Novel coronavirus vaccine and preparation method and application thereof

A coronavirus and vaccine technology, applied in the field of biomedicine, can solve the problem of no treatment for pneumonia, and achieve the effect of enhancing immunogenicity

Active Publication Date: 2021-03-16
SICHUAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the vaccine against the new coronavirus
[0006] Prevention and control of pneumonia caused by the new...

Method used

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  • Novel coronavirus vaccine and preparation method and application thereof
  • Novel coronavirus vaccine and preparation method and application thereof
  • Novel coronavirus vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089]Example 1 Design and synthesis of coding genes of fusion proteins

[0090]Section 1168 to 1569 Base base from the CDS coding sequence from Spike Protein is selected as a gene sequence (SEQ ID NO: 7) that contains a clip of a key binding site that binds to the ACE2 in the RBD, and is modified: add it to the 5 'end. Kozak sequences to increase the expression of proteins, while adding IgG secreted signal peptide sequences to enhance proteins, adding human IgG FC domain sequences (Fc (HOMO) to enhance protein immunity at 3 'ends The original, G4S is provided between the s protein receptor binding domain sequence and the human IgG Fc domain sequence, and the encoding gene of the antigen shown in SEQ ID NO: 9 is configured;

[0091]Or optimized objects in SEQ ID NO: 7, in accordance with codon preferences, select the highest frequency codon using the highest frequency, using Kesray OptimumGene codon optimization and genetic design technology (https: / / www.genscript.com.cn / / CODON_OPT_PR.HT...

Embodiment 2

[0093]Example 2 Construction of recombinant carrier

[0094]Add a NHE I enzyme dug point at the 5 'end of the encoding gene shown in SEQ ID NO: 9 or SEQ ID NO: 10, and the 3' end is added to add XHO I enzyme, using restriction endonase NHE I and XHO I Digestive Coding Gene, with Omega EZNAGelextraction Kit purification recycling;

[0095]Also use restriction endonuclease NHE I and XHO I digestion plasmid pcDNA3.1 (+), agarose gel electrophoresis, with omega e.z.n.aGel Extraction Kit recycled linear carrier, as a skeleton; use T4 DNA lid enzyme will be recycled, a linear carrier and encoding gene are connected to obtain recombinant carrier PCDNA3.1-mini-RBD-Fc, and a brief showing of recombinant carrierfigure 1 Indicated.

Embodiment 3

[0096]Example 3 Preparation and animal immunity of DNA vaccine

[0097]100 μg of recombinant carrier PCDNA3.1-mini-RBD-Fc was diluted with physiological saline to a final concentration of 1 μg / μL, and an immunization experiment was carried out as a DNA vaccine injecting mice:

[0098]After weighing the surrounding aged BALB / C mice, it was randomly divided into 2 groups (a set of experimental groups for injection plasmids, a group of control groups for injection of physiological saline), 4 per group, using abdominal injection to small Rats were anesthetized and the injected dose was 1% pentobarbital sodium 50 μl / 10g mice. After a anesthesia of 5-10 min, 100 μL of the back leg of the hind of each mouse was injected with 100 μL of recombinant carrier PCDNA3.1-mini- The RBD-Fc solution was immunized, and the first injection was injected again in the same conditions in the first injection.

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Abstract

The invention provides a novel coronavirus vaccine and a preparation method and application thereof. The encoding gene of the vaccine comprises a peptide fragment sequence, a Kozak sequence, an IgG signal peptide sequence and a human IgG Fc structural domain sequence, wherein the peptide fragment sequence contains the novel coronavirus and is combined with ACE2 in an S protein receptor binding domain RBD; the Kozak sequence and the IgG signal peptide sequence are positioned at the 5 'end of the peptide fragment sequence combined with ACE2 in the RBD; and the human IgG Fc structural domain sequence is positioned at the 3' end of the peptide fragment sequence combined with ACE2 in the RBD. The novel coronavirus vaccine has the advantages that the 1168 to 1569 sites (containing the peptide fragment combined with ACE2 in the RBD) of the encoding sequence from S protein are used as the gene sequence of antigen protein, and are modified, so that the expression ability, secretion ability and immunogenicity of the antigen protein are improved; the prepared DNA vaccine, protein vaccine and mRNA vaccine are directly injected into a human body or are injected into the human body afterbeing packaged by an adjuvant to express the corresponding antigen, and the body is induced to generate immune response; and the broad application prospect is realized in the fields of COVID-19 prevention and treatment.

Description

Technical field[0001]The present invention belongs to the field of biopharmaceutical technology, involving new coronavirus vaccines and preparation methods and applications, in particular, involving new coronavirus recombinant protein vaccines, DNA vaccines and mRNA vaccines and preparation methods and applications.Background technique[0002]The recombinant protein vaccine is to carry out in vitro recombination of the most effective antigenic epitope gene of pathogens, and constructs into bacteria, yeast, mammalian cells or insect cells, express a large number of antigenic proteins under certain induction conditions. The obtained vaccine was prepared after purification.[0003]DNA vaccine is also known as nucleic acid vaccine or genetic vaccine, and is a vaccine of the corresponding antigen from which the antigen gene is reorganized into the eukaryotic expression vector, and the exogenous gene is expressed in vitro, and the resulting antigen activates the body. Immune system, induce sp...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61P31/14C12N2770/20022C07K2317/52C07K2317/24C07K2319/00C07K2319/02C12N2770/20034A61K2039/53Y02A50/30
Inventor 宋旭刘丹赵永云李为民张天宇曾凡亚缪辉李佳李晓杰李灵
Owner SICHUAN UNIV
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