Molecular assembly, single structures and polymeric structures containing same

Abstract

The present invention provides an assembly molecule, a single structure and a multimeric structure containing the same, as well as related products, manufacturing methods and uses, which can effectively improve the binding ability to a virus by forming a multimer that inhibits binding to a receptor through the collective action of the assembly molecule, and the assembly molecule is characterized in that it is an assembly structure formed by assembling a number of binding-inhibiting molecular units that inhibit the binding between a virus and a cellular receptor.

Description

[Technical field]

[0001] The present invention is in the field of biotechnology, and in particular relates to assembled molecules, mono- and multimeric structures comprising same, and products, methods of manufacture and uses related thereto. [Background technology]

[0002] Viral cellular receptors are the gateways through which viruses enter target cells, and by specifically binding to the virus, they can mediate the entry of the virus into susceptible host cells, initiate the viral replication process, and cause harm to the host. For example, The new coronavirus infects human cells through the human body's ACE2 receptor. For the new coronavirus, the ACE2 receptor on human cells is equivalent to a "doorknob," and by binding to it with the spike protein on the virus, it opens an entrance for infection into human cells.

[0003] In the study "Structural insights into hepatitis C virus receptor binding and entry" published in Nature, researchers from the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH) of the United States disclosed the mechanism of hepatitis C virus cell entry from a structural perspective, and clearly described the interaction process between HCV envelope glycoprotein E2 and CD81, through which HCV enters and infects human cells. Scientists found that under acidic conditions, HCV E2 easily binds to the CD81 receptor, and when the virus and the receptor start to interact, the shape of HCV E2 changes (after binding to CD81, residues 418-422 in HCV E2 move, which causes the inner ring consisting of residues 520-539 to extend), which allows the virus to come into closer contact with the cell membrane and facilitates cell entry.

[0004] Hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), need to bind to receptor molecules on the cell surface to infect host cells.

[0005] Rabies virus (RV) is a member of the genus Rabies virus in the family Rhabdoviridae. It has high neuropathicity and can cause fatal encephalitis. Currently, there is no effective treatment, and the mortality rate is nearly 100%. Rabies virus encodes five proteins, and G protein plays an important role in determining the host range, neuropathicity, immunogenicity, and interaction with receptor molecules on the surface of host cells. When RV invades the neuromuscular junction, nAchR in the postsynaptic membrane and NCAM in the presynaptic membrane play an important mediating role. Neurotrophin receptor P75 is also one of the receptors for rabies virus, and when the virus invades neuronal cells, P75 can bind to the G protein of rabies virus to allow the virus to enter the cytoplasm and transmit in the reverse direction.

[0006] Currently, these viruses are generally prevented and treated with polymeric antibodies or nano antibodies. For example, Regarding antibody-based drugs, there are currently several main methods for treating novel coronavirus pneumonia: 1. Neutralizing antibodies against the S protein, which bind to the S protein on the surface of the virus particle and inhibit the binding of the S protein to ACE2, thereby inhibiting the virus from entering cells; 2. Neutralizing antibodies against the ACE2 protein, which bind to the virus receptor ACE2, thereby inhibiting the virus from entering cells; 3. ACE2 analogs, which compete with ACE2 on the surface of lung cells and bind to the S protein on the surface of virus particles. It inhibits the binding of the virus to the receptor by combining with the antibody. 4. It is an antibody against the cytokine storm, and by suppressing the cytokine storm, it achieves the purpose of treating new coronavirus pneumonia.

[0007] However, the production costs of antibody drugs are much higher than those of small molecule drugs, and they need to rely on biological pharmaceutical methods to be produced, which consumes time and resources, making it difficult to compete with small molecule drugs.

[0008] In addition, currently available antibodies are generally monovalent or bivalent, and their ability to capture viruses is still relatively insufficient. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides an assembly molecule, a single structure and a multimeric structure containing the same, as well as related products, methods of manufacture and uses, which can effectively improve the binding ability to a virus by forming a multimer that inhibits binding to a receptor through the assembly action of the assembly molecule.

[0010] To this end, the present invention provides the following technical means. [Means for solving the problem]

[0011] The assembly molecule according to the present invention is characterized in that it forms an assembly structure by assembling a plurality of binding-inhibitory molecular units that inhibit the binding between a virus and a cell receptor.

[0012] The assembly molecule of the present invention is further characterized in that the assembly molecule has a single binding site and an assembly site single binding site, the single binding site is used to bind to the binding inhibitor molecular unit to form a single structure having at least the assembly molecule and the binding inhibitor molecular unit, and the multiple single structures are assembled into an assembly structure by the assembly site, and preferably, when the assembly molecule is a peptide or protein, the single binding site is located at the N-terminus of the assembly molecule.

[0013] The assembly molecule according to the present invention is further characterized in that the assembly structure is formed by assembling any one of 2 to 10 of the single structures.

[0014] The assembling molecule according to the present invention is further characterized in that the assembling molecule is selected from any of those in Table 1.

[0015] The assembly molecule of the present invention is further characterized in that the assembly molecule is a streptavidin, preferably comprising an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO:1.

[0016] According to the assembly molecule of the present invention, the assembly molecule and the binding inhibitor molecule unit are further bonded via a linker molecule, The linker molecule comprises one or more of a fluorescent protein, human immunoglobulin G4, Fc, and HSA, for example, the linker molecule is an eGFP fluorescent protein or is modified therefrom, preferably obtained by deleting some amino acids from eGFP, or The linker molecule comprises an amino acid sequence having identity to any one of SEQ ID NOs: 2 to 6, or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any one of SEQ ID NOs: 2 to 6, and is preferably and deleting the amino acids at positions 1 to 228 of the sequence, Preferably, the N-terminus of the linker molecule is linked to the binding inhibitor molecule unit, and the C-terminus of the linker molecule is linked to the N-terminus of the assembly molecule.

[0017] The assembly molecule of the present invention is further characterized in that the binding inhibitory molecular unit inhibits the binding of the virus to the cellular receptor by binding to the site where the virus binds to the receptor, and / or the binding inhibitory molecular unit inhibits the binding of the virus to the cellular receptor by binding to the cellular receptor.

[0018] The assembly molecule of the present invention is further characterized in that the binding-inhibitory molecule unit comprises at least one inhibitory molecule that inhibits binding between a virus and a cellular receptor, and preferably the inhibitory molecule comprises an amino acid sequence having identity to any of SEQ ID NOs: 7-9 and 16-19, or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any of SEQ ID NOs: 7-9 and 16-19, and further, when a plurality of inhibitory molecules are contained, the inhibitory molecules are linked in order from the N-terminus to the C-terminus, more preferably, when the binding-inhibitory molecule unit and the assembly molecule are linked via the linker molecule, the N-terminus of the linker molecule is linked to the C-terminus of the last inhibitory molecule, and the C-terminus of the linker molecule is linked to the single binding site, and further preferably, the inhibitory molecule is a nanobody.

[0019] The assembly molecule according to the present invention is further characterized in that the cellular receptor is ACE2 and preferably the inhibitory peptide is an ACE2 analogue.

[0020] According to the assembly molecule of the present invention, the binding inhibitor molecule unit further comprises at least one first inhibitor molecule and / or at least one second inhibitor molecule, the first inhibitor molecule comprises an amino acid sequence having identity to SEQ ID NO: 7 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity thereto, and the second inhibitor molecule comprises an amino acid sequence having identity to any of SEQ ID NO: 8 and 16 to 19 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity thereto. 9%, preferably the binding inhibitor molecule unit comprises two of the first inhibitor molecules or two of the second inhibitor molecules, more preferably the two first inhibitor molecules or the two second inhibitor molecules are linked to the C-terminus of the other via the N-terminus of one, even more preferably the binding inhibitor molecule unit comprises the first inhibitor molecule and the second inhibitor molecule, and the first inhibitor molecule and the second inhibitor peptide fragment are linked to the C-terminus of the other via the N-terminus of one, more preferably the N-terminus of the second inhibitor molecule is linked to the C-terminus of the first inhibitor molecule.

[0021] The assembly molecule of the present invention is further characterized in that the single structure further comprises a leader peptide facilitating the assembly structure to be obtained by cellular expression, by cellular secretion or by cell-free in vitro expression, the leader peptide comprising an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO:10, and preferably the leader peptide is linked via its C-terminus to the N-terminus of any of the inhibitory peptide fragments.

[0022] According to the assembly molecule of the present invention, the single structure further comprises the acidic structure, the acidic structure being a negatively charged short-chain amino acid polymer, and the acidic structure is further characterized in that: (1) the acidic structure is located at the C-terminus; (2) the number of amino acids in the short chain polymer is 0 to 50, 2 to 40, 3 to 30, 2 to 20, or 2 to 10; (3) the negatively charged amino acid is aspartic acid and / or glutamic acid; Preferably, the acidic structure is characterized in that it is linked to the C-terminus of the assembly molecule.

[0023] The assembly molecule of the present invention is further characterized in that the single structure further comprises a tag protein, the tag protein comprising an amino acid sequence having identity to SEQ ID NO: 15 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity thereto, preferably the tag protein is linked to the N-terminus of the binding inhibitory molecular unit via its C-terminus, and when the binding inhibitory molecular unit is an inhibitory peptide fragment, the C-terminus of the tag protein is linked to the N-terminus of any of the inhibitory peptide fragments, and more preferably when the single structure further comprises a leader peptide, the leader peptide is linked to the binding inhibitory molecular unit via the tag protein.

[0024] The assembly molecule of the present invention is further characterized in that the virus is one or more of the following: hepatitis B virus, rabies virus, HVP and novel coronavirus.

[0025] According to the present invention, there is further provided a single structure comprising the above-mentioned assembled molecule, preferably having a size of 30 to 80 KD, and characterized in that the single structure is the above-mentioned single structure.

[0026] According to the present invention, there is further provided an aggregate structure, characterized in that a plurality of the single structures described above are aggregated to form the aggregate structure described above.

[0027] The aggregate structure according to the present invention is further characterized in that the aggregate structure is formed by assembling at least four single structures, and preferably, the aggregate structure is formed by assembling four single structures.

[0028] The structure assembly according to the present invention is further characterized in that the binding strength of the inhibitory structural unit when it is an inhibitory polypeptide is 1000 to 1 M times that of the inhibitory structural unit when it is a nanobody.

[0029] The aggregate structure according to the present invention is further characterized in that it is soluble.

[0030] The aggregate structure according to the present invention is further characterized in that the thermal stability temperature of the aggregate structure is 45° C. or higher.

[0031] According to the present invention, there is further provided a nucleic acid encoding an assembly molecule, a single structure or an assembly structure, wherein the assembly molecule is an assembly molecule as described above, the single structure is an assembly structure as described above, and the assembly structure is an assembly structure as described above.

[0032] The present invention further provides a vector comprising the aforementioned nucleic acid.

[0033] According to the present invention there is further provided a eukaryotic host cell comprising the above-mentioned nucleic acid or the above-mentioned vector and for intracellular expression, cell-secreted or in vitro cell-free synthetic expression of the single structure or the assembled structure, wherein the single structure is the above-mentioned single structure and the assembled structure is the above-mentioned assembled structure.

[0034] According to the invention there is further provided the use of a nucleic acid as defined above or a vector as defined above in a method for the production of monomeric and / or multimeric structures.

[0035] According to the present invention, there is provided the use of any of the assembled molecules, the single structures and the assembled structures in virus treatment drugs, virus detection diagnostics, medical applications, disinfection products against viruses, cosmetics, skin care products, care products, food and cleaning products, wherein the assembled molecules are the assembled molecules as described above and the single structures are the single structures as described above, The assembled structure is the above-mentioned assembled structure, and preferably, the assembled structure is synthesized in a cell-free in vitro manner, and an unpurified product is used as is.

[0036] According to the present invention, there is further provided a disinfection product, a cosmetic product, a skin care product, a care product, a food product or a cleaning product, characterized in that it comprises one or more of an aggregate molecule, a single structure and an aggregate structure, wherein the aggregate molecule is the aggregate molecule described above, the single structure is the single structure described above, and the aggregate structure is the aggregate structure described above.

[0037] According to the present invention there is further provided a pharmaceutical product comprising one or more of an aggregate molecule, a single structure and an aggregate structure and a pharma- ceutically acceptable carrier, diluent or excipient, wherein the aggregate molecule is an aggregate molecule as defined above, the single structure is an aggregate structure as defined above and the aggregate structure is an aggregate structure as defined above.

[0038] According to the present invention, there is further provided a method for producing the aforementioned assembly structure obtained by cell-free in vitro synthesis and expression, characterized in that when the product containing the assembly structure obtained by the reaction is subjected to protein purification, an ammonium sulfate precipitation method is used. Effect of the Invention

[0039] (1) The molecular assembly of the present invention can effectively assemble multiple binding inhibitor molecular units that inhibit the binding between a virus and a cellular receptor into an assembly structure, thus realizing an inhibitory action method for inhibiting the binding between a virus and a cellular receptor. The number of binding inhibitor molecular units in each assembly structure can be increased through the assembly action of the molecular assembly, and the binding between a virus and a cellular receptor can be inhibited at multiple sites. At the same time, multiple binding inhibitor molecular units can bind to multiple viruses simultaneously. The multiple assembly structures work together to generate a cross-linking effect, which can more effectively inhibit the binding between a virus and a cellular receptor. Experiments have shown that the inhibitory effect against viruses is improved after assembly compared to before assembly, and the binding strength is 1000-1M times that of nanobody. Therefore, this method requires less binding molecules and has lower production costs than single-site binding.

[0040] (2) Furthermore, when the single structures containing at least four of the above-mentioned assembly molecules are assembled together by the respective assembly molecules to form an assembly structure, each assembly structure has at least four single structures and forms at least a tetrahedral structure, so that the cross-linking effect is more effectively generated and the above-mentioned inhibitory action can be further improved. Preferably, when the assembly structure is an assembly structure in which four single structures are assembled, the size of the assembly structure is appropriate compared to an assembly structure having more than four single structures, and sufficient inhibitory action ability is guaranteed, and there is no waste of spatial binding sites and space.

[0041] (3) Furthermore, in the streptavidin according to the present invention, the single structures can be assembled by assembly molecules, and 90% or more of the assembled structures are formed by assembling four single structures.

[0042] (5) Furthermore, the binding inhibitor molecule can inhibit the binding between a virus and a cellular receptor in the form of a peptide fragment, has a smaller overall size and a more simple structure as a single structure or aggregate structure compared to antibodies, and can be suitably obtained by intracellular expression, cell secretion, or in vitro expression.

[0043] (6) Furthermore, the inhibitory molecules are multiple and block the binding of the virus to the cell receptor at multiple sites. This can increase the chance of damaging the virus, and furthermore, the first inhibitory molecule and / or the second inhibitory molecule have a stronger inhibitory effect on the binding of the virus to the cell receptor.

[0044] (7) Furthermore, it is advantageous to produce said single structure or said assembled structure by a cellular expression, cellular secretion, or cell-free in vitro expression mode, when the single structure further comprises a leader peptide facilitating that the assembled structure is obtained by cellular expression, cellular secretion, or cell-free in vitro expression.

[0045] (8) Furthermore, when the binding inhibitory molecule unit and the assembly molecule are linked by a linker molecule which is a fluorescent protein or is modified with a fluorescent protein, the virus inhibitory effect can be improved, and the single structure further contains an acidic structure, and the presence of the acidic structure can improve the inhibitory effect against viruses and improve the thermal stability of the assembly structure. [Brief description of the drawings]

[0046] [Figure 1] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Diagram 2] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Diagram 3] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 4] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Diagram 5] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 6] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 7] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 8] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 9] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 10] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 11] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 12] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 13] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 14] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 15] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 16] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 17] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 18] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 19] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 20] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 21] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 22] FIG. 13 shows the results of an experiment on the inhibition of a virus-inhibitory protein against a pseudotype virus in Example 2. [Figure 23] Electron microscope photographs before and after the action of a virus inhibitory protein on a pseudotype virus in Example 2. [Figure 24] FIG. 2 is a perspective view of an assembly structure in the first embodiment. [Diagram 25] FIG. 1 is a perspective view of the cross-linking effect of the assembly structure in Example 1 on viruses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. For specific methods or materials used in the examples, those skilled in the art can make normal substitution selections according to existing techniques based on the technical ideas of the present invention, and are not limited to the specific descriptions of the examples of the present invention.

[0048] The methods used in the examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0049] In the present invention, the term "coronavirus" refers to a coronavirus that belongs to the genus Coronavirus in the family Coronaviridae, order Nidovirales in the phylogenetic classification, and has ACE2 as a binding receptor, including, but not limited to, SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0050] The spike protein, or S protein, is a target transmembrane protein on the surface of the virus. It has two subunits, S1 and S2, and the receptor binding site (RBD) is located in the S1 subunit. It constitutes the spike on the surface of the outer membrane of the virus particle in the form of a trimer, and its main function is to recognize the receptor on the surface of the host cell and mediate fusion with the host cell.

[0051] The ACE2 of the present invention is also called ACEH, angiotensin-converting enzyme 2. ACE2 is a type I transmembrane glycoprotein consisting of 805 amino acids and having a single extracellular catalytic domain. ACE2 is a receptor protein through which coronaviruses such as SARS-Cov-2 infect human cells.

[0052] In the present invention, in vitro cell-free expression, i.e., cell-free protein synthesis system, refers to a prokaryotic or eukaryotic cell-based coupled transcription-translation system for the synthesis of polypeptides or other macromolecules in a reaction mixture containing biological extracts and / or determined reagents.

[0053] The "D2P" system in the present invention includes, but is not limited to, an IVTT reaction (in vitro transcription translation reaction). In the present invention, an IVTT reaction is preferred. The IVTT reaction corresponds to the IVTT system and is a process of transcribing and translating DNA into protein in vitro, so that such an in vitro protein synthesis system is also called the D-to-P system, D_to_P system, or DNA-to-protein system, and the corresponding in vitro protein synthesis method is also called the D2P method, D-to-P method, D_to_P method, or DNA-to-protein method, and has the same meaning as expressions such as "in vitro cell-free protein synthesis system," "in vitro expression system," "in vitro protein synthesis system," "in vitro protein synthesis reaction system," and "cell-free protein synthesis system." Protein in vitro synthesis system, in vitro protein There are various ways of describing it, such as protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. It includes in vitro translation system and in vitro transcription translation system (IVTT system). In vitro protein synthesis system is also called "protein synthesis factory". In vitro protein synthesis reaction is a reaction that synthesizes protein in an in vitro cell-free synthesis system, and includes at least the translation process.

[0054] The protein components required for the in vitro cell-free protein synthesis system of the present invention (eg, RNA polymerase) may be provided in an endogenous manner or may be added in an exogenous manner. When provided in an endogenous manner, reference can be made to the conventional literature and the genetic recombination methods provided by the cited literature, including but not limited to CN108690139A, CN109423496A, CN106978439A, CN110408635A, CN110551700A, CN110093284A, CN110845622A, CN110938649A, CN111378708A, CN111484998A, "Molecular and Cellular Biology, 1990, 10(1):353-360" and the like, specifically including but not limited to inserting the coding sequence into a free plasmid in the cell, integrating the coding gene into the cell genome, and combinations thereof. When provided in an exogenous manner, the amount used can be controlled and adjusted according to the needs of the system.

[0055] Host cells are well known in the art and include, but are not limited to, E. coli, CHO cells, Chinese hamster ovary, NS0, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, HEK-293 cells and many other cell lines, Spodoptera frugiperda or Trichoplusiani, amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungal cells, such as, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataeaminuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia guercuum), Pichia piperi (Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei reesei, Chrysosporium lucknowense, Fusarium spp. p.), Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0056] Further, the in vitro cell-free protein synthesis system includes, but is not limited to, E. coli in vitro protein synthesis system, bacterial in vitro protein synthesis system, mammalian cell (e.g., HF9, Hela, CHO, HEK293) in vitro protein synthesis system, plant cell in vitro protein synthesis system, yeast cell in vitro protein synthesis system, and insect cell in vitro protein synthesis system. The yeast cell is selected from any one or more combinations of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces. The Kluyveromyces is, for example, Kluyveromyces lactis.

[0057] The technical elements of the present invention, such as the in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, and various detection methods, can be independently selected from the following documents for suitable embodiments or implementation methods: CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496 A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, CN110964736A, etc., to the extent not inconsistent with the purpose of the present invention, these documents and the references cited therein are incorporated herein in their entirety and for all purposes.

[0058] In the present invention, inhibiting the binding of a virus to a cell receptor refers to inactivating the virus, and may be, for example, in the following manner.

[0059] In one embodiment, the binding of the virus to the cellular receptor is reduced and there are two types:

[0060] The first type belongs to competitive inhibition, that is, by using receptor analogues to bind to the virus and reduce the chances of the virus binding to the receptor cells, or by using receptor-binding molecules to reduce the number of cell receptors that can bind to the virus and reduce the chances of the virus binding to the receptor cells; Type 2: By blocking the cross-linking reaction between different viruses mediated by binding inhibitor molecular units, the chances of the virus binding to the cell receptor are reduced.

[0061] In another embodiment, the inhibitory goal is achieved by using antibodies to disrupt the functional domain that binds to the cellular receptor.

[0062] As used herein, "assembling molecules" refers to molecules that are capable of assembling together, for example, by the action of intermolecular forces or self-aggregation of particular domains, etc.

[0063] The term "single structure" as used herein refers to a single structure before assembly of an assembly molecule, that is, a structure having at least a binding inhibitor molecular unit and an assembly molecule, and is also called a monomer.

[0064] In this specification, the term "aggregated structure" refers to a structure in which a plurality of single structures are aggregated by binding between the aggregate molecules, and is also called a polymer.

[0065] Example 1 The present embodiment is intended to specifically explain the contents of the present invention.

[0066] This embodiment provides an assembly molecule characterized by being formed into an assembly structure by assembling binding-inhibitory molecular units that inhibit the binding between a virus and a cellular receptor as described above. The binding-inhibitory molecular units here have binding molecules that bind to a cellular receptor or a virus, and therefore can competitively inhibit the binding between the virus and the cellular receptor.

[0067] For ease of explanation, the assembly molecule is denoted by A in this specification, and the binding inhibitor molecular unit is denoted by B in this specification.

[0068] Through the aggregation action of the aggregation molecules, multiple binding inhibitor molecular units are aggregated, increasing the number of binding inhibitor molecular units in each aggregation structure, i.e., increasing the number of competitive inhibition sites, thereby inhibiting the binding between the virus and the cellular receptor at multiple sites. At the same time, multiple binding inhibitor molecular units can bind to multiple viruses simultaneously, generating a cross-linking effect and more effectively inhibiting the binding between the virus and the cellular receptor.

[0069] Specifically, the assembly molecule of this example has a single binding site and an assembly site.

[0070] The single binding site is used to combine with the binding inhibitor molecular unit to form a single structure, and a plurality of single structures are assembled into an assembly structure by the assembly site, i.e., the assembly sites between different single structures have the function of being able to bind to each other, for example, by intermolecular forces, etc., to generate bonds such as self-assembly.Preferably, when the assembly molecule is a peptide or protein, the single binding site is located at the N-terminus of the assembly molecule, i.e., the C-terminus of the binding inhibitor molecular unit is connected to the N-terminus of the assembly molecule.Specifically, the basic structure of the single structure is shown in Formula 1.

[0071] BA (Formula 1): Preferably, the single binding site is located at the C-terminus of the assembled molecule.

[0072] Preferably, the assembly structure is formed by assembling at least four single structures. When single structures containing at least four of the above-mentioned assembly molecules are assembled together by their respective assembly molecules to form an assembly structure, each assembly structure has at least four single structures and forms at least a tetrahedral structure, so that the cross-linking effect is more effectively generated and the above-mentioned inhibitory action can be further improved. Preferably, when an assembly structure is formed by assembling four single structures, the size of the assembly structure is appropriate compared to an assembly structure having more than four single structures, and sufficient inhibitory action ability is ensured, and there is no waste of spatial binding sites and space.

[0073] FIG. 24 is a perspective view of the assembly structure in the first embodiment.

[0074] FIG. 25 is a perspective view of the cross-linking effect of the assembly structure in Example 1 on viruses.

[0075] As shown in Figure 24, the assembly structure in the figure is composed of three single structures, and the middle part is composed of assembly sites, with each binding inhibitor molecular unit being distributed at three points to form a tetrahedral structure.

[0076] As shown in FIG. 25, the aggregate structure in the figure is formed by assembling single structures, and each aggregate structure has two binding inhibitor molecules that bind to the same virus, and another binding inhibitor molecule that binds to another virus, so that the two viruses are bridged by the binding inhibitor molecules, which continuously inhibits the contact of the other virus with the cell receptor, and improves the inhibitory effect. It is possible.

[0077] In this embodiment, the assembling molecule is selected from any of Table 1. [Table 1]

[0078] In one example, the assembled molecule is streptavidin or comprises an amino acid sequence that is identical to, or has at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to, SEQ ID NO: 1. Such assembled molecules result in a tetrameric structure (composed of four individual structures assembled together) that is 80% or more, or even 90% or more identical.

[0079] In one example, the assembly molecule and the binding inhibitor molecular unit may be linked via a linker molecule, which for convenience of illustration is designated D.

[0080] In one example, the linker molecule includes any one or more of a fluorescent protein, human immunoglobulin G4, Fc, and HSA, for example, the linker molecule is an eGFP fluorescent protein or has been modified therewith, preferably, the modification is obtained by deleting some amino acids from eGFP, for example, the linker molecule is an EGFP fluorescent protein or has been modified therewith, preferably, obtained by deleting amino acids at positions 1 to 228 from eGFP.

[0081] Preferably, the linker molecule has an amino acid sequence that has identity, or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity, to SEQ ID NO:2. or an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQIDNO:3, or an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQIDNO:4, or an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQIDNO:5, or an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQIDNO:6.

[0082] In one example, the N-terminus of the linker molecule is linked to a binding inhibitor molecule unit and the C-terminus of the linker molecule is linked to a single binding site located at the N-terminus of the assembly molecule, in which case the structure of the single structure is shown in Formula 2.

[0083] BDA (Formula 2): Preferably, the C-terminus of B is linked to the N-terminus of D and the N-terminus of D is linked to the C-terminus of A.

[0084] In one example, the binding inhibitor molecule unit includes at least one inhibitor molecule that binds to the receptor binding region of the virus, i.e., it may include one inhibitor molecule or multiple inhibitor molecules, and when multiple inhibitor molecules are included, the amino acid sequences of these inhibitor molecules may be the same or different.

[0085] When there are multiple inhibitory molecules, the opportunities for inhibiting the binding of the virus to the cell receptor at multiple sites are increased, thereby improving the aforementioned inhibitory effect.

[0086] In one example, when there are multiple inhibitory molecules, the inhibitory molecules are linked in order from the C-terminus to the N-terminus, i.e., the previous C-terminus is linked to the next N-terminus, specifically, B1-B2-B3...Bn (n is a positive integer greater than 1); more preferably, when the binding inhibitory molecule unit and the assembly molecule are linked via a linker molecule, the N-terminus of the linker molecule is linked to the C-terminus of the last inhibitory molecule, and the C-terminus of linker molecule D is linked to the assembly molecule, specifically, B1-B2-B3...Bn-DA.

[0087] In one example, the inhibitor molecule comprises an amino acid sequence having identity to any of SEQ ID NOs: 7-9 and 16-19, or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any of SEQ ID NOs: 7-9 and 16-19. Each inhibitor molecule contained in the binding inhibitor molecule unit contains any one of several amino acid sequences shown in SEQ ID NOs: 7 to 9 and 16 to 19, or contains an amino acid sequence that has at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any one of SEQ ID NOs: 7 to 9 and 16 to 19.

[0088] For example, an inhibitory molecule includes an amino acid sequence having identity to SEQIDNO:7, and for example, an inhibitory molecule is an amino acid sequence having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQIDNO:7.

[0089] SEQ ID NO:16~19 SEQ ID NO:16(Pep170): STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLGGGGSGGGGSTSGVTGGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMA YAAQPFLLRN; SEQ ID NO:17(Pep180): STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALGGGGSGGGGSTSGGVTGFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRN; SEQ ID NO:18(Pep190): STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQGGGGSGGGGSTSGGVTGRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRN; SEQ ID NO:19(Pep200): STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQGGGGSGGGGSTSGGVTGGHAGGTVDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRN.

[0090] In one example, the inhibitor molecule may be a peptide fragment or an antibody. When an antibody is used, it is preferably a nanobody, which allows the overall size of the single structure to be reduced.

[0091] In the case of inhibition using an inhibitory peptide, the size of the whole structure is smaller and more simple than antibodies, and it is preferably obtained by intracellular expression, cell secretion, or in vitro expression. Specifically, when an inhibitory peptide fragment is used, the size of the whole simple structure is in the range of 30 to 80 KD.

[0092] When there are multiple inhibitory peptide fragments, the opportunities for inhibiting the binding of the virus to the cell receptor at multiple sites are increased, thereby improving the aforementioned inhibitory effect.

[0093] In one example, the cellular receptor is ACE2.

[0094] In one example, the binding inhibitor molecule unit comprises at least one first inhibitor molecule (first inhibitor peptide fragment, for convenience of explanation, designated as B1) and / or at least one second inhibitor molecule (second inhibitor peptide fragment, for convenience of explanation, designated as B2). The first inhibitor molecule comprises an amino acid sequence having identity to SEQ ID NO: 7 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity, and the second inhibitor molecule comprises an amino acid sequence having identity to any of SEQ ID NO: 8 and 16-19 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity. The first inhibitor molecule and / or the second inhibitor molecule are each a peptide fragment, and have a particularly excellent inhibitory effect on the binding between a virus and a cell receptor, compared to a nanobody.

[0095] In one example, the binding inhibitor molecule unit comprises two first inhibitor molecules or two second inhibitor molecules, more preferably, the two first inhibitor molecules or the two second inhibitor molecules are linked via the N-terminus of one to the C-terminus of the other, i.e., for example, B1-B1 or B2-B2; In one example, when the binding inhibitor molecule includes a first inhibitor molecule and a second inhibitor molecule, the first inhibitor molecule and the second inhibitor molecule are linked to the C-terminus of the other via the N-terminus of the other, i.e., the N-terminus of the first inhibitor molecule is linked to the C-terminus of the second inhibitor molecule, or the N-terminus of the second inhibitor molecule is linked to the C-terminus of the first inhibitor molecule. It is linked to the N-terminus of the harmful molecule.

[0096] Preferably, the N-terminus of the second inhibitory molecule is linked to the C-terminus of the second inhibitory molecule, in which case the structure of the single structure is shown, for example, in Formula 3 or Formula 4.

[0097] B1-B2-A (Formula 3): Preferably, the linking method of this structure is that the C-terminus of B1 is linked to the N-terminus of B2, and the C-terminus of B2 is linked to the N-terminus of A, i.e., the previous C-terminus is linked to the next N-terminus (and so on); B1-B2-DA (Formula 4): Preferably, the linking manner of this structure is such that the C-terminus of B1 is linked to the N-terminus of B2, the C-terminus of B2 is linked to the N-terminus of D, and the C-terminus of D is linked to the N-terminus of A.

[0098] In one example, the single structure further comprises a leader peptide (shown as C for convenience of illustration) that facilitates the assembly structure being obtained by cellular expression, by cellular secretion, or by cell-free in vitro expression, the leader peptide comprising an amino acid sequence having identity to SEQ ID NO:10 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity, in which case the leader peptide is added to the structure of the single structure in addition to the binding inhibitor molecular unit and the assembly molecule, and one structure of the single structure is shown, for example, in Formula 5.

[0099] CBA (Formula 5): Preferably, the C-terminus of C is linked to the N-terminus of B and the C-terminus of B is linked to the N-terminus of A.

[0100] When there are two inhibitory molecules, the leader peptide is preferably linked to the N-terminus of any one of the inhibitory molecules, in which case the structure of the single structure is, for example, as shown in Formula 6.

[0101] C-B1-B2-A (Formula 6): The linkage of this structural formula is preferably such that the C-terminus of C is linked to the N-terminus of B1, the C-terminus of B1 is linked to the N-terminus of B2, and the C-terminus of B2 is linked to the N-terminus of A.

[0102] When a linker molecule is present, the structure of the single structure is, for example, as shown in Formula 7.

[0103] C-B1-B2-DA (Formula 7): The linkage of this structural formula is preferably such that the previous C-terminus is linked to the next N-terminus, i.e., the C-terminus of C is linked to the N-terminus of B1, the C-terminus of B1 is linked to the N-terminus of B2, the C-terminus of B2 is linked to the N-terminus of D, and the C-terminus of D is linked to the N-terminus of A.

[0104] In one example, the acidic structure includes at least two consecutive aspartic acid amino acids (designated d for convenience of illustration), and / or at least two consecutive glutamic acid amino acids (designated e for convenience of illustration), and / or at least one pair of aspartic acid amino acids and a combination of amino acids, such as 10 consecutive aspartic acids (designated 10d), 10 consecutive glutamic acids (designated 10e), 8 consecutive aspartic acids and 8 consecutive glutamic acids (designated 8d8e), 5 de (5de, one d and one e per pair for a total of 5 pairs).

[0105] In one example, the stabilizing molecule is linked to the C-terminus of the assembly molecule, in which case the basic structure of the unitary molecule is shown in Formula 8.

[0106] BAE (Formula 7): wherein preferably the previous C-terminus is linked to the next N-terminus.

[0107] Based on this, any one or more of the above C and D can be combined to obtain different structural formulas, for example, as shown in Formula 9 to Formula 11.

[0108] CBAE (Formula 9): wherein preferably the previous C-terminus is linked to the next N-terminus; BDAE (Formula 10): wherein preferably the previous C-terminus is linked to the next N-terminus, CBDAE (Formula 11): wherein preferably the previous C-terminus is linked to the next N-terminus.

[0109] When B includes B1 and B2, it is represented by, for example, formula 12.

[0110] C-B1-B2-DAE (Formula 12): wherein preferably the previous C-terminus is linked to the subsequent N-terminus.

[0111] In one example, the single structure further comprises a tag protein (designated F for ease of illustration), which is used for purification of said single structure or said collective structure, and in one example, the tag protein comprises an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 15.

[0112] Preferably, the tag protein is linked to the N-terminus of the binding inhibitor molecular unit via its C-terminus, i.e., the C-terminus of the tag protein is on the N-terminal side of the binding inhibitor molecular unit, in which case the basic structure of the single structure is shown in Formula 13.

[0113] FBA (Formula 13): wherein preferably the previous C-terminus is linked to the next N-terminus.

[0114] Based on this, any one or more of the above C, D, and E can be combined to obtain different structural formulas, for example, as shown in Formula 14 to Formula 16.

[0115] FBDA (Formula 14): wherein preferably the previous C-terminus is linked to the next N-terminus.

[0116] CFBA (Formula 15): wherein preferably the previous C-terminus is linked to the next N-terminus.

[0117] FCBD (Formula 16): Preferably, the C-terminus of the previous one is linked to the N-terminus of the next one, in which the C-terminus of F is not directly linked to the N-terminus of B, but is linked via C, i.e., the C-terminus of F is located on one side of the N-terminus of B (others being similar).

[0118] In one example, when the single structure further comprises a leader peptide, the leader peptide is linked to the binding inhibitor molecular unit via a tag protein, as shown in Formula 17.

[0119] CFBDA (Formula 17): Preferably, the C-terminus of the previous molecule is linked to the N-terminus of the next molecule; similarly, in this case, the leader peptide is not linked directly to the binding inhibitor molecule, but is linked via a tag protein.

[0120] In the above different structural formulas, when the inhibitory peptide fragment is B1 and / or B2, B1 and B2 appear simultaneously, in which case, in the previous structural formula, the positions of B1 and B2 may be B1-B2 or B2-B1, or only one of these two may appear, in which case B is represented by B1 or B2.

[0121] In one example, the virus is any one or more of the following: hepatitis B virus, rabies virus, HVP, and novel coronavirus.

[0122] In one example, the size of a single structure according to this embodiment is 30-80 KD.

[0123] In one example, the binding strength of the inhibitory structural unit when it is an inhibitory polypeptide is 1000 to 1 M times that of the inhibitory structural unit when it is a nanobody or an antibody.

[0124] In one example, the aggregate structure is soluble, for example, dissolvable in an aqueous solution.

[0125] In one example, after the above-mentioned assembly molecules are assembled into a single structure, the thermal stability temperature of the assembly structure can be close to 70°C, and preferably is 80°C or higher.

[0126] This embodiment further provides a nucleic acid encoding said single structure or encoding said assembled structure.

[0127] This example further provides a vector comprising the above-described nucleic acid.

[0128] The present embodiment further provides a eukaryotic host cell comprising said nucleic acid or vector and used for the intracellular expression, cell secretory or in vitro cell-free synthetic expression of said single construct or said assembled construct.

[0129] The present embodiment further provides the use of said nucleic acid or said vector in a method for producing monomeric and / or multimeric structures.

[0130] Due to the inhibitory advantage of the aforementioned assembly molecules forming an assembly structure by assembling single structures, the present embodiment can better exert its inhibitory effect in the following uses or products, thereby better achieving the effects of preventing, treating or detecting a specific virus.

[0131] (1) The present embodiment further provides the use of any of the aforementioned assembled molecules, the aforementioned single structures, and the aforementioned assembled structures in viral therapeutics, viral detection diagnostics, medical applications, disinfection products against viruses, cosmetics, skin care products, care products, food, and cleaning products.

[0132] (2) This embodiment further provides a disinfection product, a cosmetic product, a skin care product, a care product, a food product, or a cleaning product, characterized in that it comprises any one or more of the above-mentioned aggregate molecule, the above-mentioned single structure, and the above-mentioned aggregate structure.

[0133] (3) This embodiment further provides a pharmaceutical product comprising one or more of the aggregated molecules, the single structures, and the aggregated structures, and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0134] The disinfectant product is used in places, food or places where disinfection is required, such as air disinfection, water disinfection, food disinfection, clothing disinfection, household utensils and various tools or facilities, where the tools are, for example, vehicles, and the facilities are, for example, bus stops; The skin care products include, for example, face creams, moisturizing sprays, lotions, and ice creams. It is a product that coats the skin surface of skin such as The above-mentioned cosmetics are, for example, liquid foundation, blush, etc. The care products are, for example, care liquids, care creams, sprays, etc. for surfaces susceptible to virus infection and auxiliary products, such as, for example, eye drops, care liquids for auxiliary products such as contact lenses, etc. The cleaning products include, for example, cleaners, shampoos, body washes, and liquid laundry detergents.

[0135] The assembled structures herein are also referred to as viral inhibitory proteins, inhibitory proteins.

[0136] Preferably, the inhibitory protein is produced by D2P technology of Kangma (Shanghai) Biotechnology Co., Ltd., for example, D2P system, D2P technology, for example, includes the step of optimizing the code of the gene and cloning it into pD2P vector. The plasmid is amplified using the Ampi system and then added to the protein factory rapid reaction system (Kangma (Shanghai) Biotechnology Co., Ltd.), the volume ratio is 1:30. Thus, the reaction mixture is incubated at 30 ° C for 4 hours, and then purified by centrifugation. The supernatant of the cell-free mixture and magnetic His Monster Beads (Kangma Healthcode (Shanghai) Biotech) were rotated for 1 hour at 4° C. Wash buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole) and elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole) were used.

[0137] The Ampi system contains final concentrations of 20-30 μM random primers, 0.05-0.15 μg / mL plasmid template, 0.5-1 mM dNTPs, 2x BSA, 0.05-0.1 mg / mL phi29 DNA polymerase, and 1x phi29 reaction buffer (components: 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5).

[0138] Example 2 In this embodiment, a specific experiment is carried out using the novel coronavirus as the virus and the cellular receptor as the ACE2 as an example to illustrate the inhibitory advantages of the ensemble of various structures.

[0139] 1. Preparation of virus-inhibiting proteins according to the experimental examples, IVTT reactions: The optimized gene sequences of the target proteins (including gene sequences encoding the various virus inhibitory protein structures mentioned above) were inserted into a plasmid and then added to a homemade Kluyveromyces lactis in vitro cell-free protein synthesis system, and the proteins were prepared in vitro by Kluyveromyces lactis NRRL Y-1140. The in vitro cell-free protein synthesis system used in this example (total volume 30 μL) contains 50% (v / v) Kluyveromyces lactis cell extract, 22 mM trishydroxymethylaminomethane (pH 8), 90 mM potassium acetate, 4.0 mM magnesium acetate, 3.0 mM nucleoside triphosphate mixture, 0.16 mM amino acid mixture, 22 mM potassium phosphate, 0.003 mg / mL amylase, 3% (w / v) polyethylene glycol (PEG-8000), 340 mM maltodextrin (weighed in glucose units, corresponding to approximately 55 mg / mL), 0.04 mg / mL exogenously added RNA polymerase, and 15 ng / μL target protein DNA, etc. If the number of types of fluorescent protein DNA is greater than one, the 15 ng / μL here is the total concentration of each fluorescent protein DNA. The above reaction system was placed in an environment of 22 to 30° C. and incubated stationary for approximately 20 hours.

[0140] After the IVTT reaction, the protein was purified using His magnetic beads (a product of Kangma Biology). After elution, the buffer was replaced with PBS buffer by ultrafiltration centrifugation. After filtering through a 0.22 μm syringe filter, the purified protein was obtained and stored at 4° C. for use.

[0141] 1. The structural formulas of the virus-inhibitory proteins prepared in each experiment of this embodiment are explained below. [Table 2] TIFF2025504316000004.tif242159TIFF2025504316000005.tif241157TIFF2025504316000006.tif66158

[0142] The sequence descriptions for Table 2 are shown in Table 3. [Table 3] TIFF2025504316000008.tif242157TIFF2025504316000009.tif57158

[0143] 2. Test pseudotyped viruses and cells in preliminary experiments The pseudotyped virus of the new coronavirus used expresses the S protein on its surface and has the luciferase gene packaged inside it. After infecting cells, the luciferase protein can be expressed in the cells, and the luminescence value of the luciferase substrate can be detected by adding the substrate, which can determine the cell infection efficiency of the pseudotyped virus.

[0144] First, a preliminary experiment was performed on the cell infection of the pseudotyped virus to determine the dilution of the pseudotyped virus. The pseudotyped virus was serially diluted with a gradient of 30 times (specifically, see Table 4), and infected with HEK293T or HEK293T-ACE2 (HEK293T cells overexpressing ACE2), respectively, and two parallel experiments were performed for each infection. Finally, it was found that when the virus infected the cells, the linear window of luciferase detection was within the range of S3 to S7 (270 to 21870 times dilution). Because the supplier of the pseudotyped virus suggested infecting the cells at a dilution of 540 times, the dilution of the pseudotyped virus used in the subsequent experiments was 540 times.

[0145] Infection experiments of wild-type novel coronavirus strains into wild-type and ACE2-overexpressing HEK293T cells revealed that the efficiency of viral infection in ACE2-overexpressing cells was significantly higher than that in the wild-type cell line. Therefore, in subsequent experiments, ACE2-overexpressing HEK293T was selected as the test cell line. [Table 4]

[0146] 3.Kd evaluation Surface Plasmon Resonance (SPR) Experiments SPR was performed using a Biacore T200 biosensor (GE Healthcare, USA). Virus inhibitory proteins were immobilized on an S-series sensor chip CM5 (GE Healthcare, USA) by amine coupling. The reference strain and Omicron mutant of His-tagged RBD (Sino Biological Inc, Beijing, China) were diluted to six different concentrations (0.25, 0.5, 1, 2, 4, and 8 nM), and the RBD of Delta mutant (Sino Biological Inc, Beijing, China) was diluted to (0.5, 1, 2, 4, 8, and 16 nM). Single cycle kinetic analysis was performed at 25 °C. The binding phase was set at 120 s and 30 μL / min, and the dissociation phase was set at 300 s and 30 μL / min-20, respectively, in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween as the running buffer. The analysis cycle was repeated nine times, and the analyte concentrations were arranged in ascending order. The dissociation constant (Kd) values ​​were calculated using Biacore T200 evaluation software (2.0 version, GE Healthcare, USA).

[0147] As can be seen from the results, the structure provided in Table 2 of the present invention is tested and has strong interaction with the RBD domain of all three virus strains, i.e., it has strong binding affinity with the original, Delta and Omicron mutants. The dissociation constant (Kd) values ​​of the original, Delta and Omicron mutants can reach 1.25nM, 0.837nM and 0.656nM, respectively.

[0148] In summary, Omicron mutants pose higher health risks and surprising immune evasion capabilities, but the virus-inhibiting proteins of the present invention still bind to the RBD domain of these mutants. Has the ability to bind.

[0149] 4. For the different structures provided in Table 2, viral inhibition experiments against pseudotyped viruses were carried out, and the experimental steps are as follows:

[0150] In step (1), HEK293T-ACE2 cells to be infected were inoculated into a 48-well cell culture plate. The inoculation amount was 1.5 × 10 per well. 4 When a pseudotype virus infection experiment was performed the next day, the cell density was approximately 30%.

[0151] In step (2), the following day, the SARS-CoV-2 (2019-nCoV) S protein pseudotyped virus was removed from -80°C and thawed on ice or allowed to thaw naturally at 4°C. After complete thawing, the virus was diluted 270-fold with complete medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin, 0.75 μg / mL puromycin) to prepare a pseudotyped virus dilution solution.

[0152] In step (3), the original concentration of the KMds inhibitor was 1 μM, and it was serially diluted in complete medium in a 10-fold gradient. The gradient-diluted KMds inhibitor and the pseudotype virus dilution were mixed in a 1:1 volume ratio to prepare a pseudotype virus infection solution, and the pseudotype virus infection solution was incubated at room temperature for 1 h.

[0153] In step (4), after 1 hour, the 48-well plate with HEK293T-ACE2 cells pre-plated was removed from the incubator, and the cell density and condition were checked, after which the upper medium was aspirated off. 200 μL of pseudotype virus infection solution was added to each well along the well wall to avoid washing away the cells. The 48-well plate was then placed in the incubator and incubated for 6 hours.

[0154] In step (5), after 6 hours, the pseudotype virus infection solution was carefully removed by aspiration, and 300 μL of fresh complete medium was replaced. Then, the cells were incubated in an incubator for 48 hours.

[0155] In step (6), after 48 hours, the medium was aspirated and 200 μL of 1× PBS was carefully added to each well to wash the cells, avoiding washing away the cells. The PBS was carefully aspirated and 65 μL of 1× cell lysis solution was added to each well and incubated at room temperature for 15-20 minutes. After 15-20 minutes, the luciferase activity was detected immediately or stored at -20°C.

[0156] In step (7), 5 μL of cell lysate was mixed with 5 μL of luciferase substrate (Promega E1501 luciferase assay system), the mixture was placed in a 384-well plate, and the luciferase activity was immediately detected using a Perkin Elmer EnVision 2102 multi-microplate reader to determine the inhibition efficiency of the KMds inhibitor.

[0157] The experimental results are shown in Figs.

[0158] The horizontal axis in the figure indicates the concentration of the virus-inhibitory protein used, and the vertical axis in the figure indicates the inhibitory effect (inhibition rate) after the inhibition experiment, and the inhibition rate is calculated as follows.

number

[0159] V0 is the control RLU reading where only virus was added, and V1 is the RLU reading for the sample where virus inhibitor protein and virus were added.

[0160] The higher the value on the vertical axis, the greater the effect.

[0161] Delta indicates inhibition test against pseudotyped virus of the Delta strain, Original indicates inhibition test against pseudotyped virus of the wild-type novel coronavirus, and Omicron indicates inhibition test against pseudotyped virus of the Omicron strain.

[0162] The histograms of each group in the figure correspond, from left to right, to the results of the structure names shown in the example figures or titles. For example, in Figure 4, the example figures are Kmds008, Kmds007, Kmds001, and Kmds009-2, from left to right, and the order of the histograms of each group also corresponds to the results of the corresponding structures in this order.

[0163] result: I. Pre- and post-assembly effects: As can be seen from Figures 1 to 3 , pre-aggregation (Kmds009) is less effective than post-aggregation (Kmds009-2), pre-aggregation Kmds042 is less effective than post-aggregation (Kmds003), and pre-aggregation Kmds043 is less effective than post-aggregation (Kmds012).

[0164] II. Inhibitory effects of different inhibitory molecules: Experiments have shown that the inhibitory molecules of the present invention have specific binding ability to the S protein of the new coronavirus, (1) As shown in Figure 4, the different inhibitory molecules can inhibit the new coronavirus and Delta at low concentrations. (2) As shown in FIG. 5, even when the assembly molecules and linker molecules in 052 are removed, the inhibitory effect is still observed at low concentrations.

[0165] III. Addition of acidic structures (1) The effect of adding an acidic structure is higher than that of not adding an acidic structure. As shown in Figure 6, when an acidic structure was added to Kmds003, Kmds002 and Kmds006, the effect was higher than that of Kmds001 without the acidic structure. (2) The addition of 8d8e and 10e is more effective than the absence of the addition. As shown in Figure 6, Kmds006 has a higher inhibitory effect on the virus than Kmds003 and Kmds002, and as shown in Figure 7, Kmds012 and Kmds014 are more effective than Kmds011 and Kmds013.

[0166] IV. Linker Molecules: (1) As shown in Figure 8, when different linker molecules were used, the effects of KMds104, KMds105, KMds121, and KMds112 were similar. (2) After partial modification of eGFP, KMds045, KMds046, and KMds109 were obtained after modification of KMds003, KMds012, and KMds104, respectively. The results are shown in Figures 9 to 11. The effects before and after modification are close to each other. (3) The linker molecule was deleted, and even after deletion, it could still exert an inhibitory effect on the virus. The results are shown in Figures 12 to 14. After the Tram of KMds003, KMds012, and KMds104 was deleted, KMds036, KMds038, and KMds108 were obtained, respectively.

[0167] V. Leader Peptide: KMds003, KMds006, KMds012, KMds014, KMds044 Based on this, the leading peptide sequence was deleted to obtain KMds030, KMds031, KMds032, KMds033, and KMds104, respectively. The results are shown in Figures 15 to 22, respectively. The results show that the leader peptide does not affect the inhibitory activity of the virus-inhibitory protein against the virus.

[0168] VI. Crosslinking action: In the virus inhibition experiment, the structure with the assembly molecule was observed under an electron microscope before and after treatment with the virus. As shown in Figure 23, A in Figure 23 is the one that was not treated with the virus inhibitory protein, and the others are the images at different magnifications after treatment with the virus inhibitory protein. As can be seen from the figure, after the virus was treated with the assembly molecule under the electron microscope, large plaques appeared in the image, that is, the structure of the present invention can realize cross-linking aggregation against the virus, thereby improving the cross-linking inhibition inhibitory effect against the virus.

[0169] VII. Pseudotyped Virus IC 50 result: As can be seen from the test results, in all tests, the same inhibitor molecule was With the acidic structure, assembly molecules and linker molecules, IC 50 The IC value reaches a minimum of 42.4 pM, reaching picomolar levels, and in the absence of acidic structures, 50 The value reaches about 0.222 nM. When the linker molecule is absent, or when the assembly molecule is absent, or when both the linker molecule and the assembly molecule are absent, the inhibitory effect against the pseudotype virus is relatively decreased, and the IC 50 The values ​​can reach 1.054, 2.404 and 1.032 nM, respectively.

[0170] 5. Live Virus Testing The inhibitory ability of the viral inhibitory proteins in Table 2 against different SARS-CoV-2 virus strains was studied, including the original virus strain, Alpha, Beta, Delta and Omicron mutants.

[0171] result: The original strain was strongly inhibited by the viral inhibitor protein (IC 50 is 108.6 pM), On the other hand, the virus-inhibiting protein is expressed at extremely low concentrations (IC 50 92.8, 121.9, 61.0 and 121.9 pM, respectively), showing effective inhibitory ability against Alpha, Beta, Delta and Omicron mutants, Compared to its ability to neutralize the original SARS-CoV-2, the ability of the viral inhibitor protein to inhibit the Delta mutant is increased by about 1.8-fold, while its ability to inhibit other strains is roughly the same.

[0172] Although a large number of mutations exist in the spike protein as seen from tests on live virus, the viral inhibitory protein of the present invention still retains potent picomolar inhibitory potency in different SARS-CoV-2 mutants.

[0173] 6. Animal Experiments The SARS-CoV-2 wild-type (WT) virus strain (IVCAS 6.7512) was provided by the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences. Heterozygous B6 / JGpt-H11<em>1Cin (K18-ACE2) / Gpt mice (K18-hACE2KI mice) were purchased from Nanjing GemPharmatech.

[0174] Mice were housed and bred in individually ventilated cages (IVC) in a specific pathogen-free (SPF) environment. Animal experiments were performed by certified personnel at the Animal Experiment Center of Wuhan University and approved by the Animal Care and Use Committee (AUP#WP2021-0602). Biosafety level 3. Protocols and procedures for transmissible SARS-CoV-2 virus in the laboratory were approved by the International Bioethics Committee (IBC, committee #S01322010A).

[0175] Inactivation of all samples was performed according to standard procedures approved by IBC and samples were used to draw high level controls. Each mouse was infected with 2.5×102 PFU SARS-CoV-2. For the negative control group, SARS-CoV-2 and control buffer were premixed for 30 min.

[0176] For the 0.25 nM viral inhibitor protein premixed treatment group, SARS-CoV-2 and 0.25 nM viral inhibitor protein were premixed for 30 minutes.

[0177] For the 25 nM viral inhibitor protein premixed treatment group, SARS-CoV-2 and 25 nM viral inhibitor protein were premixed for 30 minutes.

[0178] The premix was then used to inoculate K18-hACE2 mice via the intranasal route.

[0179] The tissues were weighed and homogenized in 1000 μL of PBS in a Tissue Cell-destroyer 1000 instrument (NZK LTD). The tissue homogenate was clarified by centrifugation at 5,000 rpm for 40 seconds, and viral RNA was extracted by mixing 100 μL of the supernatant with 400 μL of Trizol LS.

[0180] A SARS-CoV-2N gene standard curve was constructed using pCMV-N plasmid and SARS-CoV-2N gene primers (F primer: ATGCTGCAATCGTGCTACAA, R primer, GACTGCCGCCTCTGCTC) to calculate the viral copy number. Data were analyzed by Student's t test using Prism version 7 (GraphPad software). P value < 0.05 was considered statistically significant.

[0181] To measure the effect of the virus-inhibitory proteins in a mouse model, K18-hACE2 mice were infected with a premix of SARS-CoV-2 and virus-inhibitors. Mice were killed on days 2 and 5 post-infection (dpi). Mice were monitored daily for weight change and mortality within 5 days.

[0182] K18-hACE2 mice inoculated intranasally with SARS-CoV-2 began to lose weight 3–4 days after infection and died 4 dpi. Compared to control mice, mice inoculated with the virus-inhibitory protein and SARS-CoV-2 premix exhibited significantly reduced weight loss and significantly improved survival rates, especially when high doses of the virus-inhibitory protein were used.

[0183] High levels of SARS-CoV-2 RNA were detected in lung tissue from K18-hACE2 mice infected with SARS-CoV-2, but viral RNA levels were low in mice infected with SARS-CoV-2 and a low dose of the viral inhibitor protein mixture, and were not detected in lung tissue from mice infected with a high dose of the viral inhibitor protein and a SARS-CoV-2 premix. In summary, these data indicate that a high dose of the viral inhibitor protein can significantly reduce weight loss, improve mouse survival, and suppress viral copy number in mice.

[0184] 7. Toxicity Experiments The toxicity test was conducted in accordance with 2.3.1 Acute Oral Toxicity Test in the Disinfection Product Inspection Technical Standard (2002 edition), Part 2 of the Disinfection Technical Standard. The abnormal toxicity test was conducted in accordance with the Disinfection Product Inspection Technical Standard (2002 edition), Part 2 of the Disinfection Technical Standard. The study was conducted in accordance with the 2020 edition of the Chinese Pharmacopoeia (Part 4), General Rules, and Principles and Testing Methods for Biological Products.

[0185] ICR mice were provided by the Animal Center of Nanjing Medical University.

[0186] Sprague Dawley (SD) rats were purchased from Oriental Breeding Co., Ltd.

[0187] New Zealand White mice were provided by Yizheng Satomo Biotechnology Co., Ltd. Animals were housed at 20°C–26°C, with a relative humidity of 40%–70% in the local barrier system.

[0188] The following toxicity experiments were carried out for the structures according to Table 2.

[0189] (1) Acute oral toxicity Twenty SPF ICR mice (18.0-22.0 g) and 20 SPF SD rats (180-220 g) were used in the acute oral toxicity test. The number of males and females was equal. Mice and rats fasted overnight were administered with the virus inhibitor protein prepared according to the structural formula in Table 3 at a dose of 5000 mg / kg bw, administered in a single dose using gastric intubation. The animals were then monitored weekly for clinical symptoms of toxicity and mortality for 14 days (days 0, 7, and 14). Behavior, number of deaths, and body weight were evaluated, respectively, and dissected at the end of the observation period.

[0190] Test results showed that administration of the virus inhibitor protein at a dose of 5000 mg / kg bw did not cause illness in overnight fasted mice and rats, and body weights remained normal. The LD50 values ​​of the virus inhibitor protein for both mice and rats were above 5000 mg / kg bw. No signs of poisoning or death were observed during the observation period.

[0191] (2) Acute inhalation toxicity test Twenty SPF ICR mice (18.0-22.0 g) were used in the acute inhalation toxicity test. The number of males and females was equal. 2.2 g of the virus inhibitor protein correspondingly prepared in Table 3 was placed in a 220 L toxic substance exposure cabinet at a concentration of 10,000 mg / m 3 The inhalation exposure time was 2 hours. Symptoms and mortality of mice were recorded over a 14-day observation period (days 0, 7, and 14).

[0192] According to the experimental records, the LC50 value of the virus-inhibiting protein after 2 hours of exposure in both female and male mice was 10,000 mg / m 3 The mice showed no abnormal symptoms and their body weights increased steadily. Therefore, under the current experimental conditions, the virus-inhibiting protein is considered to be compliant with regulations and non-toxic.

[0193] (3) Acute eye irritation test Three male New Zealand White rabbits (2.5-3.5 kg) were used for the acute eye irritation test. 0.1 mL of the virus inhibitor protein stock solution prepared according to Table 3 was dropped into the conjunctival sac of the right eye of the rabbits, and saline was dropped into the left eye as a control. The eyes were closed for 4 s, and then washed with saline after 30 s. The rabbits' conjunctiva, iris, and cornea were observed for damage and recovery for 21 days (1 hour, 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, and 28 days). The severity of corneal damage, iris damage, conjunctival congestion, and chemosis were evaluated.

[0194] According to the test results, the virus inhibitory protein tested did not show any signs of eye irritation in rabbits. As shown in Table 1, the scores of the three rabbits at 24h, 48h and 72h were all less than 1. Therefore, the irritation of the virus inhibitory protein of the present invention is classified as non-irritating.

[0195] (4) Mouse bone marrow polychromatic erythrocyte (PCE) micronucleus test Fifty ICR mice were used for the mouse bone marrow polychromatic erythrocyte micronucleus test. The number of males and females was equal. The animals were divided into five groups, with five female mice and five male mice in each group. The test groups were administered once with the correspondingly prepared virus inhibitor proteins in Table 3 at doses of 5000, 2500, and 1250 mg / kg·bw, respectively. One group was treated with the solvent, purified water, as a negative control. The other group was intraperitoneally injected with 40 mg / kg·bw cyclophosphamide (CP) as a positive control. The test groups were exposed to the virus inhibitor proteins by oral administration at 0 and 24 hours. After two exposures to the virus inhibitor proteins, the mice were killed at 6 hours and bone marrow smears were prepared. The occurrence of micronuclei in 1000 polychromatic erythrocytes (PCE) from each animal was calculated. After 200 PCEs were counted, the ratio of PCEs to normochromic erythrocytes (NCEs) was determined. Statistical analysis was performed by U test. If the incidence of micronuclei formation in the experimental group significantly increased compared with the negative control, the test agent was judged to be harmful to chromosomes in vivo, and should be judged by the dose-response relationship.

[0196] As shown in Table 2, in the micronucleus test, the micronucleus formation rates of female and male mice in the three experimental groups were not significantly different from those in the negative group (P<0.05), and were significantly different from those in the positive control group (P<0.05). In addition, the difference in the PCE / NCE ratio between any of the experimental groups and the negative control was within 20%.

[0197] (5) Abnormal toxicity experiments Ten female SPF ICR mice (18.0-22.0 g) and four standard female guinea pigs (250-350 g) were used in the abnormal toxicity tests.

[0198] Ten mice were randomly divided into a test group and a control group, with five mice in each group. The test group was intraperitoneally injected with 0.5mL of the sample, and the control group was intraperitoneally injected with 0.5mL of sodium chloride. The two groups were observed for their constitutional conditions for 7 days. Four female guinea pigs were randomly divided into a test group and a control group, with two guinea pigs in each group. The test group was intraperitoneally injected with 0.5mL of the sample, and the control group was intraperitoneally injected with 0.5mL of sodium chloride. The two groups were observed for their constitutional conditions for 7 days.

[0199] In both tests, the virus-inhibiting protein of the present invention caused no observable toxic effects.

[0200] 8. Testing the Thermal Stability of Viral Proteins The thermal stability of several proteins was tested using the Uncle device from Unchained. Tmagg indicates the temperature at which a protein becomes highly aggregated during the heating process, and the results are shown in Table 5. [Table 5]

[0201] At the same time, the Tmagg value of Kmds001 containing B1+B2 is lower than 50 degrees, but after adding the 8D8E (Kmds006) acidic structure, the Tmagg value is significantly higher than that of the non-added one (Kmds001), increasing by almost 1-fold. That is, this acidic structure can enhance the stability of the protein.

[0202] Furthermore, when tested, the average Tm of the viral inhibitory proteins of each structure in Table 2 was close to 80° C., indicating that the viral inhibitory proteins of the present invention are not affected by extreme temperatures.

[0203] 9.Accelerated stability test Stability studies were performed simultaneously at two different temperatures, 4°C and 37°C (performed in triplicate, 100uL of 10nM viral inhibitor protein).

[0204] The other group was used as a negative control (NC) without adding the virus inhibitor protein. They were sampled three times every 15 days within 90 days (days 0, 15, 30, 45, 60, 75 and 90). The inhibition rate was evaluated by pseudotype virus neutralization test and calculated by the formula: inhibition rate = (NC-sample) / NC (RFU).

[0205] As can be seen from the test results, after storage at 4°C or 37°C for more than 90 days, the inhibition efficiency of the viral inhibitory protein was almost unchanged (>99.9%), indicating that the viral inhibitory protein of the present invention is an ultra-stable SARS-CoV-2 inhibitor.

[0206] The following examples are examples of film preparations, and the virus inhibitory proteins mentioned are virus inhibitory proteins produced by referring to the structures mentioned in the method of Example 1. The original solution concentration of the virus inhibitory proteins used in each compounding component is about 2 mg / ml. It is.

[0207] The moisture absorption test method in the following examples is as follows.

[0208] The device is a humidifier, model number KW-AD01, power 5W, and capacity 100ml.

[0209] The humidifier was filled with water, and the prepared film was placed 5 to 10 cm above the atomizing nozzle of the humidifier to observe the moisture absorption and change of the film. The experimental temperature was room temperature.

[0210] 10. IVTT reactions with the structures provided in Table 3 were subjected to ammonium sulfate precipitation and then tested for their ability to bind pseudotyped viruses.

[0211] The solution after the IVTT reaction was precipitated with ammonium sulfate: centrifuged at 4000 rpm, 4°C for 10 min. 5% (w / v) ammonium sulfate powder was added to the supernatant, thoroughly stirred to dissolve, and centrifuged at 4000 rpm, 4°C for 10 min. 25% (w / v) ammonium sulfate powder was added to the supernatant, gradually added, and stirred while adding to dissolve. Centrifuged at 12000 rpm, 4°C for 10 min. The supernatant was discarded, and the precipitate was washed once with 30% (w / v) ammonium sulfate solution. Centrifuged at 12000 rpm, 4°C for 10 min. The precipitate was resuspended in PBS and thoroughly dissolved. Centrifuged at 12000 rpm, 4°C for 10 min, filtered through a 0.22 μm syringe filter, and then stored at 4°C for use.

[0212] The activity of the sample after precipitation of the IVTT reaction solution of Kmds012 with ammonium sulfate is slightly lower than that of the pure protein of Kmd012 (protein purified by magnetic beads).

[0213] As can be seen, purification of the aggregated structures of the present invention can be achieved by a simple and convenient ammonium sulfate precipitation method.

Claims

1. An assembly molecule characterized by being formed by assembling multiple binding-inhibitory molecular units that inhibit the binding between a virus and a cellular receptor into an assembly structure.

2. the assembly molecule has a single binding site and an assembly site; The single binding site is used to bind to the binding inhibitor molecular unit to form a single structure having at least an assembly molecule and a binding inhibitor molecular unit, and a plurality of the single structures are assembled into an assembly structure by the assembly site; 2. The assembly molecule of claim 1, wherein the single binding site is preferably at the N-terminus of the assembly molecule when the assembly molecule is a peptide or protein.

3. 3. The assembly molecule according to claim 1, wherein the assembly structure is formed by assembling any one of the single structures in a number ranging from 2 to 10.

4. The assembly molecule of any one of claims 1 to 3, wherein the assembly molecule is selected from any one of Table 1.

5. the assembly molecule is streptavidin; 5. The assembly molecule of claim 4, wherein the streptavidin comprises an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO:

1.

6. the assembly molecule and the binding inhibitor molecule unit are linked via a linker molecule, The linker molecule includes any one or more of a fluorescent protein, human immunoglobulin G4, Fc, and HSA. For example, the linker molecule is an eGFP fluorescent protein or is modified therefrom, preferably obtained by deleting some amino acids from eGFP; or The assembly molecule according to claim 6, characterized in that the linker molecule comprises an amino acid sequence having identity to any of SEQ ID NOs: 2 to 6, or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any of SEQ ID NOs: 2 to 6, and is preferably obtained by deleting amino acids at positions 1 to 228 of eGFP, and preferably, the N-terminus of the linker molecule is linked to the binding inhibitor molecule unit and the C-terminus of the linker molecule is linked to the N-terminus of the assembly molecule.

7. the binding-inhibitory molecular entity inhibits binding of the virus to the cell receptor by binding to the site at which the virus binds to the receptor; and / or The assembly molecule according to any one of claims 1 to 6, characterized in that the binding-inhibitory molecular unit inhibits binding between the virus and the cell receptor by binding to the cell receptor.

8. The binding inhibitory molecule unit includes at least one inhibitory molecule that inhibits binding between a virus and a cell receptor, Preferably, the inhibitory molecule has identity to any one of SEQ ID NOs: 7-9 and 16-19, or has an amino acid sequence having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any one of SEQ ID NOs: 7-9 and 16-19. Furthermore, when a plurality of inhibitory molecules are included, the inhibitory molecules are linked in order from the N-terminus to the C-terminus. More preferably, when the binding inhibitory molecule unit and the assembly molecule are linked via the linker molecule, the N-terminus of the linker molecule is the C-terminus of the last inhibitory molecule. the C-terminus of the linker molecule is linked to the single binding site; More preferably, the assembly molecule according to any one of claims 1 to 7, characterized in that the inhibitory molecule is a nanobody.

9. The assembly molecule according to any one of claims 1 to 8, characterized in that the cell receptor is ACE2.

10. The binding inhibitor molecule unit comprises at least one first inhibitor molecule and / or at least one second inhibitor molecule, the first inhibitor molecule comprising an amino acid sequence having identity to SEQ ID NO: 7 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity, and the second inhibitor molecule comprising an amino acid sequence having identity to any of SEQ ID NO: 8 and 16-19 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any of SEQ ID NO: 8 and 16-19; Preferably, the binding inhibitor molecule unit includes two of the first inhibitor molecules or two of the second inhibitor molecules, more preferably, the two first inhibitor molecules or the two second inhibitor molecules are linked by the N-terminus of one to the C-terminus of the other, More preferably, the binding inhibitor molecule unit comprises the first inhibitor molecule and the second inhibitor molecule, and the first inhibitor molecule and the second inhibitor peptide fragment are linked to the C-terminus of the other via the N-terminus of one, and more preferably, the N-terminus of the second inhibitor molecule is linked to the C-terminus of the first inhibitor molecule. This is the assembly molecule described in claim 8 or 9.

11. The single structure further comprises a leader peptide, The assembly molecule of any one of claims 1 to 10, characterized in that the leader peptide comprises an amino acid sequence having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 10, and preferably the leader peptide is linked via its C-terminus to the N-terminus of any of the inhibitory peptide fragments.

12. The single structure further comprises the acidic structure, the acidic structure being a negatively charged short chain polymer of amino acids, and the acidic structure further comprises (1) The acidic structure is located at the C-terminus, (2) the number of amino acids in the short polymer is 0 to 50, 2 to 40, 3 to 30, 2 to 20, or 2 to 10; (3) The negatively charged amino acid is aspartic acid and / or glutamic acid; The assembly molecule according to any one of claims 1 to 11, characterized in that the acidic structure is preferably linked to the C-terminus of the assembly molecule.

13. The single structure further comprises a tag protein, the tag protein comprises an amino acid sequence having identity, or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity, to SEQ ID NO: 15; Preferably, the tag protein is linked to the N-terminus of the binding inhibitor molecular unit via its C-terminus, and when the binding inhibitor molecular unit is an inhibitory peptide fragment, the C-terminus of the tag protein is linked to the N-terminus of any of the inhibitory peptide fragments; More preferably, when the single structure further comprises the leader peptide, the leader peptide is linked to the binding inhibitor molecule unit via the tag protein. The assembly molecule according to any one of claims 1 to 12.

14. The viruses include hepatitis B virus, rabies virus, HVP and the new coronavirus. The assembly molecule according to any one of claims 1 to 13, characterized in that it is one or more of the following:

15. A single structure, The assembly molecule according to any one of claims 1 to 14, Preferably, the size of the single structure is 30-80 KD; Also preferably, the unitary structure is a unitary structure according to any one of claims 2 to 14.

16. 16. An assembly structure comprising a plurality of the single structures according to claim 15 assembled together.

17. The aggregate structure is formed by aggregating any one of the single structures in a quantity of 2 to 10, 17. The assembly structure according to claim 16, wherein the assembly structure is preferably formed by assembling four of the single structures.

18. The assembly structure according to claim 16 or 17, characterized in that the binding strength of the inhibitory structural unit is 1000 to 1 M times that of a nanobody.

19. The assembly structure of any one of claims 16 to 18, wherein the assembly structure is soluble.

20. A nucleic acid encoding an assembly molecule, a single structure or an assembly structure, The assembly molecule is an assembly molecule according to any one of claims 1 to 15, The unitary structure is a unitary structure according to claim 15, The assembly structure is a nucleic acid according to any one of claims 16 to 19.

21. A vector comprising the nucleic acid of claim 20.

22. 22. The nucleic acid according to claim 20 or the vector according to claim 21, which is used for intracellular expression, cell secretory or in vitro cell-free synthetic expression of a single or assembled structure, The unitary structure is a unitary structure according to claim 15, The assembly structure is a eukaryotic host cell according to any one of claims 16 to 19.

23. Use of a nucleic acid according to claim 20 or a vector according to claim 21 in a process for the production of a single structure according to claim 15 and / or a multimeric structure according to any one of claims 16 to 19.

24. Use of any of the assembled molecules, single structures and assembled structures in virus treatment drugs, virus detection diagnostics, medical applications, disinfection products against viruses, cosmetics, skin care products, care products, food and cleaning products, The assembly molecule is an assembly molecule according to any one of claims 1 to 14, The unitary structure is a unitary structure according to claim 15, The assembly structure is an assembly structure according to any one of claims 16 to 19, Preferably, the use is carried out by directly using an unpurified product obtained by cell-free in vitro synthesis of the assembly structure.

25. The polymer may include one or more of an aggregate molecule, a single structure, and an aggregate structure; The assembly molecule is an assembly molecule according to any one of claims 1 to 14, The unitary structure is a unitary structure according to claim 15, A disinfection product, a cosmetic product, a skin care product, a care product, a food product or a cleaning product, characterized in that the collection structure is a collection structure according to any one of claims 16 to 19.

26. one or more of an aggregate molecule, a single structure, and an aggregate structure, and a pharma- ceutically acceptable carrier, diluent, or excipient; The assembly molecule is an assembly molecule according to any one of claims 1 to 14, The unitary structure is a unitary structure according to claim 15, A drug, characterized in that the aggregate structure is the aggregate structure according to any one of claims 16 to 19.

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