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COVID-19 subunit vaccine as well as preparation method and application thereof

A multivalent vaccine and vaccine composition technology, applied in the field of COVID-19 subunit vaccine and its preparation, can solve the problems of weakened effect and ineffectiveness that cannot be ruled out, and achieve the effects of stable and controllable quality, increased solubility, and short time consumption

Active Publication Date: 2021-08-31
广东克冠达医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether the vaccine prepared against the RBD of wild-type toxicity of SARS-CoV-2 has preventive or therapeutic effects on mutant strains remains to be further studied, but it cannot be ruled out that the effect is weakened or even ineffective. Therefore, it is urgent to design a vaccine that can target both wild-type and Combination vaccines for mutant SARS-CoV-2

Method used

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  • COVID-19 subunit vaccine as well as preparation method and application thereof
  • COVID-19 subunit vaccine as well as preparation method and application thereof
  • COVID-19 subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Preparation, identification and content determination of fusion protein HD-R1

[0099] According to NCBI (https: / / www.ncbi.nlm.nih.gov) the gene sequence of the RBD neutralizing epitope (artificial Synthetic, the amino acid sequence is as shown in SEQ ID No.1, which is the 319-593th position of the S protein amino acid sequence), and the gene sequence of the Fc protein in human immunoglobulin IgG (artificially synthesized, the nucleotide sequence is as in SEQ ID No. .32, the amino acid sequence is as shown in SEQ ID No.33), and the fusion protein HD-R1 (nucleotide sequence is as shown in SEQ ID No.2, and amino acid sequence is as shown in SEQ ID No.3) is prepared by genetic engineering means ).

[0100] (1) Construction and identification of PCDNA3.1-HD-R1 recombinant expression plasmid

[0101] The double-stranded DNA molecule HD-R1 was inserted between the BamHI and XhoI restriction sites of the plasmid vector pcDNA3.1, and the recombinant expression plas...

Embodiment 2

[0108] Embodiment 2: Preparation, identification and content determination of other fusion proteins

[0109] HD-R2, HD-05, HD-R3, HD-01, HD-02, HD-03, HD-04, HD-06, HD-07, HD-08, HD-09, HD-10 in Table 2 , HD-11, HD-12, HD-S1 and HD-S and other 16 kinds of fusion protein preparation, identification and content determination, except that the corresponding plasmid vector pcDNA3.1 contains the 16 kinds of fusion proteins corresponding to Table 2 Except for the difference in the nucleotide sequence relative to the amino acid sequence, the steps of its preparation, identification and content determination are similar to those in Example 1. Among them, 16 kinds of fusion proteins are obtained by linking different RBDs with corresponding tags through linkers.

[0110] Table 2: Sources and characteristic information of 17 fusion proteins

[0111]

[0112]

Embodiment 3

[0113] Example 3: Preparation of subunit vaccine and mouse immunization

[0114] (1) Dilute the fusion proteins HD-R1, HD-R2, HD-S1 and HD-S prepared in Example 1 and Example 2 with PBS buffered saline solution, and add aluminum adjuvant (Al) to fully emulsify the mixture The corresponding subunit vaccine can be obtained, and the ratio of fusion protein and aluminum adjuvant is shown in Table 3.

[0115] Table 3: Subunit vaccine ratio

[0116]

[0117] (2) For the fusion protein vaccine group and the control group above, the experiment adopted an initial immunization (1st boost) and a second boost (2nd boost) immunization scheme, and the mice were intramuscularly injected with the prepared vaccine on the 0th and 30th days, each The volume of each injection was 10 μg / 100 μL, and the control group was injected with the same volume of PBS solution; blood was collected on the 30th and 40th days, and 0.1 mL to 0.2 mL of blood was collected from each mouse, placed at 0 ° C for 6...

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Abstract

Fusion protein RBD-hFc / RBD-His obtained by combining a novel coronavirus (SARS-CoV-2) spike protein receptor binding structural domain (RBD) with a human immunoglobulin hFc domain or His tag through a genetic engineering means is convenient to extract and purify, stable and controllable in quality, short in time consumption and capable of being produced on a large scale. A vaccine composition prepared from the fusion protein and an adjuvant can increase the solubility and stability of a vaccine, enhance the immunogenicity of the vaccine and prolong the half-life period of the vaccine in vivo. The fusion protein and the vaccine composition thereof can inhibit replication and transmission of SARS-CoV-2 wild type and / or variant strains or prevent the SARS-CoV-2 wild type and / or variant strains from settling in a host, so that novel coronavirus pneumonia caused by the SARS-CoV-2 wild type and / or variant strains can be effectively prevented and / or treated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a COVID-19 subunit vaccine and its preparation method and application. Background technique [0002] SARS-CoV-2 is a positive-sense single-stranded RNA virus with a diameter of about 80-120 nm. It belongs to the genus of betacoronaviruses like SARS-CoV and MERS-CoV. Its genome is about 29.8Kb in length and encodes four structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N ) (Immunity, 2020.53(6): p.1315-1330). The S protein is located on the surface of the virus and consists of an N-terminal S1 domain and a C-terminal S2 domain. The receptor-binding domain (RBD) located in the S1 subunit can bind to angiotensin-converting enzyme 2 (ACE2) on the cell surface and mediate the process of virus entry into cells Play an important role. Studies have shown that the binding ability of SARS-CoV-2 RBD to ACE2 is about 10-20 times higher than that of SARS-CoV (Scienc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/385A61K39/215A61K39/39A61P31/14
CPCC07K14/005A61K39/12A61K39/39A61P31/14A61K39/385C12N2770/20022C12N2770/20034C07K2319/21C07K2319/30A61K2039/6056A61K2039/55505Y02A50/30
Inventor 李展如刘洪恩
Owner 广东克冠达医药科技有限公司
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