WO2021244623A1 - Ferritin-ace-2 short peptide nanodrug - Google Patents

Abstract

The present invention provides an RBD/angiotensin-converting enzyme (ACE2) repressor peptide. The RBD/ACE2 repressor peptide can inhibit the binding of an spike glycoprotein (an S protein) on the surface of the novel corona pneumonia virus (SARS-CoV-2) to ACE2, and has the binding affinity to the S protein of SARS-CoV-2. The present invention further provides a fusion protein containing the RBD/ACE2 repressor peptide and a nanoparticle containing the fusion protein. The RBD/ACE2 repressor peptide, the fusion protein, and the nanoparticle of the present invention can be used for preventing and/or treating diseases caused by Coronaviridae viruses.

Description

Ferritin-ACE-2 short peptide nanomedicine Technical field

The invention relates to the field of recombinant fusion protein therapeutic drugs. Specifically, the present invention relates to a recombinant fusion ferritin drug suitable for treating diseases caused by coronaviruses of the coronavirus family.

Background technique

Pneumonia caused by coronaviruses, especially the new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) caused by the new coronavirus (SARS-CoV-2) is a sudden disease that poses a major threat to human health.

On January 7, 2020, the Wuhan Institute of Virology of the Chinese Academy of Sciences detected SARS-CoV-2 and obtained the entire genome sequence of the virus. On February 3, by comparing SARS-CoV-2 with the partial sequence of the coronavirus detected in the laboratory early, it found that the sequence of the new coronavirus was as high as 96% with that of a coronavirus in a bat sample. SARS-CoV-2 can invade cells in the same way as SARS-CoV, that is, by binding to the human ACE2 cell receptor (A pneumonia outbreak associated with a new coronavirus of probable bat origin, Nature, Peng Zhou et.al, 2020 ).

As of April 20, 2020, the global number of new coronary pneumonia infections has exceeded 2.36 million, and the cumulative deaths have exceeded 160,000. The R0 value (basic number of infections) of SARS-CoV-2 calculated by my country's CDC is as high as 3.77 (Epidemiological and clinical features of the 2019 novel coronavirus outbreak in China, Yang Yang et.al, Medrxiv, February 12, 2020), and Because COVID-19 has a long asymptomatic incubation period and a considerable proportion of asymptomatic carriers, the huge patient population caused by it has led to a run on the medical system, and the economic recession caused by it has made the development of effective therapeutic drugs imminent. At present, many research groups at home and abroad have analyzed SARS-CoV-2 surface spike glycoprotein (S protein) receptor binding domain (RBD) and human angiotensin converting enzyme 2 through structural biology methods. (Angiotensin-converting enzyme 2, ACE2) The crystal structure of the protein complex (Structural basis for the recognition of the 2019-nCoV by human ACE2, Renhong Yan et.al, bioRxiv, February 20, 2020; Crystal structure of the 2019 -nCoV spike receptor-binding domain bound with the ACE2receptor, Jun Lan et.al, bioRxiv, February 20, 2020), revealing the interaction site between COVID-19 RBD and ACE2, which makes it possible to block SAR-2-CoV- It is possible to treat COVID-19 by the interaction between 2/ACE2 protein. The effect of blocking SAR-2-CoV-2/ACE2 related effects through small molecules is limited, and the synthesis of peptides that can specifically bind to the SAR-2-CoV-2/ACE2 region may become a treatment for the global epidemic One of the powerful means for sudden diseases.

MIT’s BLPentelute research team analyzed the co-crystal structure of the new coronavirus S protein receptor binding domain (RBD) and ACE, looking for peptide conjugates that can block the combination of the two, and obtained a peptide fragment derived from the ACE2α1 helix. Called S-protein binding protein 1 (SBP1), SBP1 is composed of 23 amino acids. It has a very high binding affinity (nanomolar level) and has the potential to block the virus from entering human cells. It is therapeutic Prospects (The first-in-class peptide binder to the SARS-CoV-2 spike protein, G. Zhang et. al, bioRxi, March 30, 2020).

Among the many antibody treatment options, repressor peptides are undoubtedly a clear stream in the exploration of diversified treatment options, and there are not many relevant studies at present. The field of coronavirus treatment still needs to explore new, more effective and safe forms of treatment. Ferritin, a substance naturally possessed by human endogenously, is similar to the repressive peptide SBP1 derived from human ACE2. It is both derived from human itself and has the advantage of low immunogenicity. In addition, ferritin has a molecular weight of about 450kDa and has a spherical cage-like structure that is self-assembled by 24 subunits. It can not only use its cage-like cavity to contain drugs (Ferritin-based drug delivery systems: Hybrid nanocarriers for vascular immunotargeting). , Makan Khoshnejad et.al, Journal of Controlled Release 282 (2018) 13-24), functional protein molecules (such as antibodies, therapeutic peptides) can also be displayed outside the ferritin cage through fusion expression to achieve enhancement, The purpose of extending the efficacy half-life of functional protein molecules (such as the applicant’s previous patent ZL201710412728.9; published an article "Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension, Kelong Fanet.al, Anal.Chem .2018"). In addition, it has been reported that because the expression of ACE2 in adipose tissue and certain cancer tissues is higher than that in the lungs, tumors and obese people are more susceptible to COVID-19 (Two things about COVID-19 Might Need Attention, Xiaodong Jia et.al, Preprints, February 23, 2020), and the unique size effect of ferritin (outer diameter 12nm) enables it to target ACE2 expression through the high permeability and retention effect of solid tumor tissue (enhanced permeability and retention effect, EPR effect) A higher amount of tumor/adipocytes, so it has the potential value to quickly alleviate the symptoms of tumor/obese COVID-19 patients.

Summary of the invention

The present invention utilizes the unique advantages of ferritin as a drug carrier, and connects the coding sequence of the RBD/ACE2 repressor peptide of SAR-2-CoV-2 to the N-terminus or C-terminus of the ferritin monomer subunit (or the C-terminus is truncated). A truncated sequence of α-helix) to construct multiple fusion proteins of ferritin and RBD/ACE2 repressor peptide. The fusion protein can self-assemble into a 24-mer, which can display multiple ferritin surfaces. Multivalent nano therapeutic drugs that block peptides and prolong the half-life of peptide therapeutics to achieve the purpose of treating new type of coronary pneumonia.

The present invention provides four RBD/ACE2 repressor peptides, all of which are derived from human ACE-2 protein and have binding affinity to the S protein of SAR-2-CoV-2.

The invention carries out Cys point mutations on the ferritin monomer subunit sequence, which can reduce the generation of aggregates and improve the soluble expression and renaturation efficiency of the protein.

The present invention provides a truncated mutant of the monomer subunit of ferritin, so that the repressor peptide can be displayed on the outer surface of ferritin when it is connected to the C-terminus of ferritin.

Specifically, the present invention proposes the following technical solutions:

In one aspect, the present invention provides a RBD/ACE2 repressor peptide.

In one aspect, the invention provides a fusion protein.

In one aspect, the present invention provides a nanoparticle containing a fusion protein.

In one aspect, the present invention provides a method for producing the aforementioned RBD/ACE2 repressor peptide, fusion protein or nanoparticle.

In one aspect, the present invention provides a fusion protein pharmaceutical composition.

In one aspect, the present invention provides a SARS-CoV-2 surface spike glycoprotein (S protein) antagonist.

In one aspect, the present invention provides a method of generating therapeutic drugs against viruses of the coronavirus family.

In one aspect, the invention provides a nucleic acid molecule.

In one aspect, the invention provides an expression construct.

In one aspect, the present invention provides a recombinant cell.

In one aspect, the present invention provides the aforementioned RBD/ACE2 repressor peptide, fusion protein, nanoparticle, pharmaceutical composition or SARS-CoV-2 surface spike glycoprotein (S protein) antagonist, nucleic acid molecule, expression construct, recombinant Use of cells in the preparation of S protein inhibitors, competitive inhibitors of the binding of ACE2 and S protein, or in the preparation of drugs for preventing and/or treating coronavirus infections or diseases caused by the coronavirus infections the use of.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The ferritin-repressor peptide fusion protein constructed in the present invention can self-assemble to form a cage protein, and multiple repressor peptides are loaded on the outer surface of ferritin nanoparticles, which can bind to the coronavirus S protein to prevent the coronavirus from entering human cells , To achieve the effect of prevention of coronavirus infection and treatment of infection. Compared with simple suppressor peptides, the product of the present invention prolongs the therapeutic half-life of suppressor peptides by fusing ferritin subunits on the one hand; on the other hand, 24 suppressor peptides can be loaded on a ferritin molecule, providing multivalence The treatment plan can greatly improve the ability to bind to the coronavirus; on the other hand, the EPR effect of ferritin can make the therapeutic drug relatively enriched in the tissues with high expression of ACE2, thereby targeted protection of some vulnerable to coronavirus infection Organ; On the other hand, the two components of the ferritin-repressor peptide fusion protein constructed in the present invention are derived from the human body's own protein, so the immunogenicity is low.

(2) The fusion protein obtained by the E. coli expression system of the present invention can self-assemble with simple and high yield to form a cage protein with binding activity. The preparation method is simple, easy to operate, and has high pharmaceutical value and industrialization value.

(3) In some technical schemes of the present invention, the Cys with active sulfhydryl reaction site in wild-type ferritin is mutated, thereby reducing the possibility of side effects caused by the active sulfhydryl group reaction in the body, and also reducing the drug preparation process. The possibility of reactive sulfhydryl groups is conducive to drug control.

Description of the drawings

In order to explain the technical solution of the present invention more clearly, the following briefly introduces the accompanying drawings that need to be used. Obviously, the drawings in the following description are only some embodiments recorded in the present invention, which are for ordinary technology in the field. As far as personnel are concerned, they can also obtain other drawings based on these drawings without creative work.

Figure 1 shows a map of plasmid pET-22b(+).

Figure 2 shows a map of the recombinant plasmid pET-22b-XYD-406-000.

Figure 3 shows a map of the recombinant plasmid pET-22b-XYD-407-000.

Figure 4 shows a map of the recombinant plasmid pET-22b-XYD-408-000.

Figure 5 shows the restriction map of recombinant plasmids pET-22b-XYD-406-000, pET-22b-XYD-407-000 and pET-22b-XYD-408-000.

Figure 6 shows the recombinant plasmids pET-22b-XYD-406-000, pET-22b-XYD-407-000 and pET-22b-XYD-408-000 transformed E. coli BL21(DE3). The growth of strains 406, 407 and 408.

Figure 7 shows the protein in the supernatant and precipitate obtained by centrifugation after lysis of strains 406, 407, and 408, where S represents the supernatant, P represents the precipitate, and 1-3 are the three of strains 406, 407, or 408, respectively. The number of a parallel sample.

Figure 8 shows the TEM results of nanoparticle samples XYD-406-000, XYD-407-000 and XYD-408-000.

Figure 9 shows the average particle size of nanoparticle sample XYD-406-000.

Figure 10 shows the average particle size of nanoparticle sample XYD-407-000.

Figure 11 shows the average particle size of nanoparticle sample XYD-408-000.

Figure 12 shows the detection result of the binding activity of nanoparticle sample XYD-406-000 and S-RBD.

Figure 13 shows the detection result of the binding activity of nanoparticle sample XYD-407-000 and S-RBD.

Figure 14 shows the detection results of the binding activity of nanoparticle sample XYD-408-000 and S-RBD.

detailed description

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are all terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "RBD/ACE2 repressor peptide" refers to the ability to inhibit the binding of new coronavirus (SARS-CoV-2) surface spike glycoprotein (S protein) to angiotensin converting enzyme 2 (ACE-2) The peptide fragment of the new coronavirus (SARS-CoV-2) surface spike glycoprotein (S protein) receptor binding domain (RBD) to prevent the new coronavirus (SARS-CoV-2) infection The human body may reduce the symptoms of those who have been infected.

As used herein, the term "nanoparticle" refers to particles formed from self-assembled monomeric subunit proteins, which can be hollow or solid structures. For example, ferritin subunit proteins self-assemble into ferritin nanoparticles with a cavity in the middle. The shape of the nanoparticles of the present invention is generally spherical or cage-like, although other shapes, such as rod-shaped, cubic, plate-shaped, oblong, oval, etc., can also be used in the practice of the present invention.

As used herein, the term "self-assembling" protein refers to a protein that can form nanoparticles by regularly arranging to form multimers while being expressed without the aid of a specific inducer.

As used herein, the term "Coronavirus" belongs to the Coronavirus family, a genus of Coronavirus, which can infect mammals and birds and cause various diseases of the respiratory system, digestion, and central nervous system. According to genomic and serological differences, coronaviruses can be divided into four different genera: α, β, γ, and δ. At present, only α and β are coronaviruses that infect humans. Up to now, six human coronaviruses (HCoV) from two genera (α and β) have been identified. α genus coronaviruses include NL63 and 229E, and β genus coronaviruses include OC43, HKU1, and acute respiratory syndrome coronavirus ( SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Novel Coronavirus (SARS-CoV-2).

As used herein, the term "ferritin" refers to an iron storage structure composed of two parts: a protein shell and an iron core. In nature, the protein shell of ferritin is a clathrin structure (about 12nm in outer diameter and about 8nm in inner diameter) formed by self-assembly of 24 subunits, and the main component of the iron core is ferrihydrite. The protein shell of ferritin without an iron core is also called "deferritin". "Ferritin" as used herein includes eukaryotic ferritin and prokaryotic ferritin, preferably eukaryotic ferritin, more preferably mammalian ferritin, such as human ferritin. Eukaryotic ferritin usually includes a heavy chain ferritin monomer subunit (H, 21kDa) and a light chain ferritin monomer subunit (L, 19kDa). The H subunit is responsible for the oxidation of Fe(II) to Fe(III) and includes catalytic iron oxidase sites, while the L subunit plays a role in iron nucleation. The H and L subunits assemble together into a 24-mer hybrid ferritin. In different tissues and organs of the body, the ratio of H and L subunits in ferritin molecules is different. However, through recombination, it is also possible to obtain "H ferritin" assembled from only H subunits or "L ferritin" assembled from L subunits only.

As used herein, the term "human heavy chain ferritin" (hereinafter abbreviated as "human HFn") refers to ferritin assembled from only the heavy chain monomer subunits of human ferritin. "Human light chain ferritin" (hereinafter abbreviated as "human LFn") refers to ferritin assembled only from the light chain monomer subunits of human ferritin.

As used herein, the term "fusion protein" refers to a natural or synthetic molecule composed of one or more of the above-mentioned molecules, in which two or more peptide or protein (including glycoprotein)-based molecules with different specificities The selected ones are fused together by chemical or amino acid-based linker molecules. The connection can be achieved by C-N fusion or N-C fusion (in the 5'→3' direction).

Table 1 Abbreviations

English abbreviations	Chinese name	English full name
Human HFn	Human heavy chain ferritin	Human Heavy Chain-Ferritin
Human LFn	Human light chain ferritin	Human Light Chain-Ferritin
SARS-CoV-2	Novel Coronavirus	Severe Acute Respiratory Syndrome Coronavirus 2
COVID-19	New coronavirus pneumonia	Corona Virus Disease-19
S-RBM	S protein receptor binding motif	S glycoprotein receptor binding motif
S-RBD	S protein receptor binding domain	S glycoprotein receptor binding Domain
ACE2	Angiotensin Converting Enzyme 2	Angiotensin converting enzyme 2
E.coli	Escherichia coli	Escherichia coli

In one aspect, the present invention provides an RBD/ACE2 repressor peptide, wherein the RBD/ACE2 repressor peptide comprises an amino acid having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 1, 2, 3, or 4. The sequence is preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more than 99% identity; more preferably, The amino acid sequence of the RBD/ACE2 repressor peptide is shown in SEQ ID NO. 1, 2, 3 or 4.

In one aspect, the present invention provides a fusion protein, wherein the fusion protein comprises the RBD/ACE2 repressor peptide.

In a specific embodiment, the fusion protein further comprises at least a part of a self-assembled, monomeric subunit.

In one aspect, the present invention provides a nanoparticle comprising a fusion protein, wherein the fusion protein comprises an RBD/ACE2 repressor peptide and at least a part of a self-assembled, monomeric subunit, and wherein the nanoparticle is on its surface The RBD/ACE2 repressor peptide is shown above. The RBD/ACE2 repressor peptide can suppress the surface spike glycoprotein (S protein) of the new coronavirus (SARS-CoV-2) and angiotensin converting enzyme 2 (ACE-2). ) Combine.

According to the present invention, the self-assembled monomeric subunit protein, monomeric subunit protein, self-assembled protein, self-assembled subunit protein, etc. of the present invention are full-length, monomeric polypeptides, or any part or variant thereof, It can guide the self-assembly of monomer self-assembled subunit proteins into nanoparticles. Such proteins are known to those skilled in the art. Examples of self-assembling proteins that can be used to prepare the nanoparticles of the present invention include, but are not limited to, ferritin monomer subunits, monomeric encapsulin proteins, monomeric 03-33 proteins, monomeric thiooxygenase reductase (SOR) proteins, monomers Body 2,4-Dioxytetrahydropteridine synthase (LS) protein, monomeric pyruvate dehydrogenase complex (PDC) protein, monomeric hydrolipoamide acetyltransferase (E2) protein, and The envelope (Env) protein of alphavirus (such as Chikungunya virus).

In a specific embodiment, the ferritin monomer subunit is any one or at least one of ferritin derived from mammalian origin, ferritin derived from amphibian, ferritin derived from bacteria or ferritin derived from plant origin. The two combined ferritin monomer subunits are preferably mammalian or bacterial ferritin monomer subunits.

In a specific embodiment, the mammalian-derived ferritin includes any one or a combination of at least two of human-derived ferritin, murine-derived ferritin, or horse spleen ferritin.

In a specific embodiment, the bacterial-derived ferritin includes Helicobacter pylori ferritin, Escherichia coli ferritin, or Pyrococcus furiosus ferritin.

In a specific embodiment, the source of the ferritin includes any one or a combination of at least two of natural extraction products, artificial synthesis products, or genetic engineering technology products.

In a specific embodiment, the ferritin monomer subunit includes a mutated amino acid sequence; preferably, the mutated amino acid is cysteine (Cys); more preferably, the cysteine is mutated to glutamic acid (Glu), serine (Ser) or alanine (Ala).

In a specific embodiment, the ferritin monomer subunit is a truncation mutant; preferably, the truncation mutant is an α-helical truncation mutant at the C-terminus of the heavy chain ferritin monomer subunit (H) Preferably, the truncation mutant is an epsilon helix truncation mutant at the C-terminus of the light chain ferritin monomer subunit (L).

In a specific embodiment, the nanoparticle comprises at least one ferritin monomer subunit, preferably, the ferritin monomer subunit is selected from heavy chain ferritin monomer subunit (H) or light chain iron Protein monomer subunit (L); preferably, the heavy chain ferritin monomer subunit (H) and/or the light chain ferritin monomer subunit (L) form a nanoparticle, more preferably, the nano The particle contains 24 ferritin monomer subunits, wherein the ratio of heavy chain ferritin monomer subunits (H) to light chain ferritin monomer subunits (L) is 0:24-24:0; preferably, The heavy chain ferritin monomer subunit (H) is a human heavy chain ferritin monomer subunit (H); preferably, the light chain ferritin monomer subunit (L) is a human light chain ferritin monomer Subunit (L).

In a specific embodiment, the heavy chain ferritin monomer subunit (H) comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 5, 6, 7, or 8, preferably having 85 %, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more 99% identical amino acid sequences; more preferably, the heavy chain iron The amino acid sequence of the protein subunit is shown in SEQ ID NO. 5, 6, 7 or 8.

In a specific embodiment, the fusion protein comprises the RBD/ACE2 repressor peptide and the heavy chain ferritin monomer subunit (H). Preferably, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. An amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more than 99% identity; more preferably, the fusion The amino acid sequence of the protein is shown in SEQ ID NO. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.

In a specific embodiment, the light chain ferritin subunit (L) comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 20, 21, 22 or 23, preferably having 85%, An amino acid sequence with 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more than 99% identity; more preferably, the light chain ferritin The amino acid sequence of the subunit is shown in SEQ ID NO. 20, 21, 22 or 23.

In a specific embodiment, the fusion protein comprises the RBD/ACE2 repressor peptide and the light chain ferritin subunit (L). Preferably, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, Preferably, an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more than 99% identity; more preferably, the The amino acid sequence of the fusion protein is shown in SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.

In one aspect, the present invention provides a method for producing the aforementioned RBD/ACE2 repressor peptide, fusion protein or nanoparticle, the method comprising combining one or more nucleic acids encoding the RBD/ACE2 repressor peptide or fusion protein The molecule is introduced into the cell, and the cell is cultured under conditions suitable for expression of the RBD/ACE2 repressor peptide, fusion protein, or formation of nanoparticles.

In one aspect, the present invention provides a pharmaceutical composition comprising the aforementioned RBD/ACE2 repressor peptide, fusion protein or nanoparticle.

In a specific embodiment, the pharmaceutical composition is a drug for coronaviruses of the coronavirus family; preferably, the viruses of the coronavirus family are selected from the group consisting of new coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, and MERS. -Cov, 229E, NL63, OC43 and HKU1.

In a specific embodiment, the pharmaceutical composition further comprises another therapeutic agent; the another therapeutic agent is selected from immunotherapeutic agents or other drugs that inhibit viruses of the coronavirus family.

In a specific embodiment, the virus of the coronavirus family is a new type of coronavirus (SARS-CoV-2); the pharmaceutical composition is a drug for the new type of coronavirus (SARS-CoV-2).

In one aspect, the present invention provides a SARS-CoV-2 surface spike glycoprotein (S protein) antagonist, which comprises the aforementioned RBD/ACE2 repressor peptide, fusion protein or nanoparticle, said repressor peptide, fusion protein or Nanoparticles work by binding to SARS-CoV-2 surface spike glycoprotein (S protein).

In one aspect, the present invention provides a method of generating a therapeutic drug for viruses of the coronavirus family, the method comprising:

a) express the aforementioned RBD/ACE2 repressor peptide or fusion protein, or form nanoparticles; and

b) Recover the RBD/ACE2 repressor peptide, fusion protein or nanoparticle.

In one aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the aforementioned RBD/ACE2 repressor peptide or fusion protein or nanoparticle.

In a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO. 35, 36 or 37, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or more 99% identical nucleotide sequences; more preferably, the nucleic acid sequence is as SEQ ID NO As shown in .35, 36 or 37.

In a specific embodiment, the nucleic acid molecule is a codon optimized nucleic acid molecule.

In one aspect, the present invention provides an expression construct comprising the aforementioned nucleic acid molecule.

In one aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or expression construct.

In one aspect, the present invention provides the aforementioned RBD/ACE2 repressor peptides, fusion proteins, nanoparticles, pharmaceutical compositions, SARS-CoV-2 surface spike glycoprotein (S protein) antagonists, therapeutic drugs, nucleic acid molecules, expression constructs Use of recombinant cells or recombinant cells in the preparation of S protein inhibitors, competitive inhibitors of ACE2 and S protein binding, or in the preparation of prevention and/or treatment of coronavirus infections or diseases caused by the coronavirus infections Preferably, the disease caused by the coronavirus infection is a disease caused by a new coronavirus infection, especially a new coronavirus pneumonia.

The RBD/ACE2 repressor peptide, fusion protein, nanoparticle, pharmaceutical composition, SARS-CoV-2 surface spike glycoprotein (S protein) antagonist, therapeutic drug, nucleic acid molecule, expression construct or recombinant cell of the present invention can be used for Prevent and/or treat infections of coronaviruses of the coronavirus family, especially for the treatment of diseases caused by new coronavirus infections, especially new coronavirus pneumonia.

Example

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the examples are only for illustrative purposes and do not have a limiting effect on the present invention. The reagents and biological materials described in the following examples can be obtained from commercial sources unless otherwise specified.

The design and experimental methods of the experimental materials used in the following examples are as follows:

1 Design of experimental materials

1.1 Design of peptides, proteins or fusion proteins

(1) The amino acid sequence of the RBD/ACE2 repressor peptide of the S protein of the candidate SARS-CoV-2 is as follows:

a) ACE2-P1:

b) ACE2-P2:

c) ACE2-P3:

d)ACE2-P4:

(2) Candidate HFn monomer subunit protein

a) Wild-type HFn monomer subunit protein, its amino acid sequence is as follows (total 183 amino acids):

b) Mutant HFn1 monomer subunit protein (mHFn1): Remove the α-helix at the C-terminus of the wild-type HFn monomer subunit protein to obtain mHFn1. Its amino acid sequence is as follows (a total of 163 amino acids):

c) Mutant HFn2 monomer subunit protein (mHFn2): The Cys at positions 91, 103, and 131 of the wild-type HFn monomer subunit protein were mutated to Glu, Ala, and Ala, respectively, to obtain mHFn2. Its amino acid sequence is as follows (total 183 Amino acids):

d) Mutated HFn3 monomer subunit protein (mHFn3): Remove the α-helix at the C-terminus of the wild-type HFn monomer subunit protein, and mutate the Cys at positions 91, 103, and 131 to Glu, Ala, and Ala, respectively, to obtain The amino acid sequence of mHFn3 is as follows (163 amino acids in total):

(3) Fusion protein of RBD/ACE2 repressor peptide and HFn monomer subunit protein

a) Amino acid sequence of ACE2-P1-mHFn2

Connect ACE2-P1 to the N-terminus of the mutant mHFn2 subunit via linker(G4S)3 to form a fusion protein:

b) Amino acid sequence of mHFn3-ACE2-P1

Connect ACE2-P1 to the C-terminus of the C-terminus truncated HFn subunit (mHFn3) via linker (G4S)3 to form a fusion protein:

c) Amino acid sequence of mHFn2-ACE2-P1

Insert ACE2-P1 into the loop of mutant mHFn2 subunits αA and αB to form a fusion protein:

d) Amino acid sequence of ACE2-P2-mHFn2

Connect ACE2-P2 to the N-terminus of the mutant mHFn2 subunit via linker(G4S)3 to form a fusion protein:

e) Amino acid sequence of mHFn3-ACE2-P2

Connect ACE2-P2 to the C-terminus of the C-terminal truncated HFn subunit (mHFn3) via linker (G4S)3 to form a fusion protein:

f) Amino acid sequence of ACE2-P3-mHFn2

Connect ACE2-P3 to the N-terminus of the mutant mHFn2 subunit via linker(G4S)3 to form a fusion protein:

g) Amino acid sequence of mHFn3-ACE2-P3

Connect ACE2-P3 to the C-terminus of the C-terminal truncated HFn subunit (mHFn3) via linker (G4S)3 to form a fusion protein:

h) Amino acid sequence of mHFn2-ACE2-P3

Insert ACE2-P3 into the loop of mutant mHFn2 subunits αA and αB to form a fusion protein:

i) Amino acid sequence of ACE2-P4-mHFn2

Connect ACE2-P4 to the N-terminus of the mutant mHFn2 subunit via linker(G4S)3 to form a fusion protein:

j) Amino acid sequence of mHFn3-ACE2-P4

Connect ACE2-P4 to the C-terminus of the C-terminal truncated HFn subunit (mHFn3) via linker (G4S)3 to form a fusion protein:

k) Amino acid sequence of mHFn2-ACE2-P4

Insert ACE2-P4 into the loop of mutant mHFn2 subunits αA and αB to form a fusion protein:

(4) Candidate LFn monomer subunit protein

a) Wild-type LFn monomer subunit protein, its amino acid sequence is as follows (a total of 174 amino acids):

b) Mutated LFn1 monomer subunit protein (mLFn1): The Cys at position 126 of wild-type LFn was mutated to Ala (LFn C126A) to obtain mLFn1. Its amino acid sequence is as follows (a total of 174 amino acids):

c) Mutant LFn2 monomer subunit protein (mLFn2): Remove the α-helix at the C-terminus of the wild-type LFn monomer subunit protein to obtain mLFn2. Its amino acid sequence is as follows (a total of 157 amino acids):

d) Mutant LFn3 monomer subunit protein (mLFn3): Remove the α-helix at the C-terminus of the wild-type LFn monomer subunit protein, and mutate the Cys at position 127 to Ala to obtain mLFn3. The amino acid sequence is as follows (total 157 amino acids):

(5) Fusion protein of RBD/ACE2 repressor peptide and LFn monomer subunit protein

a) Amino acid sequence of mLFn2-ACE2-P1

Connect ACE2-P1 to the N-terminus of the mutant mLFn2 subunit via linker(G4S)3 to form a fusion protein:

b) The amino acid sequence of mLFn3-ACE2-P1

Connect ACE2-P1 to the C-terminus of the C-terminus truncated LFn subunit (mLFn3) via linker(G4S)3 to form a fusion protein:

c) Amino acid sequence of mLFn2-ACE2-P1

Insert ACE2-P1 into the loop of mutant mLFn2 subunits αA and αB to form a fusion protein:

d) Amino acid sequence of ACE2-P2-mLFn2

Connect ACE2-P2 to the N-terminus of the mutant mLFn2 subunit via linker(G4S)3 to form a fusion protein:

e) Amino acid sequence of mLFn3-ACE2-P2

Connect ACE2-P2 to the C-terminus of the C-terminus truncated LFn subunit (mLFn3) via linker(G4S)3 to form a fusion protein:

f) Amino acid sequence of ACE2-P3-mLFn2

Connect ACE2-P3 to the N-terminus of the mutant mLFn2 subunit via linker(G4S)3 to form a fusion protein:

g) Amino acid sequence of mLFn3-ACE2-P3

Connect ACE2-P3 to the C-terminus of the C-terminus truncated LFn subunit (mLFn3) via linker(G4S)3 to form a fusion protein:

h) Amino acid sequence of mLFn2-ACE2-P3

Insert ACE2-P3 into the loop of mutant mLFn2 subunits αA and αB to form a fusion protein:

i) Amino acid sequence of ACE2-P4-mLFn2

Connect ACE2-P4 to the N-terminus of the mutant mLFn2 subunit via linker(G4S)3 to form a fusion protein:

j) Amino acid sequence of mLFn3-ACE2-P4

Connect ACE2-P4 to the C-terminus of the C-terminal truncated LFn subunit (mHFn3) via linker (G4S)3 to form a fusion protein:

k) The amino acid sequence of mLFn2-ACE2-P4

Insert ACE2-P4 into the loop of mutant mLFn2 subunits αA and αB to form a fusion protein:

1.2 Design of coding genes

According to the codon preference of the host bacteria, the coding genes of the above-mentioned polypeptides, proteins or fusion proteins are synthesized.

1.3 Construction of expression vector

The commonly used vector pET-22b(+) for expressing foreign proteins in Escherichia coli is selected, ampicillin resistance (Amp ⁺ ), Nde I and Bam H I restriction sites are selected to insert the target gene, and the recombinant pET-22b plasmid is obtained. Restriction map and gene sequencing confirmed that the expression vector was successfully constructed. Among them, the plasmid map of pET-22b(+) is shown in Figure 1.

1.4 Construction of recombinant strains

E. coli BL21(DE3) was selected as the host bacteria, the recombinant pET-22b plasmid containing the target gene was transformed into competent cells of the host bacteria, and the positive clones were screened through the ampicillin resistance plate to determine the recombinant strain.

2. Experimental method

2.1 Recombinant strain construction

2.1.1 Resuspension of recombinant plasmid

Take 10μg of recombinant pET-22b plasmid lyophilized powder, resuspend it in 200μl TE buffer, and pack 10μl/tube, save 1 tube separately, and store the rest in the refrigerator at -80℃ for later use.

2.1.2 Conversion

(1) Take out E.coli BL21(DE3) competent cells from the refrigerator at -80℃, place them on ice and melt (about 5min), in an ice bath, take 0.5-1μl plasmid resuspension and add it to 20μl feeling Mix well in the conditioned cells, and incubate them on ice for 30 min.

(2) After heat shocking the above sample in a 42°C water bath for 90 seconds, immediately place it on ice and let it stand for 2 minutes.

(3) Take 280 μL of sterile LB liquid medium, add it to the heat-shocked sample, and activate it at 37°C and 220 rpm for 1 hour.

(4) Take 150 μl of the above-mentioned transformed bacterial solution and spread it on an LB plate containing a final concentration of 100 μg/mL ampicillin (the concentration of ampicillin mother solution is 100 mg/mL), and incubate overnight in an incubator at 37° C. to observe the colony growth.

2.2 Protein expression

2.2.1 Shake flask culture

Take 3 large and plump clones on the resistant plate, respectively inoculate them in 40-60mL LB medium (shaking flask), culture at 37℃ to OD ₆₀₀ around 1.0-1.5, add 0.25～0.5mM IPTG, 25℃ Induced expression for 3-8h or induced overnight at 16°C. The expression of the target protein was detected by SDS-PAGE.

2.2.2 Test sample preparation

Bacterial lysis: Take 30mL of bacteria liquid, centrifuge at 5000-8000r/min for 10-30min, discard the supernatant, add 30mL 20mM Tris-HCl, pH8.0 buffer solution, resuspend it evenly, and break it in a high-pressure homogenizer at 800-1000bar for 3 times .

Protein purification: Centrifuge the broken bacterial solution to remove E. coli fragments. Heat the supernatant at 72°C for 15 minutes to precipitate impurities. After centrifugation to remove the precipitate, the supernatant is separated and purified with a Superdex 200 pg (GE Healthcare) column and determined by electrophoresis purity. The purified HFn can be lyophilized and stored, or stored in a pH 8.0, 50mM Tris-HCl solution.

SDS-PAGE sample preparation: Take 100μL of the above bacterial lysate, centrifuge at 8000-10000rpm for 10-20min, take 20μL of supernatant to another centrifuge tube, add 5μL of 5× loading buffer and mix well, 85 Incubate at -95°C for 5 minutes, this is the lysate supernatant sample; add 100μL of 20mM Tris-HCl, pH8.0 buffer to resuspend the pellet for the remaining pellet, add 20μL of resuspension to 5μL of 5× sample buffer and mix well ，Incubate at 85-95℃ for 5min, this is the lysate precipitation sample.

2.3 Protein activity detection method:

The indirect ELISA method was used to detect the binding activity of the purified protein with the S protein of SARS-CoV-2, thereby proving whether there is a target protein with binding activity in the refolding solution.

The experimental operation process is as follows:

1) Coating plate: Coat the sample to be tested or the ACE-2 reference product in a 96-well plate, and incubate overnight in a refrigerator at 4°C;

2) Wash the plate: Wash 3 times with PBST (take 300μl Tween-20 and add PBS 100ml and mix well);

3) Blocking: Add 300μL/well of 5% BSA blocking solution, cover with sealing film, and incubate in a 37℃ incubator for 2h;

4) Washing the plate: Wash the ELISA plate 3 times with 1×PBST in a plate washer;

5) Incubate the S-RBD-mFc protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.): Use a protein stabilizer (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., PR-SS-002) to dissolve the S-RBD-mFc protein and Dilute to a working concentration of 1.0～1.5μg/mL, 100μL/well, cover with sealing film, and incubate in a 37℃ incubator for 2h;

6) Washing the plate: Wash the ELISA plate with 1×PBST in a plate washer for 3 times;

7) Incubate the primary antibody: Dilute Anti-mFc antibody with protein stabilizer (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., PR-SS-002) (1:1000), 100μL/well, cover with sealing film, 37 Incubate in an incubator at ℃ for 1.5h;

8) Washing the plate: Wash the ELISA plate with 1×PBST in a plate washer for 3 times;

9) Incubate HRP enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody (Cytiva, goat anti-mouse) with 5% BSA (1:5000), 100μL/well, cover with sealing film, and incubate in a 37℃ incubator for 0.5 h;

10) Washing the plate: Wash the ELISA plate with 1×PBST in a plate washer for 3 times;

11) Color development: add TMB one-step color development solution, pay attention to avoid light, 100μL/well, avoid light for 5min, 10min and 30min respectively, and detect the absorbance at 650nm with a microplate reader immediately.

Example 1: Gene sequence design of ACE2-P1-mHFn2 fusion protein

Construct the ACE2-P1-mHFn2 fusion protein, and optimize the coding gene sequence according to the codon preference of E. coli to obtain the optimized ACE2-P1-mHFn2 (number: XYD-406-000) gene. The specific sequence is as follows:

Example 2: Gene sequence design of mHFn2-ACE2-P1 fusion protein

Construct the mHFn2-ACE2-P1 fusion protein, and optimize the coding gene sequence according to the codon preference of E. coli to obtain the optimized mHFn2-ACE2-P1 (number: XYD-407-000) gene. The specific sequence is as follows:

Example 3: Gene sequence design of ACE2-P2-mHFn2 fusion protein

Construct the ACE2-P2-mHFn2 fusion protein, and optimize the coding gene sequence according to the codon preference of E. coli to obtain the optimized ACE2-P2-mHFn2 (number: XYD-408-000) gene. The specific sequence is as follows:

Example 4: Construction, expression and purification of bacterial cells expressing the fusion protein of Example 1-3

1. Expression vector construction

Select the commonly used vector pET-22b(+) for expression of foreign proteins in E. coli, ampicillin resistance (Amp+), and select Nde I and BamH I restriction sites to insert target genes XYD-406-000, XYD-407-000, respectively And XYD-408-000 to get the following three plasmids: recombinant plasmid pET-22b-XYD-406-000, pET-22b-XYD-407-000 and pET-22b-XYD-408-000, the recombinant plasmid maps are respectively As shown in Figure 2-4. After the recombinant plasmid is extracted, the purity of the plasmid and the concentration of the sample are tested, and it meets the requirements.

The three recombinant plasmids obtained were double digested with Xho I and XbaI (near Nde I and BamH I, respectively), and the digested fragments contained the target gene and were about 600-800 bp in length. The digestion identification diagram is shown in Figure 5. After double enzyme digestion, the band of the target gene is about 750bp in the electrophoresis diagram, and the size is near the theoretical value, indicating that the target gene has been constructed into the expression plasmid, and the recombinant plasmid is 100% sequence correct after sequencing.

2. Resistant screening of recombinant strains

The above three recombinant plasmids were respectively transformed into E. coli BL21 (DE3) to obtain recombinant strains 406 (BP-HS-008), 407 (BP-HS-009) and 408 (BP-HS-010). Separately spread 100 μl of bacterial solution containing recombinant strains 406, 407 and 408 on an LB plate containing a final concentration of 100 μg/mL ampicillin (the concentration of ampicillin mother liquor was 100 mg/mL), and cultivate overnight in a 37° C. incubator. The growth of the colony is shown in Figure 6. The recombinant strains can grow on the resistant LB plate, and the number of clones is large. It can be judged that the recombinant strains have the corresponding resistance, and the plasmid pET-22b( +) The resistance is the same.

Take a single colony with a higher expression on the resistant plate for amplification, _{add glycerol with a final concentration of 20% when the OD 600} reaches 1.5 to 2.0, and distribute 1 mL/tube. This is a glycerol bacteria. The glycerol bacteria are stored in a refrigerator at -80°C and used for subsequent fermentation.

3. Expression of RBM-HFn fusion protein and detection of protein activity

3.1 Preparation of fermentation samples

The glycerol bacteria of the three plasmids were thawed at room temperature and inoculated at 1% in LB medium, cultured at 37℃, 220rpm shaker to OD ₆₀₀ to 1.0, added with a final concentration of 0.5mM IPTG, and induced the target protein at 25℃ Expression, induce 4-5h to terminate the culture, and obtain the fermentation broth.

3.2 Bacteria collection

The fermentation broth of the above-mentioned glycerol bacteria was taken, and the bacteria were collected by centrifugation at 4° C. and 10,000 rpm for 25 min to obtain the bacteria broths containing the respective plasmid bacteria.

3.3. Sample detection

3.3.1 Determination of the solubility of the target protein in the fermentation sample

(1) Sample preparation

Bacterial lysis: Take 30 mL of each of the three plasmid bacteria, centrifuge at 5000 r/min for 15 min, discard the supernatant, add 30 mL of 20 mM Tris-HCl, pH 8.0 buffer to resuspend, and place in a high-pressure homogenizer at 1000 bar (Yonglian Biotech, UH-03) was broken three times to obtain a cell lysate.

SDS-PAGE sample preparation: Take 100μL of the above-mentioned bacterial lysate, centrifuge at 10000rpm for 10min, take 20μL of supernatant to another centrifuge tube, add 5μL of 5× loading buffer to mix, incubate at 95℃ for 5min, this is the lysate Supernatant sample (three parallel samples for the same sample, labeled 1S, 2S, and 3S); add 100μL of 20mM Tris-HCl, pH8.0 buffer to resuspend the pellet for the remaining pellet, and add 20μL of the resuspension solution to 5μL Mix well with 5× loading buffer and incubate at 95°C for 5 minutes. This is the lysate precipitation sample (three parallel samples are set for the same sample, labeled 1P, 2P, and 3P). The sample was heated at 95°C to 100°C for 5 minutes, cooled, centrifuged, and mixed for inspection.

(2) SDS-PAGE detection

The loading volume is 10μL, the constant voltage is 90～125V, the current upper limit is 200mA, and the electrophoresis time is 60～90 minutes.

(3) Experimental results

As shown in Figure 7, the three proteins (XYD-406-000, XYD-407-000 and XYD-408-000) are all distributed in the supernatant, indicating that each recombinant strain can be soluble and expressed.

3.3.2 Morphology and particle size detection of nanoparticles

TEM detection of RBM-HFn nanoparticle morphology:

The purified protein samples XYD-406-000, XYD-407-000 and XYD-408-000 (20μL, 0.1mg/mL) were added dropwise to the treated copper mesh, using 1% uranyl acetate Stain for 1 minute, and then image with JEM-1400 80kv TEM (JEOL, Japan). Transmission electron microscopy results (Figure 8) show that the three ferritin samples are all nano-particles, and have a uniform and regular cage-like protein structure, with a diameter of about 14-17nm.

DLS particle size detection:

The instrument Nano ZSE Nanosizer (Malvern, UK) is used to detect the particle size of the sample. The parameter settings are Material as Protern and Dispersant as Tris buffer with pH 8.0 50mM. Select automatic mode scanning.

The samples are stored in pH 8.0 50mM Tris buffer, and the protein concentration is 3.78mg/mL. The results are shown in Figures 9-11. The average particle size of the nanoparticles is about 16.57nm (XYD-406-000), 17.04nm (XYD-407-000) and 14.65nm (XYD-408-000).

3.3.3 Binding activity detection-indirect ELISA method

The indirect ELISA method was used to detect the binding activity of the purified protein to the S protein, thereby proving whether there is a target protein with binding activity in the refolding solution.

The activity detection results of nanoparticle samples XYD-406-000, XYD-407-000 and XYD-408-000 are shown in Figure 12-14, respectively. The results show that the three constructed proteins all have S protein binding activity and have a concentration Dependent, XYD-408-000 has the highest activity.

Claims (14)

1. An RBD/ACE2 repressor peptide, wherein the RBD/ACE2 repressor peptide comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 1, 2, 3 or 4, preferably 85%, 90% %, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more 99% identical amino acid sequences; more preferably, the RBD/ACE2 repressor peptide The amino acid sequence is shown in SEQ ID NO. 1, 2, 3 or 4.
2. A fusion protein, wherein the fusion protein comprises the RBD/ACE2 repressor peptide of claim 1;

   Preferably, the fusion protein further comprises at least a part of self-assembled monomer subunits;

   Preferably, the monomer subunit is selected from the following group: ferritin monomer subunit, monomer encapsulin protein, monomer 03-33 protein, monomer thiooxygenase reductase (SOR) protein, monomer 2,4 -Lumazine synthase (LS) protein, monomeric pyruvate dehydrogenase complex (PDC) protein, monomeric hydrogen lipoamide acetyltransferase (E2) protein, and alphavirus (such as Chikungunya virus) envelope (Env) protein; preferably, the ferritin monomer subunit is ferritin derived from mammalian sources, ferritin derived from amphibians, ferritin derived from bacteria or plants The ferritin monomer subunits of any one or a combination of at least two of the source ferritin, preferably mammalian or bacterial ferritin monomer subunits;

   Preferably, the monomer subunit is a ferritin monomer subunit;

   Preferably, the ferritin monomer subunit is selected from a heavy chain ferritin monomer subunit or a light chain ferritin monomer subunit;

   More preferably, the heavy chain ferritin monomer subunit comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 5, 6, 7 or 8, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; preferably, the heavy chain ferritin monomer subunit The amino acid sequence is shown in SEQ ID NO. 5, 6, 7 or 8;

   More preferably, the light chain ferritin monomer subunit comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 20, 21, 22 or 23, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more 99% identical amino acid sequences; preferably, the light chain ferritin monomer subunit The amino acid sequence is shown in SEQ ID NO. 20, 21, 22 or 23;

   Preferably, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 9-19 or 24-34, preferably 85%, 90%, 95%, An amino acid sequence with 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; preferably, the fusion protein is selected from SEQ ID NO. 9-19 or 24-34.
3. A nanoparticle comprising a fusion protein, wherein the fusion protein comprises an RBD/ACE2 repressor peptide and at least a part of a self-assembled, monomeric subunit, and wherein the nanoparticle displays the RBD/ACE2 repression on its surface A peptide, the RBD/ACE2 repressor peptide can inhibit the binding of new coronavirus (SARS-CoV-2) surface spike glycoprotein (S protein) with angiotensin converting enzyme 2 (ACE-2).
4. The nanoparticle according to claim 3, wherein the RBD/ACE2 repressor peptide comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 1, 2, 3 or 4, preferably 85% , 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more 99% identical amino acid sequences; more preferably, the RBD/ACE2 represses The amino acid sequence of the peptide is shown in SEQ ID NO. 1, 2, 3 or 4.
5. The nanoparticle according to claim 3 or 4, wherein the monomer subunit is selected from the group consisting of ferritin monomer subunit, monomer encapsulin protein, monomer 03-33 protein, monomer thiooxygenase reductase (SOR) protein, monomeric 2,4-dioxotetrahydropteridine synthase (LS) protein, monomeric pyruvate dehydrogenase complex (PDC) protein, monomeric hydrolipoamide acetyl transfer Enzyme (E2) protein and the envelope (Env) protein of alphavirus (such as Chikungunya virus); preferably, the ferritin monomer subunit is derived from ferritin from mammalian sources, or from amphibians Ferritin monomer subunits of any one or a combination of ferritin, ferritin derived from bacteria or ferritin derived from plants, preferably ferritin monomer subunits derived from mammals or bacteria;

   Preferably, the mammalian-derived ferritin includes any one or a combination of at least two of human-derived ferritin, murine-derived ferritin, or horse spleen ferritin;

   Preferably, the ferritin derived from bacteria includes Helicobacter pylori ferritin, Escherichia coli ferritin or Pyrococcus furiosus ferritin;

   Preferably, the source of the ferritin includes any one or a combination of at least two of natural extraction products, artificial synthesis products, or genetic engineering technology products;

   Preferably, the ferritin monomer subunit includes a mutant amino acid sequence; preferably, the mutant amino acid is cysteine (Cys); more preferably, the cysteine is mutated to glutamic acid (Glu) , Serine (Ser) or Alanine (Ala);

   Preferably, the ferritin monomer subunit is a truncation mutant; preferably, the truncation mutant is an α-helical truncation mutant at the C-terminus of the heavy chain ferritin monomer subunit; preferably, the The truncation mutant is an epsilon helix truncation mutant at the C-terminus of the light chain ferritin monomer subunit.
6. The nanoparticle according to claim 5, which comprises at least one ferritin monomer subunit, preferably, the ferritin monomer subunit is selected from a heavy chain ferritin monomer subunit or a light chain ferritin monomer Body subunit; preferably, the heavy chain ferritin monomer subunit and/or the light chain ferritin monomer subunit form a nanoparticle, and more preferably, the nanoparticle contains 24 ferritin monomer subunits, The ratio of heavy chain ferritin monomer subunits to light chain ferritin monomer subunits is 0:24-24:0; preferably, the heavy chain ferritin monomer subunits are human heavy chain ferritin monomers Subunit; preferably, the light chain ferritin monomer subunit is a human light chain ferritin monomer subunit;

   Preferably, the heavy chain ferritin monomer subunit comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 5, 6, 7 or 8, preferably 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequence, more preferably 98% or more 99% identical amino acid sequence; more preferably, the amino acid sequence of the heavy chain ferritin subunit As shown in SEQ ID NO. 5, 6, 7 or 8; preferably, the fusion protein includes the combination with SEQ ID NO. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. Shown are amino acid sequences with 80% or more identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, and more preferably 98% or 99% identity. Amino acid sequence with more than% identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19;

   Preferably, the light chain ferritin subunit comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 20, 21, 22 or 23, preferably 85%, 90%, 95%, An amino acid sequence with 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; more preferably, the amino acid sequence of the light chain ferritin subunit is as SEQ ID NO. 20, 21, 22, or 23; preferably, the fusion protein includes the fusion protein with SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. Shown are amino acid sequences with 80% or more identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, and more preferably 98% or 99% identity. More preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.
7. A method for producing the RBD/ACE2 repressor peptide of claim 1, the fusion protein of claim 2, or the nanoparticle of any one of claims 3-6, the method comprising encoding the The RBD/ACE2 repressor peptide or one or more nucleic acid molecules of the fusion protein are introduced into cells, and cultured under conditions suitable for expressing the RBD/ACE2 repressor peptide, or expressing the fusion protein or forming the nanoparticle The cell.
8. A pharmaceutical composition comprising the RBD/ACE2 repressor peptide according to claim 1, the fusion protein according to claim 2, the nanoparticle according to any one of claims 3-6, or the nanoparticle according to claim 7 Nanoparticles produced by the method;

   Preferably, the pharmaceutical composition is a drug against coronaviruses of the coronavirus family; preferably, the viruses of the coronavirus family are selected from the group consisting of new coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, MERS-Cov, 229E , NL63, OC43 and HKU1;

   Preferably, the pharmaceutical composition further comprises another therapeutic agent; the another therapeutic agent is selected from immunotherapeutic agents or other drugs that inhibit viruses of the coronavirus family;

   Preferably, the virus of the Coronaviridae family is a novel coronavirus pneumonia virus (SARS-CoV-2); more preferably, the pharmaceutical composition is a drug for a novel coronavirus pneumonia virus (SARS-CoV-2).
9. A SARS-CoV-2 surface spike glycoprotein (S protein) antagonist, comprising the RBD/ACE2 repressor peptide according to claim 1, the fusion protein according to claim 2, and any one of claims 3-6 The nanoparticle according to item or the nanoparticle produced by the method according to claim 7, wherein the repressor peptide, fusion protein or nanoparticle functions by binding to SARS-CoV-2 surface spike glycoprotein (S protein).
10. A method for generating therapeutic drugs against viruses of the coronavirus family, the method comprising:

    a) Express the RBD/ACE2 repressor peptide of claim 1 or the fusion protein of claim 2, or form the nanoparticle of any one of claims 3-6 or the method produced by the method of claim 7 Nanoparticles; and

    b) Recover the RBD/ACE2 repressor peptide, fusion protein or nanoparticle.
11. A nucleic acid molecule comprising encoding the RBD/ACE2 repressor peptide of claim 1, the fusion protein of claim 2, the nanoparticle of any one of claims 3-6, or the method of claim 7 The nucleic acid sequence of the nanoparticle;

    Preferably, the nucleic acid sequence comprises a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO. 35, 36 or 37, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or more 99% identical nucleotide sequences; more preferably, the nucleic acid sequence is as SEQ ID NO. 35, As shown in 36 or 37;

    Preferably, the nucleic acid molecule is a codon-optimized nucleic acid molecule.
12. An expression construct comprising the nucleic acid molecule of claim 11.
13. A recombinant cell comprising the nucleic acid molecule of claim 11 or the expression construct of claim 12.
14. The RBD/ACE2 repressor peptide of claim 1, the fusion protein of claim 2, the nanoparticle of any one of claims 3-6, or the nanoparticle produced by the method of claim 7, claim The pharmaceutical composition according to claim 8, the SARS-CoV-2 surface spike glycoprotein (S protein) antagonist according to claim 9, or the therapeutic drug produced by the method according to claim 10, the nucleic acid molecule according to claim 11, Use of the expression construct of claim 12, the recombinant cell of claim 13 in the preparation of an S protein inhibitor, a competitive inhibitor of the binding of ACE2 and S protein, or in the preparation of prevention and/or treatment of coronaviruses Virus infection or use in medicine for diseases caused by the coronavirus infection; preferably, the disease caused by the coronavirus infection is a disease caused by a novel coronavirus infection, especially a novel coronavirus pneumonia.

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