CN116096736A

CN116096736A - Novel coronavirus RBD fusion protein

Info

Publication number: CN116096736A
Application number: CN202080104145.XA
Authority: CN
Inventors: 方丽娟; 张敬; 石剑; 王鑫; 罗芳; 周迟; 雷传飞; 周鹏飞; 肖庚富; 潘晓彦; 龚睿; 张哲�
Original assignee: Wuhan You Microbial Technology Co ltd
Current assignee: Wuhan You Microbial Technology Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2023-05-09
Also published as: WO2022032660A1

Abstract

本发明涉及新型冠状病毒RBD融合蛋白，RBD蛋白二聚体及其制备和用于预防新型冠状病毒SARS‑CoV‑2感染的应用。The present invention relates to novel coronavirus RBD fusion protein, RBD protein dimer and its preparation and application for preventing novel coronavirus SARS-CoV-2 infection.

Description

PCT国内申请，说明书已公开。PCT domestic application, specification has been published.

Claims

The RBD fusion protein of the novel coronavirus SARS-CoV-2 comprises, in sequence, the RBD sequence of the novel coronavirus SARS-CoV-2 (preferably said RBD sequence comprises a cysteine positioned at position 220 according to the position numbering of the sequence set forth in SEQ ID NO:1 and having activity to bind to the human ACE2 receptor; more preferably the RBD sequence comprises an amino acid sequence set forth in any of SEQ ID NO: 1-8), a proteolytic cleavage site, and a tag facilitating dimer formation.
The novel coronavirus SARS-CoV-2 RBD fusion protein according to claim 1, wherein said tag facilitating dimer formation is selected from the group consisting of a leucine zipper (preferably a sequence not comprising hhhhhhh in SEQ ID NO:30 or 31) and an Fc fragment, wherein said Fc fragment does not comprise a cysteine of a hinge region (preferably said Fc fragment is an Fc fragment derived from human IgG or horse IgG, more preferably said Fc fragment does not comprise a hinge region, even more preferably said Fc fragment comprises an amino acid sequence as set forth in any of SEQ ID NOs: 13-16, 29), preferably said leucine zipper is derived from a C-JUN or C-FOS protein, more preferably said leucine zipper is further attached at the C-terminus to a His, flag, C-myc or HA tag.
The novel coronavirus SARS-CoV-2 RBD fusion protein of claim 1 or 2, wherein said protease cleavage site and tag are linked by a linker peptide, preferably said linker peptide is selected from the group consisting of SEQ ID NO:26 27 or 28.
The novel coronavirus SARS-CoV-2 RBD fusion protein of any of claims 1-3, wherein said protease is selected from the group consisting of thrombin, enterokinase, TEV protease, and HRC-3C protease; preferably, the amino acid sequence of the thrombin, enterokinase, TEV protease or HRC-3C protease cleavage site is as shown in SEQ ID NO: 17-20; more preferably, the amino acid sequence of the protease cleavage site is not duplicated by the amino acid sequence in the RBD sequence or Fc fragment.
The novel coronavirus SARS-CoV-2 RBD fusion protein of any of claims 1-4, wherein the N-terminus of said fusion protein further comprises a signal peptide, preferably the amino acid sequence of said signal peptide comprises the amino acid sequence as set forth in SEQ ID NO: 21-23.
Novel RBD dimer of coronavirus SARS-CoV-2, characterized by the amino acid sequence according to SEQ ID NO:1, the cysteines at positions 18 and 43, the cysteines at positions 61 and 114, the cysteines at positions 73 and 207, and the cysteines at positions 162 and 170, respectively, form an intra-chain disulfide bond, and an inter-chain disulfide bond is formed between the cysteines at position 220 of the two monomeric RBDs, preferably, the monomeric RBD sequence comprises the amino acid sequence as set forth in SEQ ID NO:1 and 5-8, more preferably, the two monomeric RBD sequences of the dimer are identical.
A method for preparing the RBD fusion protein of novel coronavirus SARS-CoV-2 as claimed in any of claims 1-5 comprising sequentially ligating the RBD sequence of novel coronavirus SARS-CoV-2, a protease cleavage site and a tag facilitating dimer formation, preferably said protease cleavage site and said tag are linked by a linker peptide.
A method of preparing the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, comprising cleaving the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any of claims 1-5 with a protease, preferably the method further comprises purifying the RBD fusion protease cleavage product, preferably the purification comprises chromatography (e.g., affinity chromatography, ion exchange chromatography).
A polynucleotide encoding the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any one of claims 1-5 or the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6.
A vector comprising the polynucleotide of claim 9.
A host cell comprising the polynucleotide of claim 9 or the vector of claim 10.
Vaccine, preferably for use in the prevention of infection by a novel coronavirus, characterized in that it comprises the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 according to claim 6, or the polynucleotide according to claim 9.
The RBD fusion protein of novel coronavirus SARS-CoV-2 as claimed in any of claims 1-5, the RBD dimer of novel coronavirus SARS-CoV-2 as claimed in claim 6, or the polynucleotide as claimed in claim 9 for use in preventing infection of a human by novel coronavirus SARS-CoV-2, or for immunizing an animal, preferably a mammal, more preferably a horse, for obtaining an antiviral serum, preferably for use in preventing or treating infection of a human by novel coronavirus SARS-CoV-2.
A method for preventing or treating a novel coronavirus SARS-CoV-2 infection or obtaining neutralizing antibodies against the novel coronavirus SARS-CoV-2, comprising immunizing an animal, preferably a human or non-human mammal, more preferably a horse, with the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, or the polynucleotide of claim 9 to obtain antiviral serum.