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DNA vaccine against sars-cov-2 virus and its use

A GM-CSF, sequence technology, applied in the field of DNA vaccines, can solve safety problems and other issues

Active Publication Date: 2020-10-27
艾立克(湖北)生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, from the perspective of safety, it is also necessary to overcome the potential safety problems caused by the long-term persistence of DNA vaccines in vivo, as well as the unexpected antibody response (ie, undesired immunogenicity) produced by the body against the synergistic components. security issues

Method used

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  • DNA vaccine against sars-cov-2 virus and its use
  • DNA vaccine against sars-cov-2 virus and its use
  • DNA vaccine against sars-cov-2 virus and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1: The SARS-COV-2 S1 protein was ligated with the coding sequence of the antigen auxiliary sequence to construct a DNA vector expressing the fusion antigen protein of SARS-COV-2 S1-Hsp70

[0096] The N-terminal sequence of SARS-COV-2 S1-Hsp70 fusion antigen protein sequence SEQ ID NO: 10 is derived from the 1-667 amino acid sequence of the SARS-COV-2 S1 protein, wherein the 1-13 amino acid sequence is the S protein signal peptide sequence. Then the S1 protein is connected to the 2-625th amino acid sequence of the antigen auxiliary sequence Hsp70 through a linker composed of three glycines (see figure 1 ). The total length of the precursor of the fusion antigen protein is 1294 amino acid residues, wherein the signal peptide sequence consisting of amino acid residues 1-13 is excised in the mature fusion antigen protein.

[0097] A DNA sequence encoding a full-length 1294 amino acid residues was designed using human cell preferential expression codons, and then a...

Embodiment 2

[0098] Example 2: Ligate the coding sequence of SARS-COV-2 S1 protein with the truncated antigen auxiliary sequence Hsp70 (Δ200) to construct a DNA expression vector expressing the fusion antigen protein of SARS-COV-2 S1-Hsp70 (Δ200)

[0099] The fusion of SARS-COV-2 S1 and Hsp70 (Δ200) (compared with the antigen auxiliary sequence Hsp70 contained in the aforementioned fusion antigen protein, lacking 200 amino acid residues at the N-terminus) was prepared and tested using the same method as in Example 1 Antigen protein SARS-COV-2 S1-Hsp70 (Δ200) (see SEQ ID NO: 12), the results obtained are also consistent with the design (not shown in the figure).

Embodiment 3

[0100] Example 3: Construction of eukaryotic expression vectors for human IL-2, GM-CSF, IL-17 and IFN-γ

[0101] According to the conventional molecular cloning method in Example 1, the cDNA sequences encoding human IL-2, GM-CSF, IL-17 and IFN-γ (all these cDNA sequences can be obtained from GenBank (https: / / www.ncbi.nlm .nih.gov / nuccore), for example, human IL-2 cDNA GenBank number is NM_000586, human GM-CSF cDNA GenBank number is M11220, human IFN-γ cDNA GenBank number is NM_000619, human IL-17 cDNA GenBank number is U32659) clone to p C In the DNA-3.1 eukaryotic expression vector, p C DNA-3.1-IL-2, p C DNA-3.1-GM-CSF, p C DNA-3.1-IL-17 and p C DNA-3.1-IFN-γ expression vector plasmid. 10 mg of various cytokine expression vectors were prepared separately with the plasmid mass preparation and purification kit produced by QIAGEN. The above cytokine expression plasmids were transfected into 293 cell lines respectively, and the respective cell culture supernatants were coll...

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Abstract

The invention relates to the field of vaccines, in particular to a DNA vaccine specific for SARS-COV-2 virus. The invention discloses a fusion protein comprising the amino acid sequence of 14-1294 in SEQ ID NO:10 or a mature polypeptide encoded by SEQ ID NO:9; a fusion protein comprising 14-1294 in SEQ ID NO:12 The amino acid sequence at position 1094 or the mature polypeptide encoded by SEQ ID NO: 11; and a polynucleotide encoding the fusion protein or a nucleic acid molecule comprising the polynucleotide sequence. The invention also discloses related compositions, methods, uses and kits.

Description

technical field [0001] The invention relates to the field of vaccines, in particular to a DNA vaccine specific for SARS-COV-2 virus. Background technique [0002] SARS-COV in 2003 and MERS-COV in 2012 infected many people around the world; the outbreak of SARS-COV-2 infection at the end of 2019 also had a great impact, and there is an urgent need to develop an effective vaccine against SARS-COV-2 , such as DNA vaccines. Although various ways of enhancing the potency of antigens have been sought, efforts in this area have been limited and unpredictable. In some cases, the way of enhancing the potency of the antigen often shows the disadvantage of being difficult to apply in other cases. For example, Hsp70, which has been tried to enhance antigen potency, induces immune tolerance in autoimmune disease models (Prakken B J, Wendling U, van der Zee R, et al. Induction of IL-10 and inhibition of experimental arthritis are specific features of microbial heatshock proteins that a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/215A61K39/385A61P31/14
CPCC07K14/005A61K39/12A61K39/385A61P31/14C12N2770/20022C12N2770/20034C07K2319/31A61K2039/53A61K2039/6043
Inventor 吴炯黄勇
Owner 艾立克(湖北)生物科技有限责任公司