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Novel coronavirus S protein receptor binding domain fusion protein containing oligomerization structural domain and application of novel coronavirus S protein receptor binding domain fusion protein

A technology of oligomerization domain and receptor binding domain, which is applied in the direction of viruses/phages, medical preparations containing active ingredients, viruses, etc., and can solve unpredictable problems

Active Publication Date: 2022-04-15
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the study of the new coronavirus RBD-Fc fusion protein vaccine, the optimal RBD length for immune activity is unpredictable
In addition, two copies of the RBD tandem peptide with the highest immunogenicity are used, and Fc or other oligomerization domains are fused at the C-terminus to conduct research on protein vaccines such as RBD tetramers and hexamers, and there have been no reports yet.

Method used

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  • Novel coronavirus S protein receptor binding domain fusion protein containing oligomerization structural domain and application of novel coronavirus S protein receptor binding domain fusion protein
  • Novel coronavirus S protein receptor binding domain fusion protein containing oligomerization structural domain and application of novel coronavirus S protein receptor binding domain fusion protein
  • Novel coronavirus S protein receptor binding domain fusion protein containing oligomerization structural domain and application of novel coronavirus S protein receptor binding domain fusion protein

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Effect test

Embodiment 1

[0070] Embodiment 1: Gene synthesis and expression vector construction of RBD fusion protein

[0071] Various fusion proteins involved in the present invention are constructed in the following manner respectively:

[0072] 1) RBD-Fd: the RBD polypeptide used is the 331st to 550th position of the amino acid sequence shown in SEQ ID No.1, and the trimerization sequence shown in SEQ ID No.2 is fused at its C-terminus to obtain The amino acid sequence of the monomeric fusion protein is shown in SEQ ID No.8, and its corresponding nucleotide sequence is optimized using insect cell codons, and the GP67 coding sequence is fused upstream, and the his tag coding sequence is fused downstream to obtain the nucleotide sequence As shown in SEQ ID No.21, the pFastBac1-RBD-Fd expression plasmid was obtained by whole gene synthesis and inserted into the pFastBac1 vector through the Ba mHI / HindIII restriction site;

[0073] 2) RBDN1-Fd: the RBD polypeptide used is the 332nd to 550th position o...

Embodiment 2

[0087] Embodiment 2: the construction of the recombinant Bacmid of the gene of RBD fusion protein and recombinant baculovirus

[0088] The recombinant expression vectors pFastBac1-RBD-Fd, pFastBac1-RBDT-N1, pFastBac1-RBDN2-Fd, pFastBac1-RBDN3-Fd, pFastBac1-RBDS-Fd, pFastBac1-RBDN1-Fd-eye, pFastBac1 containing RBD fusion protein gene were used respectively -RBDN1.3-Fd-eye, pFastBac1-RS-2RBDN1.3-ColT, pFastBac1-2RBD-Fd, pFastBac1-2RBD-GCN4pII, pFastBac1-2RBD-GCN4pLI, pFastBac1-2RBD-GCN4pLL, pFastBac1-2RBD-GC N4pLLC , pFastBac1-RBD-GCN4pLLC, pFastBac1-S2P, transformed Escherichia coli DH10Bac competent, screened to obtain recombinant Bacmid, then transfected insect cells Sf9 with recombinant Bacmid, and amplified recombinant baculovirus in Sf9. The screening of recombinant Bacmid and the amplification method of recombinant baculovirus are well known, for example, patent CN101148661B.

Embodiment 3

[0089] Embodiment 3: the expression of the gene of RBD fusion protein in Sf9 cell

[0090] Sf9 cells were inoculated with recombinant baculoviruses with 14 fusion protein genes to express the fusion proteins. After culturing at 27°C for about 88 hours, the fermentation broth was harvested, centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected for expression identification and purification. The method of infection expression is public, for example, patent CN 101148661 B.

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Abstract

The invention discloses a novel coronavirus S protein receptor binding domain fusion protein containing an oligomerization structural domain and application of the novel coronavirus S protein receptor binding domain fusion protein, and particularly discloses a novel coronavirus S protein RBD fusion protein which is composed of at least one copy of RBD polypeptide derived from a novel coronavirus S protein, the oligomerization structural domain and / or an endogenous adjuvant, the endogenous adjuvant is an RS09 adjuvant, and the oligomerization structural domain is selected from a bacteriophage trimerization structural domain (Foldon domain), a GCN4 leucine zipper sequence or a human collagen trimerization structural domain. The novel coronavirus RBD fusion protein can be used for preparing vaccines for preventing novel coronavirus infection and infection-induced diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel coronavirus spike protein (S protein) receptor binding domain (RBD) fusion protein, an oligomer formed therefrom, and an RBD fusion protein or RBD fusion protein oligomerization Use of the body in the preparation of vaccines for preventing novel coronavirus infection and infection-induced diseases. Background technique [0002] The new coronavirus (SARS-CoV-2) is an enveloped positive-strand RNA virus. The virus has four structural proteins (S, M, N, E), among which the S protein is the key protein for infection and invasion. The form is distributed on the surface of the envelope, and its N-terminus contains the cell receptor binding domain (receptorbinding domain, RBD). After RBD binds to ACE2 of the host cell, the S protein undergoes allosteric changes, which initiates virus infection and invasion. The S protein is the main target antigen for coronavirus vaccine research....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61P31/14C12N2770/20022C12N2770/20034C12N2800/22C07K2319/30A61K2039/53
Inventor 许雪梅郑宁晨张婷王志荣
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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