SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof

A sars-cov-2rbd, dimer technology, applied in the field of binding domain dimer and its application, can solve the problem of no clear evidence to determine the origin and intermediate host, achieve good neutralization activity and broad application prospects Effect

Active Publication Date: 2021-11-02
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The SARS-CoV-2 virus is also believed to have originated from bats, but there is no clear evidence to identify its origin and intermediate host

Method used

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  • SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof
  • SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof
  • SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, prepare SARS-CoV-2 RBD

[0057] 1. Preparation of dimers

[0058] 1. Use the pcDNA3.1(+) vector as the starting vector to construct the recombinant plasmid.

[0059] The recombinant plasmid is shown in sequence 4 of the sequence listing. In sequence 4 of the sequence listing, the 910th-1728th nucleotide constitutes the fusion gene.

[0060] The fusion gene expresses the fusion protein shown in sequence 3 of the sequence list. In sequence 3 of the sequence listing, amino acid residues 1-33 constitute the signal peptide, amino acid residues 34-256 constitute the SARS-CoV-2 RBD, amino acid residues 257-264 constitute the Strep-tag II tag, Amino acid residues 265-272 constitute the FLAG tag.

[0061] The signal peptide in the fusion protein is recognized by the receptor on the endoplasmic reticulum membrane and combined with it, then the fusion protein reaches the lumen of the endoplasmic reticulum through the channel formed by the protein in the endoplasmic...

Embodiment 2

[0108] Example 2, the binding activity of SARS-CoV-2 RBD dimer and ACE2

[0109] Angiotensin converting enzyme 2 (angiotensin I converting enzyme, ACE2) is the receptor of SARS-CoV-2. ACE2 is shown in sequence 8 of the sequence listing.

[0110] 1. Preparation of ACE2 solution

[0111] For the preparation method of ACE2, refer to Step 2 of Example 1.

[0112] The difference lies in the replacement of the DNA molecule encoding the SARS-CoV-2 RBD with the DNA molecule encoding ACE2.

[0113] ACE2 solution is obtained.

[0114] 2. SPR test

[0115] ACE2 was coated on the CM5 chip, and then SARS-CoV-2 RBD dimer solution was added to detect the binding activity of SARS-CoV-2 RBD dimer to ACE2. see results Figure 4 .

[0116] 3. Gel filtration experiment

[0117] Mix 0.5ml of SARS-CoV-2 RBD dimer solution (2mg / ml protein concentration) and 0.5ml ACE2 solution (2mg / ml protein concentration) and incubate on ice for 2 hours to obtain a mixed solution (COMPLEX). The SARS-CoV-2RB...

Embodiment 3

[0119] Neutralizing activity of serum after embodiment 3, SARS-CoV-2 RBD immunization

[0120] 1. Group immunization

[0121] balB / C mice (Weitong Lihua Company) were divided into 6 groups, 5 in each group, and were immunized as follows:

[0122] The first group: the first immunization was carried out on the first day, the second immunization was carried out on the 15th day, the third immunization was carried out on the 29th day, and the fourth immunization was carried out on the 43rd day; the immunization method was intramuscular injection; The immune volume of mice is 40 μl, and the immune substance is “white emulsion formed by mixing 20 μl 1mg / ml RBD dimer solution and 20 μl Freund’s complete adjuvant”; The substance is "white emulsion formed by mixing 20 μl 1mg / ml RBD dimer solution with 20 μl Freund's incomplete adjuvant";

[0123] The second group: the first immunization was carried out on the 1st day, the second immunization was carried out on the 15th day, the third ...

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Abstract

The invention discloses an SARS-CoV-2 Spike protein receptor binding domain dimer and an application thereof, and particularly relates to an SARS-CoV-2 RBD dimer (SARS-CoV-2 RBD dimer) or an SARS-CoV-2 RBD monomer (SARS-CoV-2 RBD monomer) and an application of the SARS-CoV-2 RBD dimer or the SARS-CoV-2 RBD monomer or the SARS-CoV-2 RBD monomer or the SARS-CoV-2 RBD monomer or the SARS-CoV-2 RBD monomer in neutralizing the SARS-CoV-2, and the SARS-CoV-2 RBD dimer or the SARS-CoV-2 RBD monomer in neutralizing the SARS- the invention provides a dimer of SARS-CoV-2 RBD, and a preparation method of the dimer, a protein represented by Sequence 1 in the sequence table, a protein represented by Sequence 3 in the sequence table, and the protein shown as the 34th-272th amino acid residues in the sequence 3 of the sequence table. The invention has important value and wide application prospect in research and development of drugs, vaccines and the like for treating and preventing SARS-CoV-2.

Description

technical field [0001] The present invention relates to SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof, be specifically related to SARS-CoV-2 RBD dimer (SARS-CoV-2 RBD dimer) or SARS-CoV-2RBD monomer (SARS-CoV-2 -CoV-2 RBD monomers) and their application in neutralizing SARS-CoV-2, wherein SARS-CoV-2 RBD dimers have more significant activity. Background technique [0002] The disease caused by the novel coronavirus (SARS-CoV-2) is named as Coronavirus Disease 2019 (COVID-19). [0003] The novel coronavirus manifests as asymptomatic carriage, acute respiratory distress syndrome (ARD), and pneumonia. Because the disease can be transmitted from person to person, it has attracted great attention around the world. [0004] Gene sequence comparison shows that SARS-CoV-2 belongs to βcoronavirus, and has high similarity with other two highly pathogenic coronaviruses in terms of gene composition, structure and function. These two highly pathogenic co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C07K19/00C12N15/50C12N15/62C12N15/85C12N15/866A61K39/215A61P31/14A61P11/00
CPCC07K14/005C12N15/85C12N15/86A61K39/12A61P31/14A61P11/00C12N2770/20022C12N2770/20034C12N2770/20051C07K2319/02C07K2319/43C07K2319/22C07K2319/21C12N2800/107C12N2800/105C12N2710/14043Y02A50/30
Inventor 张林琦单思思史宣玲
Owner TSINGHUA UNIV
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