recombinant S protein and a pharmaceutically acceptable adjuvant. The obtained S protein of the trimer conformation with biological activity is used for preparing a trimer subunit vaccine, after a mouse is immunized, the mouse can be induced to generate a neutralizing antibody which aims at SARS-CoV-2 and has an immune protection effect, and after the immunized mouse is subjected to lethal challenge infection of novel coronavirus, the protection efficiency of 100% can be provided.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a novel coronavirus S protein and a subunit vaccine thereof. Background technique [0002] On January 30, 2020, WHO declared the global outbreak caused by the novel coronavirus (SARS-CoV-2) as a public health emergency of international concern. Due to the extremely high transmission potential of the virus, as of March 20, 2021, more than 123 million cases of SARS-CoV-2 infection have been reported worldwide, resulting in 2,718,896 patient deaths. In response to the global pandemic of novel coronavirus pneumonia, governments, companies and academia are formulating various treatment and prevention strategies, with the most important being the development and application of vaccines and antiviral drugs. Vaccines are the most effective means of combating infection with all viral diseases and the most effective measure to protect the uninfected population. [0003] The types of SARS-CoV-2 ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61P31/14
CPCC07K14/005C12N15/85C12N5/0682A61K39/12A61P31/14C12N2770/20022C12N2770/20034C07K2319/02C07K2319/03C07K2319/00C12N2800/107C12N2800/22C12N2510/02Y02A50/30
Inventor 徐可蓝柯
Owner WUHAN UNIV
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