Novel coronavirus multivalent antigen as well as preparation method and application thereof

A coronavirus, a new type of technology, applied in the field of biomedicine, can solve problems such as reducing the protective effect of vaccines

Pending Publication Date: 2022-04-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There have been some mutations in the current viral strains, especially some existing vaccines have proved that the mutant strain 5

Method used

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  • Novel coronavirus multivalent antigen as well as preparation method and application thereof
  • Novel coronavirus multivalent antigen as well as preparation method and application thereof
  • Novel coronavirus multivalent antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design of novel coronavirus broad-spectrum protective vaccine RBD-tr2-WTV2

[0052] Analysis of new coronavirus mutant strains: In January and February 2020, the D614G mutation of the new coronavirus S protein appeared. In June 2020, the D614G mutant virus strain became the main epidemic strain in the world. Cell and animal experiments showed that the D614G mutation enhanced Compared with the original strain, the infection of D614G new coronavirus will not enhance the pathogenicity. In December 2020, a new mutant strain 501Y.V1 (also called VOC-202012 / 01, belonging to B.1.1.7lineage) appeared, which has 23 mutations compared with the original strain, which may have a greater impact on the S protein The three mutations are N501Y, 69-70del (deletion) and P681H, where the N501Y mutation enhances the binding ability of the S protein to the receptor ACE2 protein, and 501Y.V1 has a stronger transmission ability, but does not enhance the pathogenicity. Also in Dece...

Embodiment 2

[0055] Example 2: Detection of protein expression by Western blot

[0056] The amino acid sequence of the designed RBD-tr2-WTV2 antigen is SEQ ID NO: 4 (signal peptide and histidine sequence are not given in the sequence), and the nucleotide sequence for expressing the antigen is SEQ ID NO: 5, nucleotide The sequence elements from N-terminal to C-terminal are (1) Kozak sequence gccacc; (2) sequence encoding MERS-S protein signal peptide: MIHSVFLLMFLLTPTES (SEQID NO: 6); (3) New coronavirus (GenBank on NCBI: QHR63250 ) S protein RBD (R319-K537); (4) New coronavirus (GenBank on NCBI: QHR63250) S protein RBD (R319-K537), which contains three point mutations are K417N, E484K, N501Y; (5) 6 histidine; (6) stop codon. The gene sequence was synthesized in Suzhou Jinweizhi Co., Ltd., and cloned into pCAGGS plasmid through EcoRI and XhoI restriction sites to obtain pCAGGS-RBD-tr2-WTV2 plasmid.

[0057] The pCAGGS plasmid expressing RBD-tr2-WTV2 was transfected into HEK293T cells, and ...

Embodiment 3

[0058] Example 3: Expression and purification of RBD-tr2-WTV2

[0059] Use HEK293F cells suitable for mass expression of proteins to express RBD-tr2-WTV2 antigen: transfect HEK293F cells with the plasmid pCAGGS-RBD-tr2-WTV2, collect the supernatant after 5 days, centrifuge to remove the precipitate and filter through a 0.22 μm filter membrane, further Remove impurities. The cell supernatant was adsorbed by a nickel affinity column (Histrap, GE Healthcare) at 4°C, and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specific binding proteins. Then use buffer B (20mM Tris, 150mM NaCl, pH 8.0, 1000mM imidazole) to gradiently elute the target protein from Histrap, and set the ratio of buffer B to 2%, 5%, 10%, 20% in turn , 30%, 50%, 100%, see the results figure 2 . Concentrate the eluted protein solution corresponding to 10% imidazole with a 10kD concentrator tube, and change the liquid more than 30 times until the final volume of buffer A is less than 1ml....

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Abstract

The invention relates to a novel coronavirus multivalent antigen as well as a preparation method and application thereof. The invention discloses a novel coronavirus antigen. The amino acid sequence comprises an amino acid sequence arranged according to a (A-B)-(A-B ') style or an amino acid sequence arranged according to a (A-B)-C-(A-B') style, A-B represents a partial amino acid sequence or a full-length amino acid sequence of a receptor binding region of the surface spike protein of the novel coronavirus, C represents a connecting amino acid sequence, and C represents a connecting amino acid sequence; a-B'represents an amino acid sequence obtained by substituting an amino acid sequence of A-B with one or more amino acids in K417N, E484K or N501Y. Compared with two original strain RBD protein tandem repeat dimers, the novel coronavirus multivalent antigen disclosed by the invention can activate a broad-spectrum protection antibody, and has a very good prevention effect on the original strains and current epidemic strains.

Description

[0001] cross reference [0002] This application claims the priority rights of the invention patent application submitted on March 1, 2021 with the application number 202110225584.2 and the title of the invention is "a novel coronavirus multivalent antigen, its preparation method and application", the entire content of which is by reference Incorporated into this article. technical field [0003] The invention relates to the field of biomedicine, in particular to a novel coronavirus multivalent antigen, its preparation method and application. Background technique [0004] Novel coronavirus pneumonia (new coronary pneumonia, COVID-19) is caused by infection with a new type of coronavirus (new coronavirus, SARS-CoV-2). The new coronavirus belongs to the β-coronavirus genus of the Coronaviridae family, has an envelope, and is a positive-strand RNA virus. The spike (S) protein on the surface of the virus is responsible for recognition, binding and membrane fusion with the rece...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61P31/14C12N2770/20022C07K2319/00C12N2770/20034A61K2039/53
Inventor 高福戴连攀徐坤高萍安亚玲
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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