WO2023126009A1 - 聚合分子、包括其的单一结构和多聚结构 - Google Patents

聚合分子、包括其的单一结构和多聚结构 Download PDF

Info

Publication number
WO2023126009A1
WO2023126009A1 PCT/CN2023/070015 CN2023070015W WO2023126009A1 WO 2023126009 A1 WO2023126009 A1 WO 2023126009A1 CN 2023070015 W CN2023070015 W CN 2023070015W WO 2023126009 A1 WO2023126009 A1 WO 2023126009A1
Authority
WO
WIPO (PCT)
Prior art keywords
blocking
molecule
polymeric
binding
virus
Prior art date
Application number
PCT/CN2023/070015
Other languages
English (en)
French (fr)
Inventor
郭敏
徐丽琼
刘章
张俊
伍志
于雪
Original Assignee
康码（上海）生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 康码（上海）生物科技有限公司 filed Critical 康码（上海）生物科技有限公司
Priority to KR1020247025893A priority Critical patent/KR20240128089A/ko
Publication of WO2023126009A1 publication Critical patent/WO2023126009A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a polymeric molecule, its single structure and multimeric structure, and related products, preparation methods and uses.
  • the cell receptor of the virus is the portal for the virus to invade the target cell. It can specifically bind to the virus, mediate the virus to invade the susceptible host cell and start its replication process, and cause harm to the host, such as:
  • the new coronavirus will use the human body's ACE2 receptor to infect human cells.
  • the receptor ACE2 on human cells is equivalent to a "door handle". The door of the cell.
  • HCV E2 under acidic conditions, HCV E2 easily binds to the CD81 receptor, and once the virus and the receptor begin to interact, HCV E2 changes shape (after binding to CD81, residues 418-422 in HCV E2 is displaced, which allows the extension of the inner loop consisting of residues 520-539), bringing the virus into tighter contact with the cell membrane, thereby facilitating its entry into the cell.
  • Hepatitis B virus (HBV) and its satellite virus hepatitis D virus (HDV) must bind to cell surface receptor molecules to infect host cells.
  • Rabies virus a member of the Rhabdoviridae genus Lyssavirus, is highly neurotropic and can cause fatal encephalitis. There is currently no effective treatment, and the fatality rate is almost 100%. Rabies virus encodes five proteins, among which G protein plays an important role in determining the host range of the virus, neurotropic virulence, immunogenicity of the virus, and interaction with host cell surface receptor molecules.
  • G protein plays an important role in determining the host range of the virus, neurotropic virulence, immunogenicity of the virus, and interaction with host cell surface receptor molecules.
  • nAchR located in the postsynaptic membrane and NCAM located in the presynaptic membrane play an important mediating role.
  • Neurotrophin receptor P75 is also a receptor of rabies virus. When the virus enters neuron cells, P75 can combine with rabies virus G protein to allow the virus to enter the cytoplasm for reverse transmission.
  • the invention provides a polymeric molecule, including its single structure and multimeric structure, as well as related products, preparation methods and uses, through the polymerization of polymeric molecules to form polymers that block the binding with receptors, so as to effectively improve the Ability to bind viruses.
  • the present invention provides the following technical solutions.
  • the present invention provides a polymeric molecule, which is characterized in that it is used to polymerize multiple blocking binding molecular units for blocking the binding between viruses and cell receptors into a polymeric structure.
  • the improved polymeric molecule of the present invention also has such a feature: wherein, the polymeric molecule has a single binding site and a single binding site of the polymeric site, and the single binding site is used to combine with the blocking binding molecular unit to form a polymeric molecule having at least As for the single structure of the block binding molecular unit, multiple single structures are aggregated into a polymer structure through the polymerization site.
  • the polymer molecule is a peptide or protein
  • the single binding site is set at the N-terminal of the polymer molecule.
  • the improved polymeric molecule of the present invention also has the following feature: wherein, the polymeric structure is formed by polymerizing any one of 2-10 of the single structures.
  • the improved polymer molecule of the present invention also has the following characteristics: wherein, the polymer molecule is selected from any item in Table 1.
  • the improved polymeric molecule of the present invention also has such a feature: wherein the polymeric molecule is streptavidin, preferably, the streptavidin comprises an identity with SEQ ID NO: 1 or at least 50%, 60% %, 70, 80%, 85%, 90%, 95% or 99% identical amino acid sequences.
  • the improved polymer molecule of the present invention also has the following characteristics: wherein, the polymer molecule is combined with the blocking binding molecular unit through a linker molecule,
  • the linker molecule contains any one or more of fluorescent proteins, human immunoglobulin G4, Fc, and HSA.
  • the linker molecule is eGFP fluorescent protein or obtained through its transformation, preferably, by modifying eGFP Deletion of some amino acids results in; or:
  • the linker molecule comprises any one of SEQ ID NO:2-6 having identity or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% of any one of SEQ ID NO:2-6.
  • the amino acid sequence with % identity is preferably obtained by deleting the 1-228th amino acid of eGFP;
  • the N-terminus of the linker molecule is linked to the blocking binding molecular unit, and the C-terminus of the linker molecule is linked to the N-terminus of the polymer molecule.
  • the improved polymeric molecule of the present invention also has such a feature: wherein, the blocking binding molecular unit blocks the binding between the virus and the receptor by binding to the site where the virus binds to the cell receptor, and/or blocks the binding of the binding molecule The unit blocks the binding between the virus and the cellular receptor by binding to the cellular receptor.
  • the improved polymeric molecule of the present invention also has such a feature: wherein, the blocking binding molecular unit contains at least one blocking molecule that blocks the binding between the virus and the cell receptor, preferably, the blocking The molecule contains any one of SEQ ID NO: 7-9 and 16-19, or at least 50%, 60%, 70, 80%, 85%, 90% of any of SEQ ID NO: 7-9 and 16-19 %, 95% or 99% identical amino acid sequence; further, when multiple blocking molecules are contained, the blocking molecules are sequentially connected from N-terminal to C-terminal, more preferably, when the blocking molecules When the binding molecular unit and the polymer molecule are connected through the linker molecule, the N-terminal of the linker molecule is connected with the C-terminal of the last blocking molecule, and the C-terminal of the linker molecule is connected with the single The binding site is linked, and preferably, the blocking molecule is a Nanobody.
  • the improved polymeric molecule of the present invention also has such a feature: wherein the cell receptor is ACE2, preferably, the blocking peptide is an ACE2 analogue.
  • the improved polymer molecule of the present invention also has the following characteristics: wherein, the blocking binding molecular unit contains at least one first blocking molecule and/or at least one second blocking molecule, and the first blocking molecular needle comprises An amino acid sequence having identity to SEQ ID NO: 7 or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity thereto, the second blocking molecule comprising an amino acid sequence identical to SEQ ID NO: 8 and any one of 16-19 has identity or has at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 8 and 16-19 Amino acid sequence, preferably, the blocking binding molecular unit contains two of the first blocking molecules or two of the second blocking molecules More preferably, between the two first blocking molecules or between two The second blocking molecules are connected through the N-terminal of one and the C-terminal of the other; preferably, the blocking binding molecular unit contains the first blocking molecule and the second blocking molecule The first blocking molecule is connected to
  • the improved polymeric molecule of the present invention also has such a feature: wherein, the single structure further contains a leader peptide for promoting the expression of the polymeric structure through cells, secreted by cells, or expressed without cells in vitro, and the leader peptide includes An amino acid sequence that is identical to SEQ ID NO: 10 or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identical to it, preferably, the leader peptide is blocked by any one of the C-terminal The N-terminus of the broken peptide is connected.
  • the improved polymer molecule of the present invention also has the following characteristics: wherein, the single structure also contains the acidic structure, the acidic structure is a negatively charged amino acid short-chain polymer, and further, the acidic structure has the following A combination of one or more of the characteristics:
  • the acidic structure is arranged at the C-terminus
  • the number of amino acids in the short-chain polymer is 0-50, 2-40, 3-30, 2-20 or 2-10;
  • the negatively charged amino acid is aspartic acid and/or glutamic acid
  • the acidic structure is linked to the C-terminus of the polymer molecule.
  • the improved polymeric molecule of the present invention also has such a feature: wherein, the single structure also contains a tag protein, and the tag protein includes a protein that is identical to SEQ ID NO: 15 or has at least 50%, 60%, 70, 80%, 85% of it. %, 90%, 95% or 99% identical amino acid sequence, preferably, the tag protein is connected between the C-terminal and the N-terminal of the blocking binding molecular unit, when the blocking binding molecular unit is a blocking peptide, The C-terminus of the tag protein is connected to the N-terminus of any blocking peptide; more preferably, when the single structure also contains a leader peptide, the leader peptide is connected between the tag protein and the blocking binding molecular unit.
  • the improved polymeric molecule of the present invention also has the feature that the virus is one or more of hepatitis B virus, rabies virus, HVP and novel coronavirus.
  • the present invention also provides a single structure, which is characterized in that it contains: the aforementioned polymer molecule, preferably, the size of the single structure is 30-80KD, and the single structure is the single structure mentioned above.
  • the present invention also provides a polymeric structure, which is characterized in that it is formed by polymerizing a plurality of the aforementioned single structures, and the polymeric structure is the aforementioned polymeric structure.
  • the polymer structure provided by the present invention also has the feature that the polymer structure is formed by the polymerization of at least four single structures, preferably, the polymer structure is formed by the polymerization of four single structures.
  • the polymeric structure provided by the present invention also has such a feature: wherein, the blocking structural unit is a blocking polypeptide whose binding force is 1000-1M times that of the nanobody.
  • the polymeric structure provided by the present invention also has the feature that the polymeric structure is soluble.
  • the polymeric structure provided by the present invention also has the feature that the thermal stability temperature of the polymeric structure is greater than or equal to 45°C.
  • the present invention also provides a nucleic acid encoding a polymeric molecule, a single structure or a polymeric structure, wherein the polymeric molecule is the aforementioned polymeric molecule; the single structure is the aforementioned single structure; and the polymeric structure is the aforementioned polymeric structure.
  • the present invention also provides a vector comprising the aforementioned nucleic acid.
  • the present invention also provides a eukaryotic host cell, which contains the aforementioned nucleic acid or the aforementioned vector, and is used for intracellular expression, cell secretion, or in vitro cell-free synthesis to express a single structure or a polymeric structure, wherein the single structure is the aforementioned single structure; the polymeric structure is the aforementioned polymeric structure.
  • the present invention also provides an application of the aforementioned nucleic acid or the aforementioned vector in a method for preparing a single structure and/or a multimeric structure.
  • the present invention also provides the application of any one of a polymeric molecule, a single structure and a polymeric structure in the following: medicines for treating viruses, virus detection and diagnosis, medical use, disinfection products for viruses, cosmetics, skin care products , care products, food, cleaning products, wherein the polymer molecule is the aforementioned polymer molecule; the single structure is the aforementioned single structure;
  • the polymeric structure is the aforementioned polymeric structure; preferably, the unpurified product obtained through cell-free in vitro synthesis of the polymeric structure is directly used for application.
  • the present invention also provides a disinfection product, cosmetics, cosmetics, skin care products, care products, food or cleaning products, characterized in that it includes: one or more of any one of polymeric molecules, single structures and polymeric structures , wherein, the polymer molecule is the aforementioned polymer molecule; the single structure is the aforementioned single structure; and the polymer structure is the aforementioned polymer structure.
  • the present invention also provides a medicine, which is characterized in that it includes: one or more of a polymeric molecule, a single structure and a polymeric structure, and a pharmaceutically acceptable carrier, diluent or excipient, wherein the polymeric molecule is the aforementioned polymeric molecule; the single structure is the aforementioned single structure; and the polymeric structure is the aforementioned polymeric structure.
  • the present invention also provides a method for preparing a polymeric structure, which is obtained through cell-free in vitro synthesis and expression, and is characterized in that ammonium sulfate precipitation is used for protein purification of the product including the polymeric structure obtained from the reaction, wherein the polymeric structure is the aforementioned aggregate structure.
  • the polymer molecule provided by the present invention can effectively polymerize a plurality of blocking binding molecular units that block the binding between the virus and the cell receptor into a polymer structure, so as to realize the blocking between the virus and the cell receptor.
  • the way of binding blocking action due to the polymerization of polymer molecules, increases the number of blocking binding molecular units of each polymer structure, which can block the combination between virus and cell receptors from multiple points, and at the same time due to multiple
  • a blocking binding molecular unit can bind multiple viruses at the same time, and the combination of multiple polymeric structures will produce cross-linking, which can more effectively block the binding between viruses and cell receptors.
  • each polymer structure has at least four single structures, at least a tetrahedral structure is formed, thereby being able to More effectively realize the cross-linking effect, further improve the above-mentioned blocking effect, and preferably, when it is a polymer structure with four single-structure polymers, compared with more than four polymer structures, the size of the polymer structure is appropriate, and sufficient Blocking ability without generating steric binding sites and wasting space;
  • the streptavidin provided by the present invention can allow a single structure to be polymerized by polymerizing molecules, and more than 90% of it is a polymeric structure formed by the polymerization of four single structures;
  • blocking binding molecules can block the binding between viruses and cell receptors in the form of peptides.
  • the size of the entire single structure or polymeric structure is smaller, and the structure is more simple , preferably obtained by in vivo cell expression, cell secretion or in vitro expression;
  • Cell-expressed, cell-secreted or in vitro cell-free expressed leader peptides are more conducive to the production of the aforementioned single structure or the aforementioned polymeric structure through cell expression, cell secretion or in vitro cell-free expression;
  • the virus blocking effect can be improved; further, the single structure also contains an acidic structure, and the acidic structure Existence, can improve the blocking effect on viruses, and can improve the thermal stability of the polymeric structure.
  • Fig. 1-Fig. 22 is the result figure of the inhibition experiment of the virus blocking protein involved in embodiment 2 to pseudovirus;
  • Fig. 23 is in embodiment 2, the electron micrograph before and after the effect of virus blocking protein on pseudovirus;
  • Figure 24 is a three-dimensional schematic diagram of a polymeric structure in Example 1;
  • Fig. 25 is a three-dimensional schematic diagram of the cross-linking effect of a polymeric structure in Example 1 on viruses.
  • Coronavirus in the present invention refers to a coronavirus that belongs to the genus Coronavirus (Coronavirus) of the order Nidovirales (Nidovirales) Coronaviridae (Coronavirus) in systematic classification, and uses ACE2 as the coronavirus binding receptor, including but Not limited to SARS-CoV, MERS-CoV, SARS-CoV-2, etc.
  • Spike protein also known as S protein, is a kind of iconic transmembrane protein on the surface of the virus. It has two subunits: S1 and S2, and the receptor binding site (RBD) is located on the S1 subunit. It forms the spikes 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 host cell surface receptors and mediate fusion with the host cells.
  • RBD receptor binding site
  • ACE2 of the present invention is also called ACEH, called angiotensin-converting enzyme 2.
  • ACE2 consists of 805 amino acids and is a type I transmembrane glycoprotein with a single extracellular catalytic domain.
  • ACE2 is a receptor protein for SARS-Cov-2 and other coronaviruses to infect human cells.
  • in vitro cell-free expression that is, a cell-free protein synthesis system
  • a transcription-translation coupling system based on prokaryotic or eukaryotic cells, and refers to a system containing biological extracts and/or defined reagents Synthesis of peptides or other macromolecules in reaction mixtures.
  • the "D2P" system in the present invention includes but not limited to IVTT reaction (in vitro transcription translation reaction).
  • IVTT reaction in vitro transcription translation reaction.
  • IVTT reaction is preferred.
  • IVTT reaction corresponding to the IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro.
  • in vitro protein synthesis system in vitro 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) and other description methods.
  • CFS system cell-free system
  • CFPS System cell-free protein synthesis system
  • ITT system in vitro transcription translation system
  • the in vitro protein synthesis reaction refers to the reaction of synthesizing protein in an in vitro cell-free synthesis system, at least including the translation process.
  • the protein components (for example, RNA polymerase) required in the in vitro cell-free protein synthesis system of the present invention can be provided by endogenous means or added by exogenous means.
  • endogenous means you can refer to documents including but not limited to CN108690139A, CN109423496A, CN106978439A, CN110408635A, CN110551700A, CN110093284A, CN110845622A, CN110938649A, CN111378708A, CN111484998A, "Molecular and Cellular Biology, 1990,10(1):353-360"
  • the genetic modification methods provided in the existing literatures and their references specifically include but are not limited to: inserting coding sequences into intracellular episomal plasmids, integrating coding genes into cellular genomes, and combinations thereof. When provided by external sources, the dosage can be controlled and adjusted according to the needs of the system.
  • Host cells are well known in the art, including but not limited to Escherichia coli, CHO cells, Chinese hamster ovary, NSO, SP2 cells, HeLa cells, small hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (eg, 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 including, for example, Pichia finlandica, Pichia trehalophila, Pichia koclamae ), Pichia membranaefaciens, Pichia minuta (Ogataeaminuta, Pichia lindneri), Pichia opuntiae, resistant Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanol Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.
  • Kluyveromyceslactis Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrellapatens, and Neurospora crassa ( Neurospora crassa).
  • in vitro cell-free protein synthesis system including but not limited to Escherichia coli in vitro protein synthesis system, bacterial in vitro protein synthesis system, mammalian cell (such as HF9, Hela, CHO, HEK293) in vitro protein synthesis system, plant cell in vitro protein synthesis system system, yeast cell in vitro protein synthesis system, and insect cell in vitro protein synthesis system.
  • the yeast cells are selected from the group consisting of any one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces. Kluyveromyces is, for example, Kluyveromyces lactis.
  • Embodiments or implementation methods including but not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN10 9423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN11055 1785A, CN110819647A, CN110845622A, CN110938649A,
  • the mentioned blocking of the binding between the virus and the cell receptor refers to the inactivation of the virus, for example, in the following ways:
  • the first type belongs to competitive blocking, that is, using an analogue to the receptor to bind to the virus, reducing the chance of the virus binding to the recipient cell; or reducing the ability to be bound by the virus through molecules that bind to the receptor cell receptors, which reduces the chance of the virus binding to recipient cells;
  • the second type through blocking the cross-linking effect formed by blocking the binding molecular unit between different viruses, reducing the chance of binding between the virus and the cell receptor;
  • Another way is to use antibodies to destroy the functional region that binds to cell receptors to achieve the purpose of blocking.
  • polymeric molecule herein refers to a molecule that can be aggregated together through, for example, intermolecular forces or self-association of specific domains.
  • single structure refers to a single structure before the polymerization of polymer molecules, that is, a structure with at least blocking molecular units and polymer molecules, also known as a monomer.
  • polymeric structure herein refers to the structure formed by the aggregation of multiple single structures through the combination of polymer molecules, also known as a polymer.
  • This embodiment provides a polymeric molecule, which is characterized in that: the blocking binding molecular unit used to block the binding between the virus and the cell receptor is polymerized into a polymeric structure.
  • the blocking binding molecular unit here has binding molecules that bind to cell receptors or viruses, so as to competitively block the binding of viruses to cell receptors.
  • polymer molecule herein is represented by A
  • blocking binding molecular unit herein is represented by B.
  • multiple blocking molecular units are polymerized, which increases the number of blocking binding molecular units of each polymerization structure, that is, increases the competitive blocking point, and can block from multiple points.
  • the combination between viruses and cell receptors can simultaneously bind multiple viruses due to multiple blocking binding molecular units, resulting in cross-linking, which can more effectively block the combination between viruses and cell receptors.
  • the polymer molecule of this embodiment has a single binding site and a polymer site
  • a single binding site is used to combine with the blocking binding molecular unit to form a single structure, and multiple single structures are aggregated into a polymeric structure through the polymerization site, that is, the polymerization site between different single structures has the function of being able to bind together, for example, through molecular Interaction forces, etc., occur in bonds such as self-association.
  • the polymeric molecule is a peptide or protein
  • the single binding site is set at the N-terminal of the polymeric molecule, that is, the C-terminal of the binding molecular unit is connected to the N-terminal of the polymeric molecule by blocking.
  • the basic structure of the single structure is as formula one:
  • the single binding site is set at the C-terminus of the polymer molecule.
  • the polymeric structure is formed by the polymerization of at least four single structures.
  • at least four single structures containing the aforementioned polymeric molecules are aggregated into a polymeric structure through their respective polymeric molecules, since each polymeric structure has at least four single structures, Form at least a tetrahedral structure, so that the cross-linking effect can be more effectively realized, and the above-mentioned blocking effect can be further improved, and preferably, when it is a polymeric structure with four single-structure aggregates, compared with a polymeric structure with more than four, the aggregated
  • the size of the structure is appropriate, and sufficient blocking ability is ensured, and no space binding sites and space waste are generated.
  • Figure 24 is a three-dimensional schematic diagram of a polymeric structure in Example 1;
  • 25 is a three-dimensional schematic diagram of the cross-linking effect of the polymeric structure on the virus in Example 1.
  • the polymerization structure in the figure is formed by the polymerization of three single structures, and the middle part is the aggregation of the polymerization site, and each blocking binding molecular unit is distributed at three points, forming a tetrahedral structure.
  • the polymeric structure in the figure is composed of a single single structure.
  • Each polymeric structure has two blocking binding molecules that bind to the same virus, and another blocking binding molecule that binds to other viruses. Therefore, the two By blocking the binding molecules to form cross-links between two viruses, it can continuously block the access of other viruses to cell receptors, thereby improving the blocking effect.
  • polymer molecule is selected from any item in Table 1:
  • the polymeric molecule is streptavidin, or contains an amino acid that is identical to or at least 50%, 60%, 70, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 Sexual amino acid sequence.
  • the tetrameric structure (polymerization of four single structures) produced by the polymerization can reach more than 80%, or even more than 90%.
  • the polymer molecule and the binding-blocking molecular unit can be additionally combined through a linker molecule, and for the convenience of illustration, the linker molecule is represented by D.
  • the linker molecule contains any one or more of fluorescent proteins, human immunoglobulin G4, Fc, and HSA.
  • the linker molecule is eGFP fluorescent protein or obtained through its transformation.
  • the transformation is Obtained by deleting part of amino acids of eGFP, such as EGFP fluorescent protein or obtained through its transformation, preferably, obtained by deleting amino acids 1-228 of eGFP. .
  • the linker molecule comprises an amino acid sequence identical to SEQ ID NO: 2 or at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identical thereto, or to SEQ ID NO: 3
  • the N-terminus of the linker molecule is connected to the blocking binding molecular unit, and the C-terminus of the linker molecule is connected to a single binding site of the polymer molecule set at the N-terminus.
  • the structure of the single structure is shown in Formula 2:
  • B-D-A (Formula 2): Preferably, the C-terminal of B is connected to the N-terminal of D, and the N-terminal of D is connected to the C-terminal of A.
  • the blocking binding molecule unit contains at least one blocking molecule that binds to the receptor binding region of the virus. That is, one blocking molecule may be included, or multiple blocking molecules may be included. When multiple blocking molecules are included, the amino acid sequences of these blocking molecules may be the same or different.
  • the chances of multi-point blocking the binding between the virus and the cell receptor can be increased, and the aforementioned blocking effect can be improved.
  • the blocking molecules are sequentially connected from the C-terminal to the N-terminal, that is, the C-terminal of the previous one is connected to the N-terminal of the next one, specifically B1-B2-B3 ...Bn (n is a positive integer greater than 1); more preferably, when blocking the connection between the binding molecular unit and the polymer molecule through a linking molecule, the N-terminal of the linking molecule is connected to the C-terminal of the last blocking molecule, The C-terminal of the connecting molecule D is connected to the polymer molecule, specifically B1-B2-B3...Bn-D-A.
  • the blocking molecule contains any one of SEQ ID NO:7-9 and 16-19 that is identical or at least 50%, 60%, 70, 80% identical to any of SEQ ID NO:7-9 and 16-19. %, 85%, 90%, 95% or 99% identical amino acid sequences.
  • the specific explanation is:
  • Each blocking molecule contained in the blocking binding molecular unit may contain any one of several amino acid sequences shown in SEQ ID NO: 7-9 and 16-19, or contain the same amino acid sequence as SEQ ID NO: 7-9 and 16- Amino acid sequences having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identity to any of 19:
  • the blocking molecule contains an amino acid sequence identical to SEQ ID NO: 7, and for example, the blocking molecule is at least 50%, 60%, 70, 80%, 85%, 90%, 95%, or 99% identical amino acid sequence.
  • the blocking molecule can be a peptide or an antibody.
  • antibodies preferably nanobodies
  • the size of the overall single structure can be reduced.
  • the size of the entire structure is smaller, and the structure is more simple, and it is more suitable to be obtained through cell expression in vivo, cell secretion or in vitro expression.
  • the size range of the entire single structure is 30-80KD.
  • the chance of multi-point blocking the binding between the virus and the cell receptor can be increased, and the aforementioned blocking effect can be improved.
  • the cellular receptor is ACE2.
  • the blocking binding molecule unit contains at least one first blocking molecule (first blocking peptide, denoted by B1 for convenience of illustration) and/or at least one second blocking molecule (second blocking peptide segment, for the convenience of explanation, denoted by B2).
  • the first blocking molecular needle comprises an amino acid sequence identical to SEQ ID NO: 7 or having at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identical thereto, said
  • the second blocking molecule comprises an amino acid that is at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identical to any of SEQ ID NO: 8 and 16-19 sequence.
  • the first blocking molecule and/or the second blocking molecule which are respectively peptides, have a particularly better blocking effect on the binding between viruses and cell receptors than nanobodies.
  • the blocking binding molecule unit contains two first blocking molecules or two second blocking molecules, and more preferably, there is a passage between the two first blocking molecules or between the two second blocking molecules.
  • the N-terminus of one is connected to the C-terminus of the other, i.e. for example B1-B1 or B2-B2;
  • the blocking binding molecule contains a first blocking molecule and a second blocking molecule
  • the first blocking molecule and the second blocking molecule are connected through the N-terminal of one and the C-terminal of the other, That is, either the N-terminus of the first blocking molecule is connected to the C-terminus of the second blocking molecule, or the N-terminus of the second blocking molecule is connected to the N-terminus of the first blocking molecule.
  • the N-terminal of the second blocking molecule is connected to the C-terminal of the second blocking molecule.
  • the structure of the single structure is, for example, shown in formula 3 or formula 4:
  • connection method of this structure is that the C-terminal of B1 is connected with the N-terminal of B2, and the C-terminal of B2 is connected with the N-terminal of A, that is, the C-terminal of the previous one and the N-terminal of A
  • N-terminal connection (the same below);
  • B1-B2-D-A (Formula 4): Preferably, the structure is connected in such a way that the C-terminal of B1 is connected to the N-terminal of B2, the C-terminal of B2 is connected to the N-terminal of D, and the C-terminal of D is connected to the N-terminal of A. end connection.
  • the single structure also contains a leader peptide (represented by C for convenience of description) for promoting the expression of the polymer structure by cells, cell secretion or in vitro cell-free expression.
  • the leader peptide includes the same sequence as SEQ ID NO: 10
  • a leader peptide is added, and an example of a single structure is shown in formula 5:
  • C-B-A (Formula 5): Preferably, the C-terminal of C is connected to the N-terminal of B, and the C-terminal of B is connected to the N-terminal of A.
  • the leader peptide is connected to the N-terminus of any blocking molecule.
  • the structure of the single structure is shown in formula 6, for example:
  • C-B1-B2-A (Formula 6): The connection of this structural formula, preferably, the C-terminal of C is connected with the N-terminal of B1, the C-terminal of B1 is connected with the N-terminal of B2, and the C-terminal of B2 is connected with the N-terminal of A end connection.
  • connection of this structural formula is preferably also that the C-terminal of the previous one is connected with the N-terminal of the latter, that is, the C-terminal of C is connected with the N-terminal of B1, and the C-terminal of B1 is connected
  • the C-terminal is connected to the N short chain of B2
  • the C-terminal of B2 is connected to the N-terminal of D
  • the C-terminal of D is connected to the N-terminal of A.
  • the acidic structure contains at least two consecutive aspartic acid amino acids (denoted by d for ease of illustration), and/or at least two consecutive amino acids of glutamic acid (denoted by e for ease of illustration), And/or at least one group of aspartic acid amino acids and combinations of amino acids.
  • an acidic structure contains 10 consecutive aspartic acids (denoted by 10d), 10 consecutive glutamic acids (denoted by 10e), 8 consecutive aspartic acids and 8 consecutive glutamic acids (denoted by 8d8e means), 5 d-e (5de, a d and an e are a group, a total of 5 groups).
  • the stabilizing molecule is connected to the C-terminus of the polymeric molecule.
  • the basic structure of the single structure is shown in formula 8:
  • the single structure also contains a tag protein (represented by F for convenience of illustration), and the tag protein is used for the aforementioned purified single structure or the aforementioned polymer structure.
  • the tag protein includes an or an amino acid sequence at least 50%, 60%, 70, 80%, 85%, 90%, 95% or 99% identical thereto.
  • the tag protein is connected to the N-terminal of the blocking binding molecular unit through the C-terminal, that is, the C-terminal of the tagging protein is on the N-terminal side of the blocking binding molecular unit.
  • the basic structure of the single structure is as shown in formula 10 Three shown:
  • F-B-A (Formula 13): In the formula, preferably, the C-terminal of the previous one is connected to the N-terminal of the latter.
  • F-C-B-D (Formula 16): In the formula, preferably, the C-terminal of the previous one is connected to the N-terminal of the latter. At this time, although the C-terminal of F is not directly connected to the N-terminal of B, it is connected through C, That is, the C-terminal of F is located on the side of the N-terminal of B (other similar explanations).
  • the leader peptide is connected between the tag protein and the blocking binding molecular unit, as shown in formula 17:
  • C-F-B-D-A (Formula 17):
  • the C-terminal of the previous one is connected to the N-terminal of the latter.
  • the leader peptide is not directly connected to the blocking binding molecule, but is connected through the tag protein.
  • B1 and B2 when the blocking peptides are B1 and/or B2, B1 and B2 appear at the same time.
  • the positions of B1 and B2 can be B1-B2 or B2-B1; Only any one of the two may appear, and B is represented by B1 or B2 at this time.
  • the aforementioned virus is any one or more of hepatitis B virus, rabies virus, HVP and novel coronavirus.
  • the size of the single structure involved in this embodiment is 30-80KD.
  • the blocking structural unit is a blocking polypeptide with a binding capacity 1000-1M times that of a nanobody or an antibody.
  • the polymeric structure is soluble, eg, in an aqueous solution.
  • the thermal stability temperature of the polymer structure can be close to 70°C, preferably greater than or equal to 80°C.
  • This embodiment also provides a nucleic acid encoding the aforementioned single structure or encoding the aforementioned polymeric structure.
  • This embodiment also provides a vector containing the aforementioned nucleic acid.
  • This embodiment also provides a eukaryotic host cell, which contains the aforementioned nucleic acid or vector, for intracellular expression, cell secretion, or in vitro cell-free synthesis to express the aforementioned single structure or the aforementioned polymeric structure.
  • This embodiment also provides an application of the aforementioned nucleic acid or the aforementioned vector in a method for preparing a single structure and/or a multimeric structure.
  • this embodiment can better play a blocking role in the following applications or products, so as to better achieve the prevention or treatment of specific viruses Or detection, etc.:
  • This embodiment also provides the application of any one of the aforementioned polymeric molecules, the aforementioned single structure, and the aforementioned polymeric structure in the following: medicines for treating viruses, virus detection and diagnosis, medical applications, and for targeting viruses Disinfection products, cosmetics, skin care products, care products, food, cleaning products.
  • This embodiment also provides a disinfection product, cosmetics, skin care products, care products, food or cleaning products, characterized in that it includes any of the aforementioned polymeric molecules, the aforementioned single structure, and the aforementioned polymeric structure. one or more of an item;
  • This embodiment also provides a medicine, including: one or more of the aforementioned polymeric molecules, the aforementioned single structure and the aforementioned polymeric structures, and a pharmaceutically acceptable carrier, diluent or excipient agent.
  • the above-mentioned disinfection products such as air cleaning and disinfection, water cleaning and disinfection, food disinfection, clothing disinfection, household utensils and various tools or facilities, etc., all places, practices or places that may need to be disinfected, tools such as vehicles, facilities such as bus stops ;
  • the above-mentioned skin care products such as face cream, spray water, toner, eye cream and other products that coat the skin surface;
  • care products are, for example, care solutions, care creams, sprays, etc. for surfaces or auxiliary products that are susceptible to virus infection, such as eye drops, auxiliary products such as contact lens care solutions, etc.;
  • the above-mentioned cleaning products such as detergent, shampoo, shower gel, laundry detergent, etc.
  • the polymeric structure herein is also referred to herein as virus blocking protein, blocking protein.
  • the blocking protein is produced by the D2P technology of Kangma (Shanghai) Biotechnology Co., Ltd., such as the D2P system, and the D2P technology is produced, such as including the following steps: the gene is coded and optimized and cloned into the pD2P vector.
  • the plasmid was amplified using the Ampi system, and then added to the Protein Factory Rapid Reaction System (Conma (Shanghai) Biotechnology Co., Ltd.), with a volume ratio of 1:30. This reaction mixture was incubated at 30 °C for 4 hours and then collected and purified by centrifugation.
  • the supernatant of the cell-free mixture was rotated with magnetic His Monster Beads (Kangma Healthcode (Shanghai) Biotech) at 4°C for 1 hr. with wash buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole) and with elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole).
  • wash buffer 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole
  • elution buffer 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole.
  • the Ampi system is: random primers at a final concentration of 20-30 ⁇ M, plasmid template at 0.05-0.15 ⁇ g/mL, dNTP at 0.5-1 mM, 2 ⁇ BSA, phi29 DNA polymerase at 0.05-0.1 mg/mL, 1 ⁇ phi29 reaction buffer Solution (composition is 50mM Tris-HCl, 10mM MgCl2, 10mM (NH4) 2SO4, 4mM DTT, pH7.5).
  • the virus is a new coronavirus and the cell receptor is ACE2 as an example to illustrate the blocking advantages of polymeric structures of various structures.
  • the optimized gene sequence of the target protein (including the gene sequence encoding the structure of the aforementioned various viral blocking proteins) was inserted into the plasmid, and then added to the self-made Kluyveromyces lactis in vitro cell-free protein synthesis system, with gram lactic acid Ruver yeast (Kluyveromyceslactis NRRL Y-1140) was prepared in vitro.
  • the in vitro cell-free protein synthesis system used in this example (total volume 30 ⁇ L): including Kluyveromyces lactis cell extract 50% (v/v), 22 mM tris(pH8), 90 mM potassium acetate, 4.0 mM Magnesium acetate, 3.0mM nucleoside tri-19 phosphate mixture, 0.16mM amino acid mixture, 22mM potassium phosphate, 0.003mg/mL amylase, 3% (w/v) polyethylene glycol (PEG-8000), 340mM maltodextrin ( Measured in glucose units, corresponding to about 55 mg/mL), 0.04 mg/mL exogenously added RNA polymerase, and 15 ng/ ⁇ L target protein DNA, etc.
  • 15ng/ ⁇ L is the total concentration of each fluorescent protein DNA.
  • the above reaction system was placed in an environment of 22-30° C., and incubated for about 20 hours.
  • His magnetic beads (comma biological product) were used for purification. After the protein was eluted, it was replaced by ultrafiltration and centrifugation into PBS buffer. After filtering with a 0.22 ⁇ m syringe filter, the purified protein was obtained and stored at 4°C until use.
  • the new coronavirus pseudovirus we used expressed the S protein on the surface and packaged the luciferase gene inside. After infecting cells, the luciferase protein can be expressed in the cells, and by adding its substrate and detecting the luminescence value of the luciferase substrate, the efficiency of pseudovirus infection of cells can be obtained.
  • a pre-experimental test is carried out on the cell infection of the pseudovirus to determine the dilution of the pseudovirus. Dilute the pseudovirus step by step in a 30-fold gradient (see Table 4 for details), and infect HEK293T or HEK293T-ACE2 (HEK293T cells overexpressing ACE2), respectively, and do two parallel experiments for each infection, and finally find that the virus infects the cells.
  • the linear window of luciferase detection is in the range of S3-S7 (diluted 270-21870 times).
  • the pseudovirus supplier recommended to infect the cells at a dilution of 540 times, so the dilution of the pseudovirus we used in the experiment was 540 times.
  • SPR Surface Plasmon Resonance
  • the binding phase was set at 120s and 30 ⁇ L/min, and the dissociation phase was set at 300s and 30 ⁇ L/min-20 as the running buffer.
  • Kd Dissociation constant
  • virus-blocking proteins of the present invention are still qualified to bind to the RBD domain of these variants.
  • the inoculum amount is 1.5 ⁇ 10 4 cells per well; when the pseudovirus infection experiment is carried out the next day, the cell density is about 30%.
  • the original concentration of KMds blocking agent was 1 ⁇ M, which was serially diluted with complete medium according to a 10-fold gradient. Mix the serially diluted KMds blocking agent and the pseudovirus diluent 1:1 to form a pseudovirus infection solution, and incubate the pseudovirus infection solution at room temperature for 1 hour.
  • the abscissa in the figure represents the concentration of the virus blocking protein used; the ordinate in the figure represents the inhibition effect (inhibition rate) after the blocking experiment, and the inhibition rate is calculated as follows:
  • V0 is the RLU reading of the control with only added virus
  • V1 is the RLU reading of the sample with added virus blocking protein and virus.
  • Delta means to conduct inhibition test against the Delta strain pseudovirus
  • Original means to conduct inhibition test against the new crown wild-type pseudovirus
  • Omicron to conduct inhibition test against the Omicron strain pseudovirus
  • Each group of column graphs in the figure corresponds to the result graph of the structure name displayed in the legend or title in order from left to right: for example, in Figure 4, the legends are Kmds008, Kmds007, Kmds001 and Kmds009-2 from left to right, Then the order of each group of histograms also corresponds to the results of the corresponding structure in this order.
  • the effect before polymerization is worse than that after polymerization (Kmds009-2); the effect of Kmds042 before polymerization is worse than that after polymerization (Kmds003); the effect of Kmds043 before polymerization is worse than that after polymerization (Kmds012);
  • the blocking effect of different blocking molecules, the experimental surface, the blocking molecule of the present invention has specific binding ability with the S protein of the new coronavirus:
  • Kmds006 has a better blocking effect on the virus than Kmds003 and Kmds002; as shown in Figure 7, Kmds012 and Kmds014 are better than Kmds011 and Kmds013;
  • the lowest IC50 value can reach 42.4pM, reaching the level of picomolar; without acidic structure, the IC50 value can reach about 0.222nM;
  • the original strain was strongly inhibited by the virus blocking protein (IC50 was 108.6pM);
  • virus blocking protein can show effective inhibitory ability to Alpha, Beta, Delta and Omicron variants at very low concentrations (IC50 are 92.8, 121.9, 61.0 and 121.9pM, respectively);
  • the ability of the virus blocking protein to inhibit the Delta variant increased by about 1.8 times, and the ability to inhibit other strains was almost the same.
  • the virus blocking protein of the present invention still maintains a strong inhibitory ability at the picomolar level in different SARS-CoV-2 variants.
  • the wild-type (WT) strain of SARS-CoV-2 (IVCAS 6.7512) was provided by the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences.
  • 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 Experimentation Center of Wuhan University, approved by the Institutional Animal Care and Use Committee (AUP#WP2021-0602). Protocols and procedures for infectious SARS-CoV-2 virus in animal biosafety level 3 laboratory facilities have been approved by the Institutional Biosafety Committee (IBC, Protocol #S01322010A).
  • SARS-CoV-2 was premixed with 0.25nM virus blocking protein for 30 minutes;
  • SARS-CoV-2 was premixed with 25nM virus blocking protein for 30 minutes;
  • K18-hACE2 mice were then inoculated with the premix by intranasal route.
  • Tissues were weighed and homogenized in 1000 ⁇ L PBS in a Tissue Cell-destroyer 1000 instrument (NZK LTD). The tissue homogenate was clarified by centrifugation at 5,000 rpm for 40 s, and 100 ⁇ L of the supernatant was mixed with 400 ⁇ L of Trizol LS to extract viral RNA.
  • mice were infected with a premix of SARS-CoV-2 and viral blocking. Mice were sacrificed on days 2 and 5 post-infection (dpi). Over the course of 5 days, mice were monitored for body weight change and mortality 326 days a day.
  • K18-hACE2 mice inoculated intranasally with SARS-CoV-2 began to lose weight 3 to 4 days after infection and died at 4 dpi;
  • mice vaccinated with the virus-blocking protein and SARS-CoV-2 premix experienced significant weight loss and significantly improved survival compared with control mice, especially when high doses of the virus-blocking protein were used.
  • ICR mice were provided by the Animal Center of Nanjing Medical University.
  • Sprague Dawley (SD) rats were purchased from Pizhou Dongfang Breeding Co., Ltd.
  • New Zealand white rabbits were provided by Yizheng Anlimao Biotechnology Co., Ltd. The animals are kept at 20°C-26°C, and the relative humidity in the local barrier system is 40%-70%.
  • SPF ICR mice (18.0-22.0g) and 20 SPF SD rats (180-220g) were used for acute oral toxicity test. There are equal numbers of males and females.
  • the virus-blocking protein prepared corresponding to the structural formula in Table 3 was administered to mice and rats fasted overnight at a dose of 5000 mg/kg ⁇ bw, and a single dose was used by gastric intubation. Animals were then monitored weekly for clinical signs of toxicity and mortality for 14 days (Day 0, Day 7 and Day 14). Behavior, mortality and body weight were assessed separately and they were dissected at the end of the observation period.
  • mice Twenty SPF ICR mice (18.0-22.0g) were used for acute inhalation toxicity test. There are equal numbers of males and females. Put 2.2g of the correspondingly prepared virus blocking protein in Table 3 into a 220L poison exposure cabinet, and the concentration is assumed to be 10,000mg/m3. The inhalation exposure time was set at 2 hours. Symptoms and deaths of mice were recorded during 14 days of observation (0 days, 7 days and 14 days).
  • the LC50 value of virus blocking protein under 2h exposure is higher than 10,000mg/m3.
  • the mice showed no signs of abnormality, and a steady weight gain was normal. Therefore, under the current experimental conditions, the virus blocking protein complies with regulations and is considered non-toxic.
  • Three male New Zealand rabbits (2.5-3.5kg) were used for the acute eye irritation test.
  • 0.1 mL of the virus blocking protein stock solution prepared correspondingly in Table 3 was instilled into the conjunctival sac of the right eye of the rabbit, and physiological saline was instilled into the left eye as a control. Close the eyes for 4 seconds, and rinse with normal saline after 30 seconds.
  • the injury and recovery of the rabbit's conjunctiva, iris and cornea were observed for 21 days (1 hour, 24 hours, 48 hours, 72 hours, 7 days, 14 days and 28 days). The severity of corneal injury, iris injury, conjunctival hyperemia, and conjunctival edema was scored.
  • the tested virus-blocking proteins showed no signs of eye irritation in rabbits.
  • the scores of the three rabbits at 24h, 48h and 72h were all less than 1, as shown in Table 1. Accordingly, the irritative properties of the virus blocking proteins of the present invention are classified as non-irritative.
  • ICR mice Fifty ICR mice were used for the micronucleus test of polychromatic erythrocytes in mouse bone marrow. The number of males is equal to the number of females. Animals were divided into five groups of five female mice and five male mice each. The test group was given the corresponding virus blocking protein prepared in Table 3 once at doses of 5000, 2500 and 1250 mg/kg ⁇ bw. One set served as a negative control and was treated with solvent-purified water. The other group was used as a positive control, and 40 mg/kg ⁇ bw cyclophosphamide (CP) was intraperitoneally injected. The test group was exposed to the virus-blocking protein toxin by oral administration at 0 hours and 24 hours.
  • CP cyclophosphamide
  • mice were sacrificed 6 hours after the second exposure to the virus blocking protein, and bone marrow smears were prepared. The occurrence of micronuclei in 1000 polychromatic erythrocytes (PCE) per animal was counted. Once 200 PCEs were counted, the ratio of PCE to normochromic erythrocytes (NCE) was determined. Statistical analysis was performed by U test. When the incidence of micronucleus formation in the experimental group is significantly increased compared with the negative control, it is judged that the test agent is harmful to chromosomes in vivo, which should occur in a dose-response relationship.
  • PCE polychromatic erythrocytes
  • NCE normochromic erythrocytes
  • the micronucleus formation rates of female and male mice had no significant difference among the three experimental groups compared with the negative group (P ⁇ 0.05), but significantly different (P ⁇ 0.05) compared with the positive control group , as shown in Table 2.
  • the difference in PCE/NCE ratio between any experimental group and the negative control was within 20%.
  • mice were randomly divided into a test group and a control group, with 5 mice in each group.
  • the test group was intraperitoneally injected with 0.5mL sample, and the control group was intraperitoneally injected with 0.5mL sodium chloride.
  • the physical condition of the two groups was observed for 7 days.
  • Four female guinea pigs were randomly divided into a test group and a control group, with 2 guinea pigs in each group.
  • the test group was intraperitoneally injected with 0.5mL sample, and the control group was intraperitoneally injected with 0.5mL sodium chloride.
  • the physical condition of the two groups was observed for 7 days.
  • virus blocking proteins provided by the present invention did not cause observable toxic effects.
  • Tmagg characterizes the temperature at which the protein undergoes high aggregation during heating, and the results are shown in Table 5.
  • the Tmagg value of Kmds001 containing B1+B2 at the same time is lower than 50 degrees, but after adding 8D8E (Kmds006) acidic structure, the Tmagg value is much higher than that of Kmds001 without tail (Kmds001), which is nearly doubled. In other words, this acidic structure can increase the stability of the protein.
  • the average Tm of the virus blocking protein of each structure in Table 2 is close to 80°C, indicating that the virus blocking protein provided by the present invention can be protected from extreme temperatures.
  • the test results show that after storage at 4°C or 37°C for more than 90 days, the inhibition efficiency of the virus blocking protein is hardly damaged (>99.9%), indicating that the virus blocking protein of the present invention is an ultra-stable SARS-CoV -2 blocker.
  • the following examples are film examples, wherein the virus blocking protein involved is the virus blocking protein prepared by referring to the various structures involved in the method in Example 1, wherein the virus blocking protein used in each formula
  • the stock solution concentration of protein is about 2mg/ml.
  • the moisture absorption test method in the following examples is as follows:
  • the humidifier is filled with water, and the prepared film is placed 5-10 cm above the atomization nozzle hole of the humidifier, and the moisture absorption of the film is observed, and the test temperature is room temperature.
  • sample activity of the IVTT reaction solution of Kmds012 after ammonium sulfate precipitation is slightly lower than that of Kmd012 pure protein (magnetic bead purified protein).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Inorganic Chemistry (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Pest Control & Pesticides (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • Pulmonology (AREA)
  • Microbiology (AREA)
  • Nutrition Science (AREA)
  • Dermatology (AREA)

Abstract

本发明提供一种聚合分子、包括其的单一结构和多聚结构、以及相关的产品、制备方法和用途，通过聚合分子的聚合作用形成阻断与受体结合的多聚体，以能有效提高对病毒的结合能力，其中的聚合分子，其特征在于：用于将对病毒与细胞受体之间的结合进行阻断的多个阻断结合分子单元聚合为聚合结构。

Description

聚合分子、包括其的单一结构和多聚结构 技术领域
本发明属于生物技术领域，具体涉及一种聚合分子、包括其的单一结构和多聚结构、以及相关的产品、制备方法和用途。
背景技术
病毒的细胞受体是病毒侵入靶细胞的门户,能与病毒特异性的结合,介导病毒入侵易感宿主细胞而启动其复制过程，而对宿主造成危害，例如：
新冠病毒会利用人体的ACE2受体感染人类细胞，对于新冠病毒来说，人体细胞上的受体ACE2相当于一个“门把手”，通过病毒上的刺突蛋白会与之结合，从而打开感染人体细胞的大门。
发表在《Nature》上的一项题为“Structural insights into hepatitis C virus receptor binding and entry”的研究中，来自美国国家卫生研究院下属的国家过敏与传染病研究所(NIAID)的研究人员从结构上揭示丙型肝炎病毒进入细胞的机制，清晰的描述了HCV包膜糖蛋白E2和CD81之间的相互作用过程既HCV进入并感染人类细胞的过程。科学家发现，在酸性条件下，HCV E2很容易与CD81受体结合，而一旦病毒和受体开始相互作用，HCV E2就会改变形状(在与CD81结合后，HCV E2中的残基418-422被移位，这使得由残基520-539组成的内环得以延伸)，使病毒与细胞膜更紧密地接触，从而促进其进入细胞。
乙型肝炎病毒(HBV)及其卫星病毒丁型肝炎病毒(HDV)必须通过结合细胞表面受体分子，才能实现对宿主细胞的感染。
狂犬病病毒(Rabies Virus，RV)是弹状病毒科狂犬病毒属的成员,具有高度的嗜神经性并且能引起致命的脑炎。目前没有有效的治疗方法,病死率几乎100％。狂犬病病毒编码五种蛋白，其中G蛋白在决定病毒的宿主范围、嗜神经毒力、病毒的免疫原性及与宿主细胞表面受体分子的相互作用等方面起着重要的作用。RV在侵入神经肌肉接头时,位于突触后膜的nAchR和位于突触前膜的NCAM起到重要的介导作用。神经营养素受体P75同样作为狂犬病病毒的一个受体，当病毒进入神经元细胞后，P75可与狂犬病病毒G蛋白结合从而使病毒进入细胞浆内进行逆向传递。
目前的针对这些病毒，一般是通过大分子抗体或纳米抗体等类型进行防治，例如：
对于抗体类药物，目前治疗新冠肺炎主要有以下几种策略：1、针对S蛋白的中和抗体，结合病毒颗粒表的S蛋白，阻断S蛋白与ACE2的结合，从而阻断病毒进入细胞。2、针对ACE2蛋白的中和抗体，通过结合病毒的受体ACE2，阻断病毒进入细胞。3、ACE2类似物，与肺细胞表面ACE2竞争结合病毒颗粒表面的S蛋白，阻断病毒与受体结合。4、针对细胞因子风暴的抗体，通过抑制细胞因子风暴，从而达到治疗新冠肺炎的目的。
但抗体药物的生产成本比小分子药物要高很多，需要依赖生物制药方法生成，耗时耗材，所以还难以与小分子药物进行竞争。
另外，目前一般是单价或二价的抗体，相对来说，对病毒的捕获能力还不够。
发明内容
本发明提供一种聚合分子、包括其的单一结构和多聚结构、以及相关的产品、制备方法和用途，通过聚合分子的聚合作用形成阻断与受体结合的多聚体，以能有效提高对病毒的结合能力。
为此，本发明提供了以下的技术方案。
本发明提高一种聚合分子，其特征在于：用于将对病毒与细胞受体之间的结合进行阻断 的多个阻断结合分子单元聚合为聚合结构。
本发明提高的聚合分子，还具有这样的特征：其中，聚合分子具有单一结合部位和聚合部位单一结合部位，所述单一结合部位用于与所述阻断结合分子单元结合而形成至少具有聚合分子与阻断结合分子单元的单一结构，多个单一结构通过聚合部位聚合为聚合结构，优选地，聚合分子为肽或蛋白时，单一结合部位设置在聚合分子的N端。
本发明提高的聚合分子，还具有这样的特征：其中，所述聚合结构由数量为2-10中任意一种的所述单一结构聚合而成。
本发明提高的聚合分子，还具有这样的特征：其中，聚合分子选自表1中的任意一项。
本发明提高的聚合分子，还具有这样的特征：其中，聚合分子为链霉亲和素，优选地，所述链霉亲和素包含与SEQIDNO:1具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。
本发明提高的聚合分子，还具有这样的特征：其中，所述聚合分子与所述阻断结合分子单元之间通过连接分子结合，
所述连接分子包含有荧光类蛋白、人免疫球蛋白G4、Fc以及HSA中的任意一种或多种，例如，所述连接分子是eGFP荧光蛋白或经其改造得到，优选地，通过对eGFP删除部分氨基酸得到；或：
所述连接分子包含与SEQIDNO:2-6中任意一个具有一致性或与SEQIDNO:2-6中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列优选地，通过对eGFP删除其第1-228位的氨基酸得到；
优选地，所述连接分子的N端与所述阻断结合分子单元连接，所述连接分子的C端与所述聚合分子N端连接。。
本发明提高的聚合分子，还具有这样的特征：其中，阻断结合分子单元通过与病毒与细胞受体结合的部位结合而阻断病毒与受体之间的结合，和/或阻断结合分子单元通过与细胞受体结合而阻断病毒与细胞受体之间的结合。
本发明提高的聚合分子，还具有这样的特征：其中，所述阻断结合分子单元含有至少一个对病毒与细胞受体之间的结合进行阻断的阻断分子，优选地，所述阻断分子含有与SEQIDNO:7-9以及16-19中任意一个具有一致性或与与SEQIDNO:7-9以及16-19中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列；进一步地，当含有多个阻断分子时，所述阻断分子之间从N端到C端顺序连接，更优选地，当所述阻断结合分子单元和所述聚合分子之间通过所述连接分子连接时，所述连接分子的N端与最末一个所述阻断分子的C端连接，所述连接分子的C端与所述单一结合部位连接，再一优选地，所述阻断分子为纳米抗体。
本发明提高的聚合分子，还具有这样的特征：其中，细胞受体为ACE2，优选地，阻断肽为ACE2类似物。
本发明提高的聚合分子，还具有这样的特征：其中，所述阻断结合分子单元含有至少一个第一阻断分子和/或至少一个第二阻断分子，所述第一阻断分子针包含与SEQIDNO:7具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，所述第二阻断分子包含与SEQIDNO:8以及16-19中任意一个具有一致性或与SEQIDNO:8以及16-19中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，优选地，所述阻断结合分子单元含有两个所述第一阻断分子或两个所述第二阻断分子更优选地，两个所述第一阻断分子之间或两个所述第二阻断分子之间通过一个的N端和另一个的C端连接；再一优选地，所述阻断结合分子单元含有所述第一阻断分子和所述第二阻断分子所述第一阻断分子与所述第二阻断肽段之间通过一个的N端和另一个的C端连接，更优选地，所述第二阻断分子的N端和所述第一阻断分子的C端连接。
本发明提高的聚合分子，还具有这样的特征：其中，单一结构还含有用于促进聚合结构通过细胞进行表达得到、细胞进行分泌得到或体外无细胞进行表达得到的先导肽，所述先导肽包含与SEQIDNO:10具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序，优选地，先导肽通过C端任意一个阻断肽段的N端连接。
本发明提高的聚合分子，还具有这样的特征：其中，所述单一结构还含有所述酸性结构，所述酸性结构为带有负电的氨基酸短链聚合物，进一步地，所述酸性结构具有以下特征中的一个或多个的组合：
(1)所述酸性结构设置在C末端；
(2)所述的短链聚合物的氨基酸的个数为0-50、2-40、3-30、2-20或2-10；
(3)所述的带有负电的氨基酸为天冬氨酸和/或谷氨酸，
优选地，所述酸性结构与所述聚合分子的C端连接。
本发明提高的聚合分子，还具有这样的特征：其中，单一结构还含有标签蛋白，所述标签蛋白包含与SEQIDNO:15具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，优选地，标签蛋白通过C端与阻断结合分子单元的N端之间连接，当阻断结合分子单元为阻断肽段时，标签蛋白的C端与任意一个阻断肽段的N端连接；更优选地，当单一结构还含有先导肽时，先导肽通过标签蛋白与阻断结合分子单元之间连接。
本发明提高的聚合分子，还具有这样的特征：其中，病毒为乙肝病毒、狂犬病毒、HVP以及新冠病毒中的一种或多种。
本发明还提供一种单一结构，其特征在于，含有：前述的聚合分子，优选地，单一结构的大小为30-80KD，单一结构为前述涉及的单一结构。
本发明还提供一种聚合结构，其特征在于：由多个前述的单一结构聚合而成，聚合结构为前述涉及的聚合结构。
本发明提供的聚合结构，还具有这样的特征：其中，聚合结构为至少四个单一结构聚合而成，优选地，聚合结构为四个单一结构聚合而成。
本发明提供的聚合结构，还具有这样的特征：其中，阻断结构单元为阻断多肽的结合力是为纳米抗体的1000-1M倍。
本发明提供的聚合结构，还具有这样的特征：其中，聚合结构是可溶性地。
本发明提供的聚合结构，还具有这样的特征：其中，聚合结构具有的热稳定温度大于等于45℃。
本发明还提供一种核酸，其编码聚合分子、单一结构或聚合结构，其特征在于：其中，聚合分子为前述的聚合分子；单一结构为前述的单一结构；聚合结构为前述的聚合结构。
本发明还提供一种载体，其含有前述的核酸。
本发明还提供一种真核宿主细胞，其包含前述的核酸或前述的载体，用于细胞内表达、细胞分泌性或体外无细胞合成表达单一结构或聚合结构，其中，单一结构为前述的单一结构；聚合结构为前述的聚合结构。
本发明还提供一种前述的核酸或前述的载体在制备单一结构和/或多聚结构的方法中的应用。
本发明还提供一种聚合分子、单一结构以及聚合结构中的任意一项在以下中的应用：在治疗病毒的药品、病毒检测诊断、医学用途、用于针对病毒的消毒产品、化妆品、护肤品、护理品、食品、清洁产品，其中，聚合分子为前述的聚合分子；单一结构为前述的单一结构；
聚合结构为前述的聚合结构；优选地，直接采用经无细胞体外合成聚合结构得到的未纯化的产物进行应用。
本发明还提供一种消毒产产品、化妆品、化妆品、护肤品、护理品、食品或清洁产品， 其特征在于，包括：聚合分子、单一结构以及聚合结构中的任意一项的一种或多种，其中，聚合分子为前述的聚合分子；单一结构为前述的单一结构；聚合结构为前述的的聚合结构。
本发明还提供一种药品，其特征在于，包括：聚合分子、单一结构以及聚合结构中的一种或多种和医药学上可接受的载剂、稀释剂或赋形剂，其中，聚合分子为前述的的聚合分子；单一结构为前述的单一结构；聚合结构为前述的聚合结构。
本发明还提供一种聚合结构的制备方法，采用无细胞体外合成表达得到，其特征在于：对反应得到的包括聚合结构的产物进行蛋白纯化时，采用硫酸铵沉淀法，其中，聚合结构为前述的聚合结构。
发明作用与效果
(1)本发明提供的聚合分子，能有效将对病毒与细胞受体之间的结合进行阻断的多个阻断结合分子单元聚合为聚合结构，这样实现阻断病毒与细胞受体之间结合的阻断作用的方式，由于聚合分子的聚合作用，提高了每个聚合结构的阻断结合分子单元的个数，能从多点阻断病毒与细胞受体之间的结合，同时由于多个阻断结合分子单元，能同时结合多个病毒，多个聚合结构配合，会产生交联作用，更有效阻断病毒与细胞受体之间的结合，通过实验可以看出，聚合后相比聚合前，对病毒的阻断效果提高了，并且结合力是纳米抗体的1000-1M倍，由此，这种方式，相比单个位点结合来说，需要的结合分子的量也更少，生产成本也更低；
(2)进一步地，当至少四个含有前述聚合分子的单一结构通过各自的聚合分子相互聚合为聚合结构时，由于每个聚合结构具有至少四个单一结构，形成至少四面体的结构，从而能更有效实现交联效果，进一步提高上述的阻断作用，而优选地，当为四个单一结构聚合的聚合结构时，相比大于四个的聚合结构，聚合结构的大小适宜，且保证足够的阻断作用能力，又不产生空间结合位点和空间浪费；
(3)进一步地，本发明提供的链霉亲和素，能让单一结构通过聚合分子的聚合，90％以上为四个单一结构聚合形成的聚合结构；
(5)进一步地，阻断结合分子是可以通过肽段的形式进行病毒和细胞受体之间的结合阻断，相比抗体类，整个单一结构或聚合结构的大小更小，而且结构更单一，更适宜通过细胞体内表达、细胞分泌或体外表达获得；
(6)进一步地，阻断分子为多个，可以增加多点阻断病毒与细胞受体之间的结合的机会；进一步地，第一阻断分子和/或第二阻断分子，对病毒与细胞受体之间的结合的阻断效果更好；
(7)进一步地，当单一结构还含有用于促进聚合结构通过
细胞表达、细胞分泌或体外无细胞表达的先导肽时，更有利于通过细胞表达、细胞分泌或体外无细胞表达的方式生产前述的单一结构或前述的聚合结构；
(8)进一步地，当所阻断结合分子单元与聚合分子之间通过荧光蛋白或其改造得到连接分子连接时，能提高病毒阻断效果；进一步地，单一结构还含有酸性结构，该酸性结构的存在，能提高对病毒的阻断效果，并且能提高聚合结构的热稳定性。
附图说明
图1-图22为实施例2涉及的病毒阻断蛋白对假病毒的抑制实验结果图；
图23为实施例2中，病毒阻断蛋白对假病毒作用前后的电镜图；
图24为实施例1中一种聚合结构的三维示意图；
图25为实施例1中的一种聚合结构对病毒的交联作用三维示意图。
具体实施方式
以下结合附图来说明本发明的具体实施方式。对于实施例中所用到的具体方法或材料，本领域技术人员可以在本发明技术思路的基础上，根据已有的技术进行常规的替换选择，而不仅限于本发明实施例的具体记载。
实施例中所使用的方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径获得。
本发明中的“冠状病毒”是指冠状病毒在系统分类上属套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)，且以ACE2作为结合受体的冠状病毒，包括但不限于SARS-CoV、MERS-CoV、SARS-CoV-2等。
刺突蛋白，也即S蛋白，为病毒表面的一类标志性跨膜蛋白，有两个亚基：S1和S2，受体结合位点(RBD)位于S1亚基上。它以三聚体的形式组成病毒粒子外膜表面的刺突，其主要功能是识别宿主细胞表面受体，介导与宿主细胞的融合。
本发明的ACE2也称为ACEH，称为血管紧张素转化酶2。ACE2由805个氨基酸组成，是具有单一胞外催化结构域的I型跨膜糖蛋白。ACE2为SARS-Cov-2等冠状病毒侵染人体细胞的受体蛋白。
在本发明中，体外无细胞表达，也即无细胞蛋白质合成系统，是一种基于原核或真核细胞的转录-翻译偶联的体系，是指在包含生物提取物和/或确定的试剂的反应混合物中多肽或其他大分子的合成。
本发明中“D2P”体系包括但不限于IVTT反应(体外转录翻译反应)。本发明中，优选IVTT反应。IVTT反应，对应IVTT体系，是在体外将DNA转录翻译为蛋白质(Protein)的过程，因此，我们还将这类的体外蛋白合成体系称为、D-to-P体系、D_to_P体系、DNAto-Protein体系；相应的体外蛋白合成方法，还称为D2P方法、D-to-P方法、D_to_P方法、DNA-to-Protein方法，其与“体外无细胞蛋白合成体系”、“体外表达系统”、“体外蛋白合成体系”、“体外蛋白质合成反应体系”、“无细胞蛋白合成体系”等表述具有相同的含义。蛋白质体外合成系统、体外蛋白合成体系、无细胞系统、无细胞蛋白合成体系、无细胞体外蛋白合成体系、体外无细胞蛋白合成体系、体外无细胞合成体系、CFS体系(cell-free system)、CFPS体系(cell-free protein synthesis system)等描述方式。包括体外翻译体系、体外转录翻译体系(IVTT体系)等。我们还将体外蛋白合成系统称为“蛋白质合成工厂”(Protein Factory)。体外蛋白合成反应，是指在体外无细胞合成体系中合成蛋白的反应，至少包括翻译过程。
本发明体外无细胞蛋白合成体系中需要的蛋白组分(举例如RNA聚合酶)，可以通过内源方式提供，也可以通过外源方式添加。通过内源方式提供时，可以参考包括但不限于文献CN108690139A、CN109423496A、CN106978439A、CN110408635A、CN110551700A、CN110093284A、CN110845622A、CN110938649A、CN111378708A、CN111484998A、“Molecular andCellular Biology,1990,10(1):353-360”等现有文献及其引用文献提供的基因改造方法，具体地，包括但不限于：将编码序列插入到细胞内游离型质粒，将编码基因整合入细胞基因组，及其组合方式。通过外源方式提供时，用量可以根据体系所需进行控制和调节。
宿主细胞是本领域中众所周知的，包括但不限于大肠杆菌、CHO细胞、中国仓鼠卵巢、NS0、SP2细胞、海拉细胞(HeLa cell)、小仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、HEK-293细胞和许多其它细胞系。草地贪夜蛾(Spodoptera frugiperda)或粉纹夜蛾(Trichoplusiani))、两栖动物细胞、细菌细胞、植物细胞和真菌细胞。真菌细胞包含酵母和丝状真菌细胞，丝状真菌细胞包含例如，毕赤酵母、芬兰毕赤酵母(Pichia  finlandica)、喜海藻糖毕赤酵母(Pichia trehalophila)、科克拉马毕赤酵母(Pichia koclamae)、膜醭毕赤酵母(Pichia membranaefaciens)、微小毕赤酵母(Pichia minuta)(甲醇诱导型酵母(Ogataeaminuta)、林氏毕赤酵母(Pichia lindneri))、仙人掌毕赤酵母(Pichia opuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、松栎毕赤酵母(Pichia g uercuum)、皮杰普毕赤酵母(Pichia pijperi)、树干毕赤酵母(Pichiastiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母菌(Pichia sp.)、酿酒酵母(Saccharomyces cerevisiae)、酵母菌(Saccharomyces sp.)、多形汉逊酵母(Hansenulapolymorpha)、克鲁维酵母菌(Kluyveromyces sp.)、乳酸克鲁维酵母(Kluyveromyceslactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、卢克诺文思金孢子菌(Chrysosporium lucknowense)、镰刀菌(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、镰孢霉(Fusarium venenatum)、小立碗藓(Physcomitrellapatens)以及粗糙脉孢菌(Neurospora crassa)。
进一步地，体外无细胞蛋白合成体系，包括但不限于大肠杆菌体外蛋白合成体系、细菌体外蛋白合成体系、哺乳动物细胞(如HF9、Hela、CHO、HEK293)体外蛋白合成体系、植物细胞体外蛋白合成体系、酵母细胞体外蛋白合成体系、昆虫细胞体外蛋白合成体系。酵母细胞选自下组：酿酒酵母、毕氏酵母、克鲁维酵母中的任意一个或多个的组合。克鲁维酵母例如为乳酸克鲁维酵母。
本发明的体外蛋白合成体系、模板、质粒、目标蛋白、体外蛋白合成反应(孵育反应)、各种制备方法、各种检测方法等技术要素，还可以各自独立地从下述文献中选择合适的实施方式或实施方法，包括但不限于CN111484998A、CN106978349A、CN108535489A、CN108690139A、CN108949801A、CN108642076A、CN109022478A、CN109423496A、CN109423497A、CN109423509A、CN109837293A、CN109971783A、CN109988801A、CN109971775A、CN110093284A、CN110408635A、CN110408636A、CN110551745A、CN110551700A、CN110551785A、CN110819647A、CN110845622A、CN110938649A、CN110964736A等文献。除非和本发明目的相冲突，否则，这些文献及其引用文献以全部内容、全部目的被引用。
本发明，提及的对病毒与细胞受体之间的结合进行阻断，指的是使得所述病毒失活，例如可以是以下方式：
一种方式：减少病毒与细胞受体之间的结合，包括：
第一种：属于竞争性阻断，也即用一种与受体的类似物，来与病毒结合，减少病毒结合受体细胞的机会；或者通过与受体结合的分子，减少能被病毒结合的细胞受体，也就减少了病毒结合受体细胞的机会；
第二种：通过不同病毒之间以阻断结合分子单元为媒介形成的交联作用的阻挡，减少病毒与细胞受体之间的结合机会；
另一种方式利用抗体对与细胞受体结合的功能区域进行破坏，达到阻断的目的。
本文的“聚合分子”，指能通过例如分子间作用力，或者特定结构域等自缔合的作用，聚合到一起的分子。
本文的“单一结构”指聚合分子聚合之前的单个结构，也即至少具有阻断结合分子单元和聚合分子的结构，也称为单体。
本文的“聚合结构”指通过聚合分子之间的结合，将多个单一结构聚合到一起形成的结构，也称为多聚体。
实施例1
本实施例，对本发明的内容进行具体解释说明。
本实施例提供一种聚合分子，其特征在于：用于将对病毒与细胞受体之间的结合进行前述的阻断的阻断结合分子单元聚合为聚合结构。这里的阻断结合分子单元具有与细胞受体或病毒结合的结合分子，从而能竞争性阻断病毒与细胞受体的结合。
为了便于说明，本文的聚合分子，用A表示，本文的阻断结合分子单元用B表示。
通过聚合分子的聚合作用，将多个阻断结构分子单元进行聚合，提高了每个聚合结构的阻断结合分子单元的个数，也即增加了竞争阻断的点，能从多点阻断病毒与细胞受体之间的结合，同时由于多个阻断结合分子单元，能同时结合多个病毒，产生交联作用，更有效阻断病毒与细胞受体之间的结合。
具体地，本实施例的聚合分子具有单一结合部位和聚合部位，
单一结合部位用于与阻断结合分子单元结合而组成单一结构，多个单一结构通过聚合部位聚合为聚合结构，也即不同单一结构之间的聚合部位具有能结合到一起的作用，例如通过分子间作用力等，发生例如自缔合的结合。优选地，聚合分子为肽或蛋白时，单一结合部位设置在聚合分子的N端，也即通过阻断结合分子单元的C端与聚合分子的N端连接。具体地，该单一结构的基本结构如式一：
B-A(式一)：优选地，单一结合部位设置在聚合分子的C端。
优选地，聚合结构为至少四个单一结构聚合而成，当至少四个含有前述聚合分子的单一结构通过各自的聚合分子相互聚合为聚合结构时，由于每个聚合结构具有至少四个单一结构，形成至少四面体的结构，从而能更有效实现交联效果，进一步提高上述的阻断作用，而优选地，当为四个单一结构聚合的聚合结构时，相比大于四个的聚合结构，聚合结构的大小适宜，且保证足够的阻断作用能力，又不产生空间结合位点和空间浪费。
图24为实施例1中一种聚合结构的三维示意图；
图25为实施例1中聚合结构对病毒的交联作用三维示意图。
如图24，图中的聚合结构，是三个单一结构聚合而成，中间部分是聚合部位聚合到一起，各个阻断结合分子单元分布在三个点，形成四面体的结构。
如图25所示，图中聚合结构由单个单一结构聚合而成，每个聚合结构除了两个阻断结合分子分别结合同一病毒外，另外一个阻断结合分子去结合其他病毒，由此，两个病毒之间通过阻断结合分子形成交联，可以不断阻挡其他病毒对细胞受体的接触，从而提高阻断作用。
本实施例中，聚合分子选自表1中的任意一项：
Figure PCTCN2023070015-appb-000001
Figure PCTCN2023070015-appb-000002
在一示例中，聚合分子为链霉亲和素，或含有与SEQIDNO:1具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。通过这样的聚合分子，使得聚合产生的四聚结构(四个单一结构聚合成)达到80％以上，甚至是90％以上。
在一示例中，聚合分子与阻断结合分子单元之间可以另外通过连接分子结合，为便于说明，连接分子用D表示。
在一示例中，连接分子包含有荧光类蛋白、人免疫球蛋白G4、Fc以及HSA中的任意一种或多种，例如，连接分子是eGFP荧光蛋白或经其改造得到，优选地，改造是通过对eGFP删除部分氨基酸得到，例如是EGFP荧光蛋白或经其改造得到，优选地，通过对eGFP删除其第1-228位的氨基酸得到。。
一优选地，连接分子包含与SEQIDNO:2具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，或与SEQIDNO:3具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，或与SEQIDNO:4具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，或与SEQIDNO:5具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，或与SEQIDNO:6具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。
在一示例中，连接分子的N端与阻断结合分子单元连接，连接分子的C端与聚合分子的设置在N端的单一结合部位连接，此时单一结构的结构如式二所示：
B-D-A(式二)：优选地，B的C端与D的N端连接，D的N端与A的C端连接。
在一示例中，阻断结合分子单元含有至少一个与病毒的受体结合区域进行结合的阻断分子。也即可能包括一个阻断分子，也可能是多个阻断分子，当包括多个时，这些阻断分子的氨基酸序列可能是相同的，也可能是不同的。
当阻断分子为多个时，则可以增加多点阻断病毒与细胞受体之间的结合的机会，提高前述的阻断作用。
在一示例中，当阻断分子为多个时，阻断分子之间从C端到N端顺序连接，也即前一个的C端和后一个的N端连接，具体为B1-B2-B3…Bn(n为大于1的正整数)；更优选地，当阻断结合分子单元和聚合分子之间通过连接分子连接时，连接分子的N端与最末一个阻断分子的C端连接，连接分子D的C端与聚合分子连接，具体为B1-B2-B3…Bn-D-A。
在一示例中，阻断分子含有与SEQIDNO:7-9以及16-19中任意一个具有一致性或与SEQIDNO:7-9以及16-19中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。具体解释就是：
阻断结合分子单元中含有的每个阻断分子，可能含有SEQIDNO:7-9以及16-19中所示几种氨基酸序列中的任意一种，或是含有与SEQIDNO:7-9以及16-19中的任意一种具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列：
例如，阻断分子含有与SEQIDNO:7具有一致性的氨基酸序列，又例如，阻断分子是与SEQIDNO:7具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。
SEQIDNO:16-19，具体显示如下：
SEQIDNO:16(Pep170)：
Figure PCTCN2023070015-appb-000003
SEQIDNO:17(Pep180)：
Figure PCTCN2023070015-appb-000004
SEQIDNO:18(Pep190)：
Figure PCTCN2023070015-appb-000005
SEQIDNO:19(Pep200)：
Figure PCTCN2023070015-appb-000006
在一示例中，阻断分子可以为肽段或抗体。当采用抗体时，优选为纳米抗体，可以降低整体单一结构的大小。
当采用阻断肽进行阻断，相比抗体类，整个结构的大小更小，而且结构更单一，更适宜通过细胞体内表达、细胞分泌或体外表达获得。具体地，采用阻断肽段，整个单一结构的大小范围为30-80KD。
当阻断肽段为多个时，则可以增加多点阻断病毒与细胞受体之间的结合的机会，提高前述的阻断作用。
在一示例中，细胞受体为ACE2。
在一示例中，阻断结合分子单元含有至少一个第一阻断分子(第一阻断肽段，为了便于说明，用B1表示)和/或至少一个第二阻断分子(第二阻断肽段，为了便于说明，用B2表示)。所述第一阻断分子针包含与SEQIDNO:7具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，所述第二阻断分子包含与SEQIDNO:8以及16-19中任意一个具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。第一阻断分子和/或第二阻断分子，分别为肽段，相比纳米抗体，对病毒与细胞受体之间的结合的阻断效果尤其好。
在一示例中，阻断结合分子单元含有两个第一阻断分子或两个第二阻断分子，更优选地，两个第一阻断分子之间或两个第二阻断分子之间通过一个的N端和另一个的C端连接，也即例如B1-B1或B2-B2；
在一示例中，当阻断结合分子含有第一阻断分子和第二阻断分子时，第一阻断分子与第 二阻断分子之间通过一个的N端和另一个的C端连接，也即要么第一阻断分子的N端与第二阻断分子的C端连接，要么，第二阻断分子的N端与第一阻断分子的N端连接。
优选地，第二阻断分子的N端和第二阻断分子的C端连接，此时，单一结构的结构例如为式三或式四所示：
B1-B2-A(式三)：优选地，该结构的连接方式为，B1的C端与B2的N端连接，B2的C端与A的N端连接，也即前一个的C端和后一个的N端连接(下同)；
B1-B2-D-A(式四)：优选地，该结构的连接方式为，B1的C端与B2的N端连接，B2的C端与D的N端连接，D的C端与A的N端连接。
在一示例中，单一结构还含有用于促进聚合结构通过细胞进行表达得到、细胞分泌得到或体外无细胞进行表达得到的先导肽(为便于说明，用C表示)，先导肽包含与SEQIDNO:10具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，此时，单一结构的结构在阻断结合分子单元和聚合分子的基础上，增加了先导肽，单一结构的一个结构例举如式五所示：
C-B-A(式五)：优选地，C的C端与B的N端连接，B的C端和A的N端连接。
有两个阻断分子时，优选地，先导肽与任意一个阻断分子的N端连接，此时，单一结构的结构例如式六所示：
C-B1-B2-A(式六)：该结构式的连接，优选地，C的C端与B1的N端连接，B1的C端与B2的N端连接，B2的C端与A的N端连接。
当有连接分子时，单一结构的结构例如式七所示：
C-B1-B2-D-A(式七)：该结构式的连接，优选地，也是前一个的C端与后一个的N端连接，也即，C的C端与B1的N端连接，B1的C端与B2的N短链连接，B2的C端与D的N端连接，D的C端与A的N端连接。
在一示例中，酸性结构含有至少两个连续的天冬氨酸氨基酸(为便于说明，用d表示)，和/或至少两个连续的谷氨酸氨基酸(为便于说明，用e表示)，和/或至少一组天冬氨酸氨基酸和氨基酸的组合。例如，酸性结构含有10个连续的天冬氨酸(用10d表示)、10个连续的谷氨酸(用10e表示)，8个连续的天冬氨酸和8个连续的谷氨酸(用8d8e表示)，5个d-e(5de，一个d和一个e是一组，总共5组)。
在一示例中，稳定分子与聚合分子的C端连接，此时，单一结构的基本结构如式八所示：
B-A-E(式七)：式中，优选地，前一个的C端与后一个的N端连接；
在此基础上，再组合前述的C、D中的任意一个或多个，得到不同的结构式，例如式九-式十一所示：
C-B-A-E(式九)：式中，优选地，前一个的C端与后一个的NC端连接；
B-D-A-E(式十)：式中，优选地，前一个的C端与后一个的N端连接；
C-B-D-A-E(式十一)：式中，优选地，前一个的C端与后一个的N端连接；
当B包括B1和B2时，例如式十二所示：
C-B1-B2-D-A-E(式十二)：式中，优选地，前一个的C端与后一个的N端连接。
在一示例中，单一结构还含有标签蛋白(为便于说明，用F表示)，标签蛋白为了前述的纯化单一结构或前述的聚合结构用，在一示例中，标签蛋白包含与SEQIDNO:15具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。
优选地，标签蛋白通过C端与阻断结合分子单元的N端之间连接，也即标签蛋白的C端在阻断结合分子单元N端一侧，此时，单一结构的基本结构如式十三所示：
F-B-A(式十三)：式中，优选地，前一个的C端与后一个的N端连接。
在此基础上，再组合前述的C、D以及E中的任意一个或多个，得到不同的结构式，例 如式十四-式十六所示：
F-B-D-A(式十四)：式中，优选地，前一个的C端与后一个的N端连接。
C-F-B-A(式十五)：式中，优选地，前一个的C端与后一个的N端连接。
F-C-B-D(式十六)：式中，优选地，前一个的C端与后一个的N端连接，此时，F的C端虽然没有直接与B的N端连接，但通过C进行了连接，也即F的C端位于B的N端的一侧(其他类似解释)。
在一示例中，当单一结构还含有先导肽时，先导肽通过标签蛋白与阻断结合分子单元之间连接，如式十七所示：
C-F-B-D-A(式十七)：优选地，前一个的C端与后一个的N端连接，同样地，此时是先导肽没有直接与阻断结合分子之间连接，而是通过标签蛋白进行连接。
以上不同的结构式中，当阻断肽段为B1和/或B2时，B1和B2同时出现，此时前面的结构式中，B1和B2的位置可以为B1-B2，也可以为B2-B1；也可以只出现这两个中的任意一个，此时B用B1或B2表示。
在一示例中，前述的病毒为为乙肝病毒、狂犬病毒、HVP以及新冠病毒中的任意一种或多种。
在一示例中，本实施例涉及的单一结构的大小为30-80KD。
在一示例中，阻断结构单元为阻断多肽的结合力是为纳米抗体或抗体的1000-1M倍。
在一示例中，聚合结构是可溶性地，例如溶于水溶液。
在一示例中，前述的聚合分子聚合单一结构后，能使得聚合结构具有的热稳定温度接近70℃，优选地，大于等于80℃。
本实施例还提供一种核酸，其编码前述的单一结构或编码前述的聚合结构。
本实施例还提供一种载体，其含有前述的核酸。
本实施例还提供一种真核宿主细胞，其包含前述的核酸或载体，用于细胞内表达、细胞分泌性或体外无细胞合成表达前述的单一结构或前述的聚合结构。
本实施例还提供一种前述的核酸或前述的载体在制备单一结构和/或多聚结构的方法中的应用。
由于前述的聚合分子聚合单一结构形成聚合结构带来的阻断优势，本实施例在以下应用或产品中，能更好地发挥阻断作用，从而更好地实现对特定病毒的预防、或治疗或检测等作用：
(1)本实施例还提供前述的聚合分子、前述的单一结构以及前述的聚合结构中的任意一项在以下中的应用：在治疗病毒的药品、病毒检测诊断、医学用途、用于针对病毒的消毒产品、化妆品、护肤品、护理品、食品、清洁产品。
(2)本实施例还提供一种消毒产产品、化妆品、护肤品、护理品、食品或清洁产品，其特征在于，包括：前述的聚合分子、前述的单一结构以及前述的聚合结构中的任意一项的一种或多种；
(3)本实施例还提供一种药品，包括：前述的聚合分子、前述的单一结构以及前述的聚合结构中的一种或多种和医药学上可接受的载剂、稀释剂或赋形剂。
上述的消毒产品例如空气清洁消毒、水的清洁消毒、食品消毒、衣服消毒、家庭用具以及各种工具或设施等一切可能需要消毒的地方、实务或场所，工具比如交通工具，设施比如公共汽车站；
上述的护肤品例如涂脸霜、喷雾水、爽肤水、眼霜等涂覆皮肤表面的产品；
上述的化妆品例如粉底液、腮红等；
上述的护理品例如容易感染病毒的表面或辅助用品的护理液、护理膏、喷雾等，例如滴眼液、辅助用品例如隐形眼镜护理液等；
上述的清洁产品，例如洗洁剂、洗发水、沐浴露、洗衣液等。
本文的聚合结构，本文也称为病毒阻断蛋白、阻断蛋白。
优选地，阻断蛋白通过康码(上海)生物科技有限公司D2P技术进行生产，如D2P系统，D2P技术进行生产，如包括如下步骤：该基因经过编码优化并克隆到pD2P载体中。质粒是使用Ampi系统扩增，然后添加到蛋白质工厂快速反应中系统(康码(上海)生物科技有限公司)，体积比为1:30。这将反应混合物在30℃下孵育4小时，然后通过离心收集纯化。将无细胞混合物的上清液与磁性His Monster Beads(Kangma Healthcode(Shanghai)Biotech)在4℃下旋转1小时。用洗涤缓冲液(50mM Tris-HCl,pH 8.0,500mM NaCl，10mM咪唑)并用洗脱缓冲液(50mM Tris-HCl，pH 8.0，500mM NaCl、250mM咪唑)。
Ampi系统为：终浓度为20-30μM的随机引物，0.05-0.15μg/mL的质粒模板，0.5-1mM的dNTP，2×BSA，0.05-0.1mg/mL的phi29DNA聚合酶，1×phi29反应缓冲液(成分为50mM Tris-HCl,10mM MgCl2,10mM(NH4)2SO4,4mM DTT,pH7.5)。
实施例2
本实施例以病毒为新冠病毒、细胞受体为ACE2为例的具体实验，说明各种结构的聚合结构的阻断优势。
1.实验例涉及的病毒阻断蛋白的制备IVTT反应：
将优化后的目标蛋白的基因序列(包括编码前述各种病毒阻断蛋白结构的基因序列)插入到质粒中，然后加入到自制的乳酸克鲁维酵母体外无细胞蛋白合成体系中，以乳酸克鲁维酵母(KluyveromyceslactisNRRL Y-1140)制备体外。本实施例所用体外无细胞蛋白合成体系(总体积30μL)：包括乳酸克鲁维酵母细胞提取物50％(v/v)，22mM三羟甲基氨基甲烷(pH8)，90mM醋酸钾，4.0mM醋酸镁，3.0mM核苷三19磷酸混合物，0.16mM氨基酸混合物，22mM磷酸钾，0.003mg/mL淀粉酶，3％(w/v)聚乙二醇(PEG-8000)，340mM麦芽糊精(以葡萄糖单元计量，对应约55mg/mL)，0.04mg/mL外源添加的RNA聚合酶，以及15ng/μL目标蛋白DNA等。当荧光蛋白DNA的种类大于1时，这里的15ng/μL为各荧光蛋白DNA的总浓度。将上述反应体系置于22-30℃的环境中，静置孵育约20h。
IVTT反应后，纯化使用His磁珠(康码生物产品)纯化，蛋白洗脱后，超滤离心置换至PBS缓冲液中。用0.22μm的针筒滤器过滤后，得到纯化蛋白，4度保存待用。
1.以下对本实施例各种实验涉及的制备的病毒阻断蛋白结构式进行说明：
Figure PCTCN2023070015-appb-000007
Figure PCTCN2023070015-appb-000008
Figure PCTCN2023070015-appb-000009
Figure PCTCN2023070015-appb-000010
表2中涉及的序列说明，如表3：
Figure PCTCN2023070015-appb-000011
Figure PCTCN2023070015-appb-000012
Figure PCTCN2023070015-appb-000013
2.预实验测试假病毒与细胞
我们所使用的新冠假病毒表面表达了S蛋白，内部包装了荧光素酶基因。在感染细胞后，可在细胞内表达荧光素酶蛋白，通过加入其底物，检测荧光素酶底物的发光值，可以得到假病毒感染细胞的效率。
首先对假病毒的细胞感染进行预实验测试，确定假病毒的稀释度。将假病毒以30倍的梯度，逐级稀释(具体见表4)，并分别感染HEK293T或HEK293T-ACE2(过表达ACE2的HEK293T细胞)，每种感染做两个平行实验，最后发现病毒感染细胞时，荧光素酶检测的线性窗口在S3-S7范围内(稀释270-21870倍)。假病毒供应商建议的以540倍的稀释度感染细胞，所以后续我们在实验中采用的假病毒稀释度为540倍。
通过使用野生型新冠毒株对野生型和过表达ACE2的HEK293T细胞的感染实验发现，在过表达ACE2的细胞中，病毒感染的效率明显高于野生型的细胞株，因此，后续实验中，选用了过表达ACE2的HEK293T作为测试细胞株。
Figure PCTCN2023070015-appb-000014
Figure PCTCN2023070015-appb-000015
3.Kd评估
表面等离子共振(SPR)实验SPR研究使用Biacore T200生物传感器(GE Healthcare，美国)进行。将病毒阻断蛋白通过胺偶联固定在S系列传感器芯片CM5(GE Healthcare，美国)上。His-tagged RBD的参考菌株和Omicron变体(Sino Biological Inc，北京，中国)被稀释至六种浓度(0.25、0.5、1、2、4和8nM)，Delta变体的RBD(Sino Biological Inc，北京,中国)稀释至(0.5,1,2,4,8和16nM)。在25℃下进行单循环动力学分析。在10mM HEPES、pH 7.4、150mM NaCl、3mM EDTA和0.05％Tween下，结合相设置为120s和30μL/min，解离相分别设置为300s和30μL/min-20作为运行缓冲区。重复9次分析循环，分析物浓度按升序排列。使用Biacore T200评估软件(2.0版，GE Healthcare，美国)计算解离常数(Kd)值。
结果显示，测试本发明表2中提供的结构，与所有三种病毒株的RBD结构域有强烈的相互作用，即原始、Delta和Omicron变体具有牢固的结合亲和力。对原始、Delta和Omicron变体的解离常数(Kd)值分别能达到1.25nM、0.837nM和0.656nM。
总而言之，尽管Omicron变体具有更高的健康风险和惊人的免疫逃避，但本发明的病毒阻断蛋白仍然有资格与这些变体的RBD结构域结合。
4.对表2提供的不同结构进行的针对假病毒的病毒阻断实验，实验步骤如下：
(1)将待感染HEK293T-ACE2细胞细胞接种于48孔细胞培养板中。接种量为1.5×10 4个细胞每孔；次日进行假病毒感染实验时，细胞密度约在30％左右。
(2)次日，从-80℃取出SARS-CoV-2(2019-nCoV)S蛋白假病毒，置于冰上融化或4℃条件下自然融化。待完全融化后，用完全培养基(DMEM，10％胎牛血清，1％双抗，0.75μg/mL嘌呤霉素)进行270倍稀释成假病毒稀释液。
(3)KMds阻断剂原始浓度为1μM，将其按10倍梯度用完全培养基连续稀释。将梯度稀释的KMds阻断剂与假病毒稀释液1：1体积混合成假病毒感染液，并将假病毒感染液在室温孵育1小时。
(4)1小时后，从培养箱取出提前铺好HEK293T-ACE2细胞的48孔板，确认细胞密度及状态后，吸去上层培养基。沿孔壁将200μL假病毒感染液加入每个孔中，避免冲起细胞。将48孔板放入培养箱中培养6小时。
(5)6小时后，小心吸出假病毒感染液，并更换300μL新鲜的完全培养基。继续在培养箱中培养48小时。
(6)48小时后，吸出培养基，并在每孔中小心加入200μL 1×PBS润洗细胞，避免冲起细胞。小心吸出PBS，并于每孔中加入65μL 1×细胞裂解液并在室温孵育15-20分钟。15-20分钟后，立即检测荧光素酶的活性，或置于-20℃保存。
(7)将5μL细胞溶解液与5μL荧光素酶底物(Promega E1501萤光素酶检测系统)混合，将混合液加入384孔板中，立即用Perkin Elmer EnVision 2102多功能酶标仪上检测荧光素酶的活性，并判定KMds阻断剂的抑制效率。
实验结果如图1-图23显示：
其中，图中的横坐标表示用的病毒阻断蛋白的浓度；图中的纵坐标则表示阻断实验后抑制效果(抑制率)，抑制率计算如下：
Figure PCTCN2023070015-appb-000016
其中，V0为仅加了病毒的对照的RLU读数；V1为加有病毒阻断蛋白和病毒的样品RLU读数。
纵坐标数值越高，表示效果越好。
Delta表示针对Delta株假病毒进行抑制测试，Original表示针对新冠野生型假病毒进行抑制测试，Omicron针对奥密克戎株假病毒进行抑制测试)；
图中每组柱形图分别按从左到右的顺序对应图例或标题中显示的结构名称的结果图：例如图4中，图例从左到右分别是Kmds008、Kmds007、Kmds001以及Kmds009-2，则每组柱形图的顺序也分别按这个顺序对应相应结构的结果。
结果显示：
I.聚合前后效果：
如图1-3显示，聚合前(Kmds009)比聚合后(Kmds009-2)效果差；聚合前Kmds042比聚合后(Kmds003)效果差；聚合前Kmds043比聚合后(Kmds012)效果差；
II.不同阻断分子的阻断效果，实验表面，本发明的阻断分子与新冠病毒的S蛋白具有特异性结合能力：
(1)如图4所示，不同阻断分子均能在较低浓度抑制新冠和德塔；
(2)如图5所示，0052去除聚合分子和连接分子，仍能在较低浓度有抑制效果。III.加酸性结构：
(1)加酸性结构效果比不加好：如图6，Kmds003、Kmds002和Kmds006分别加了酸性结构的，效果比未加酸性结构的Kmds001好；酸性结构酸性结构；
(2)加8d8e、10e效果好：如图6显示，Kmds006比Kmds003和Kmds002对病毒的阻断效果好；如图7显示，Kmds012和Kmds014比Kmds011和Kmds013效果好；
IV.连接分子：
(1)如图8用不同的连接分子，效果接近：KMds104,KMds105，KMds121，KMds 112；
(2)对eGFP进行部分改造，与未改造前对比：KMds003，KMds012，KMds104
改造后分别得到KMds045，KMds046，KMds109，结果分别如图9-11所示，改造前后效果接近；
(3)对连接分子进行删除，删除后，依然能对病毒起到抑制作用，结果如图12-14：KMds003，KMds012，KMds104的Tram被删除后，分别得到KMds036，KMds038，KMds108。
V.先导肽：
将KMds003，KMds006，KMds012，KMds014，KMds044基础上删去leading peptide序列，分别得到KMds030，KMds031，KMds032，KMds033，KMds104，结果分别如图15-22所示，根据结果，先导肽对病毒阻断蛋白对病毒的抑制性没有影响；
VI.交联作用：
对病毒阻断实验中的具有聚合分子的结构处理病毒前后做了电镜观察，如图23中， 图23中A是未经病毒阻断蛋白处理的，其他是经病毒阻断蛋白处理后不同放大倍数的图，从图中可见，电镜下，经具有聚合分子处理病毒后，图片中显示出现大的斑块，也就是，我们的结构能实现对病毒的交联集聚，从而能提高对病毒的交联阻断抑制作用。
VII.假病毒IC 50结果：
测试结果显示，在所有测试中，针对同样的阻断分子：
具有酸性结构、聚合分子以及连接分子的，IC50值最低的能达到为42.4pM，达到皮摩尔的级别；不存在酸性结构，IC50值能达到为0.222nM左右；
没有连接分子、没有聚合分子、连接分子和聚合分子都没有的，对假病毒的抑制作用相比来说，有降低，其中IC50值分别能达到1.054、2.404和1.032nM。
5.活病毒测试
我们研究了表2中的病毒阻断蛋白对不同SARS-CoV-2毒株的抑制能力，包括原始毒株、Alpha、Beta、Delta和Omicron变体。结果显示：
原始菌株被病毒阻断蛋白强烈抑制(IC50为108.6pM)；
并且另一方面，病毒阻断蛋白可以在极低浓度(IC50分别为92.8、121.9、61.0和121.9pM)下显示出对Alpha、Beta、Delta和Omicron变体的有效抑制能力；
与中和原始SARS-CoV-2的能力相比，病毒阻断蛋白抑制Delta变体的能力增加了约1.8倍，抑制其他菌株的能力几乎相同。
对活病毒的测试表明，尽管刺突蛋白存在大量突变，但本发明的病毒阻断蛋白在不同的SARS-CoV-2变体中仍保持皮摩尔水平的强大抑制能力。
6.动物实验
SARS-CoV-2野生型(WT)毒株(IVCAS 6.7512)由中国科学院武汉病毒研究所国家病毒资源中心提供。杂合B6/JGpt-H11em1Cin(K18-ACE2)/Gpt小鼠(K18-hACE2KI小鼠)购自南京GemPharmatech。
在特定的无病原体(SPF)环境中，在单独通风的笼子(IVC)中饲养和繁殖小鼠。动物实验由武汉大学动物实验中心的认证人员执行，经机构动物护理和使用委员会(AUP#WP2021-0602)批准。动物生物安全三级实验室设施下传染性SARS-CoV-2病毒的方案和程序已获得机构生物安全委员会(IBC，协议#S01322010A)的批准。
所有样品的灭活均按照IBC批准的标准程序进行，用于将样品从高密封中取出。每只小鼠都感染了2.5×102PFU SARS-CoV-2。对于阴性对照组，将SARS-CoV-2与对照缓冲液预混合30分钟。
对于0.25nM病毒阻断蛋白预混治疗组，将SARS-CoV-2与0.25nM病毒阻断蛋白预混30分钟；
对于25nM病毒阻断蛋白预混治疗组，将SARS-CoV-2与25nM病毒阻断蛋白预混30分钟；
然后用预混物通过鼻内途径接种K18-hACE2小鼠。
将组织称重并在Tissue Cell-destroyer 1000仪器(NZK LTD)中在1000μL PBS中匀浆。通过在5,000rpm下离心40秒来澄清组织匀浆，将100μL上清液与400μL Trizol LS混合以提取病毒RNA。
使用pCMV-N质粒和SARS-CoV-2N基因引物(F引物：ATGCTGCAATCGTGCTACAA；R引物：GACTGCCGCCTCTGCTC)构建SARS-CoV-2N基因标准曲线，计算病毒拷贝数。使用Prism版本7(GraphPad软件)通过学生测试分析数据。P值<0.05被认为具有统计学意义。
为了测试病毒阻断蛋白在小鼠模型中的作用，我们用SARS-CoV-2和病毒阻断的预混物感染了K18-hACE2小鼠。在感染后第2天和第5天(dpi)处死小鼠。在5天的时间过程中，每天326监测小鼠的体重变化和死亡率。
鼻内接种SARS-CoV-2的K18-hACE2小鼠在感染后3至4天开始体重减轻，并在4dpi时死亡；
与对照小鼠相比，接种病毒阻断蛋白和SARS-CoV-2预混物的小鼠显着减轻体重减轻并显着提高存活率，尤其是使用高剂量病毒阻断蛋白时。
在感染SARS-CoV-2的K18-hACE2的肺组织中检测到高水平的SARS-CoV-2RNA，而在感染SARS-CoV-2和低剂量病毒阻断蛋白混物病毒RNA的小鼠中病毒RNA水平较低，在感染高剂量病毒阻断蛋白和SARS-CoV-2预混物的小鼠的肺组织中甚至检测不到水平。总之，这些数据表明高剂量病毒阻断蛋白可以显着减轻体重减轻，提高小鼠存活率，并抑制小鼠的病毒拷贝数。
7.毒性实验
毒理学试验按《消毒技术标准》进行(2002年版)第二部分《消毒产品检验技术标准》2.3.1急性经口毒性试验。异常毒性试验按《中华人民共和国药典2020年版(第四部分)》《通则》《生物制品原则、检验方法》，异常毒性试验。
ICR小鼠由南京医科大学动物中心提供。
Sprague Dawley(SD)大鼠购自邳州东方繁育有限公司。
新西兰大白兔由益正安里茂生物科技有限公司提供。动物饲养于20℃-26℃，局部屏障系统内相对湿度为40％-70％。
针对表2中涉及的结构，进行以下毒性实验：
(1)急性经口毒性
20只SPF ICR小鼠(18.0～22.0g)和20只SPF SD大鼠(180～220g)用于急性经口毒性试验。雄性和雌性的数量是相等的。对过夜禁食的小鼠和大鼠施用表3中的结构式对应制备的病毒阻断蛋白，剂量5000mg/kg·bw，使用胃插管以单次。接下来每周监测动物的毒性和死亡率的临床症状14天(第0天、第7天和第14天)。行为、死亡人数和体重分别为评估，并在观察期结束时对它们进行解剖。
根据试验结果，给予5000mg/kg·bw剂量的病毒阻断蛋白不会导致过夜禁食的小鼠和大鼠发病，并且体重正常。病毒阻断蛋白对小鼠和大鼠的LD50值均在5000mg/kg·bw以上。观察期间未发现中毒和死亡迹象。
(2)急性吸入毒性试验
20只SPF ICR小鼠(18.0～22.0g)用于急性吸入毒性试验。雄性和雌性的数量是相等的。将2.2g表3中对应制备的病毒阻断蛋白置于220L毒物暴露柜，浓度假定为10,000mg/m3。这吸入的暴露时间设定为2小时。老鼠的症状和死亡是在14天观察期间(0天、7天和14天)记录。
根据试验记过，在雌性和雄性小鼠中，在2h暴露下的病毒阻断蛋白LC50值均高于10,000mg/m3。小鼠没有表现出异常迹象，现体重稳定增加是正常的。因此，在目前的实验条件下，病毒阻断蛋白符合法规，被认为是无毒的。
(3)急性眼刺激试验
三只雄性新西兰兔(2.5～3.5kg)用于急性眼刺激试验。将0.1mL表3中对应制备的病毒阻断蛋白原液滴入兔右眼结膜囊，左眼滴入生理盐水作为对照。闭眼4s，30s后用生理盐水冲洗。观察家兔结膜、虹膜和角膜的损伤和恢复情况，为期21天(1小时、24小时、 48小时、72小时、7天、14天和28天)。对角膜损伤、虹膜损伤、结膜充血和结膜水肿的严重程度进行评分。
根据试验结果，测试的病毒阻断蛋白在兔子身上没有表现出眼刺激的迹象。三只兔子在24h、48h和72h的评分均小于1，如表1所示。因此，本发明病毒阻断蛋白的刺激性归类为无刺激性。
(4)小鼠骨髓多染红细胞(PCE)微核试验
将50只ICR小鼠用于小鼠骨髓多染红细胞微核试验。男性的数量等于女性的数量。将动物分成五组，每组五只雌性小鼠和五只雄性小鼠。试验组分别以5000、2500和1250mg/kg·bw的剂量分别给予一次表3中对应制备的病毒阻断蛋白。一组作为阴性对照并用溶剂纯化水处理。另一组作为阳性对照，腹腔注射40mg/kg·bw的环磷酰胺(CP)。测试组在0小时和24小时通过口服给药暴露于病阻断蛋白毒。第二次暴露于病毒阻断蛋白后6小时处死小鼠，制备骨髓涂片。计算每只动物1000个多染红细胞(PCE)中微核的出现。一旦计数了200个PCE，就确定了PCE与正色素红细胞(NCE)的比率。通过U检验进行统计分析。当与阴性对照相比，实验组微核形成的发生率显着增加时，判断试验剂对体内染色体有害，这应以剂量-反应相关性发生。
对于微核试验，雌性和雄性小鼠的微核形成率在三个实验组中与阴性组相比无显着差异(P<0.05)，与阳性对照组相比差异显着(P<0.05)，如如表2所示。此外，任何实验组与阴性对照之间的PCE/NCE比率差异均在20％以内。
(5)异常毒性实验
10只雌性SPF ICR小鼠(18.0～22.0g)和4只标准级雌性豚鼠(250～350g)进行异常毒性试验。
10只小鼠随机分为试验组和对照组，每组5只。试验组腹腔注射0.5mL样品，对照组腹腔注射0.5mL氯化钠。观察两组体质情况7d。将4只雌性豚鼠随机分为试验组和对照组，每组2只豚鼠。试验组腹腔注射0.5mL样品，对照组腹腔注射0.5mL氯化钠。观察两组体质情况7d。
在所有测试中，本发明提供的病毒阻断蛋白均没有引起可观察到的毒性作用。
8.测试病毒蛋白的热稳定性
使用Unchained公司的Uncle设备，测试了几种蛋白的热稳定性。Tmagg表征蛋白在加热过程中发生高聚的温度，结果见表5。
Figure PCTCN2023070015-appb-000017
同时含有B1+B2的Kmds001 Tmagg值低于50度，但上加8D8E(Kmds006)酸性结构后，Tmagg值比不加尾(Kmds001)高很多，增加了近1倍。也就是说这个酸性结构能够增加蛋白的稳定性。
另外，经测试，表2中各个结构的病毒阻断蛋白的平均Tm接近80℃，表明本发明提供的病毒阻断蛋白可免受极端温度的影响。
9.加速稳定性试验
稳定性研究在两个不同的温度下同时进行，4℃和37℃(三个重复，100uL的10nM病毒阻断蛋白)。
另一组没有加病毒阻断蛋白作为阴性对照(NC)。在90天内(0天、15天、30天、45天、60天、75天和90天)，每15天对它们进行3次取样。通过假病毒中和试验评估抑制百分比，并通过以下公式计算：抑制百分比＝(NC-样品)/NC(RFU)。
试验结果显示：在4℃或37℃下储存超过90天后，病毒阻断蛋白的抑制效率几乎没有受损(>99.9％)，表明本发明的病毒阻断蛋白是一种超稳定的SARS-CoV-2阻断剂。
以下实施例为膜剂实施例，其中涉及到的病毒阻断蛋白，为实施例1中的方法制备参照涉及到的各个结构制备得到的病毒阻断蛋白，其中，各个配方中采用的病毒阻断蛋白的原液浓度为2mg/ml左右。
以下实施例中的吸湿实验方法如下：
设备：加湿器，型号：KW-AD01；功率5W，容量100ml。
加湿器中加满水，将制备得到的膜剂置于加湿器的雾化喷孔上方5-10cm，观察膜剂吸湿变化情况，实验温度为室温。
10.测试表3提供的一个结构的IVTT反应液经过硫酸铵沉淀处理后，与假病毒的结合能力。
IVTT反应后的溶液经硫酸铵沉淀处理：4000rpm 4度离心10min。上清液加入5％(w/v)的硫酸铵粉末，充分搅拌溶解，4000rpm 4度离心10min。上清液中继续加入25％(w/v)的硫酸铵粉末，缓慢加入，边加入边搅拌使其溶解。12000rpm 4度离心10min。弃上清，沉淀用30％(w/v)的硫酸铵溶液漂洗1次。12000rpm 4度离心10min。沉淀用PBS重悬，充分溶解。12000rpm，4度离心10min，用0.22μm的针筒滤器过滤后，4度保存待用。
Kmds012的IVTT反应液经硫酸铵沉淀后的样品活性略低于Kmd012纯蛋白(磁珠纯化蛋白)。
可见，我们再对本发明提供的聚合结构进行纯化，可以采用简单方便的硫酸铵沉淀法进行。

Claims (26)

  1. 一种聚合分子，其特征在于：
    用于将对病毒与细胞受体之间的结合进行阻断的多个阻断结合分子单元聚合为聚合结构。
  2. 根据权利要求1所述的聚合分子，其特征在于：
    其中，所述聚合分子具有单一结合部位和聚合部位，
    所述单一结合部位用于与所述阻断结合分子单元结合而形成至少具有聚合分子与阻断结合分子单元的单一结构，多个所述单一结构通过所述聚合部位聚合为聚合结构，
    优选地，所述聚合分子为肽或蛋白时，所述单一结合部位在所述聚合分子的N端。
  3. 根据权利要求1或2所述的聚合分子，其特征在于：
    其中，所述聚合结构由数量为2-10中任意一种的所述单一结构聚合而成。
  4. 根据权利要求1-3任意一项所述的聚合分子，其特征在于：
    其中，所述聚合分子选自表1中的任意一项。
  5. 根据权利要求4所述的聚合分子，其特征在于：
    其中，所述聚合分子为链霉亲和素，
    优选地，所述链霉亲和素包含与SEQIDNO:1具有一致性或与 其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列。
  6. 根据权利要求6所述的聚合分子，其特征在于：
    其中，所述聚合分子与所述阻断结合分子单元之间通过连接分子结合，
    所述连接分子包含有荧光类蛋白、人免疫球蛋白G4、Fc以及HSA中的任意一种或多种，例如，所述连接分子是eGFP荧光蛋白或经其改造得到，优选地，通过对eGFP删除部分氨基酸得到；或：
    所述连接分子包含与SEQIDNO:2-6中任意一个具有一致性或与SEQIDNO:2-6中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列优选地，通过对eGFP删除其第1-228位的氨基酸得到；
    优选地，所述连接分子的N端与所述阻断结合分子单元连接，所述连接分子的C端与所述聚合分子N端连接。
  7. 根据权利要求1-6任意一项所述的聚合分子，其特征在于：
    其中，所述阻断结合分子单元通过与所述病毒与所述细胞受体结合的部位结合而阻断所述病毒与所述受体之间的结合，和/或
    所述阻断结合分子单元通过与所述细胞受体结合而阻断所述病 毒与所述细胞受体之间的结合。
  8. 根据权利要求1-7任意一项所述的聚合分子，其特征在于：
    其中，所述阻断结合分子单元含有至少一个对病毒与细胞受体之间的结合进行阻断的阻断分子，
    优选地，所述阻断分子含有与SEQIDNO:7-9以及16-19中任意一个具有一致性或与与SEQIDNO:7-9以及16-19中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列；进一步地，当含有多个阻断分子时，所述阻断分子之间从N端到C端顺序连接，更优选地，当所述阻断结合分子单元和所述聚合分子之间通过所述连接分子连接时，所述连接分子的N端与最末一个所述阻断分子的C端连接，所述连接分子的C端与所述单一结合部位连接，
    再一优选地，所述阻断分子为纳米抗体。
  9. 根据权利要求1-8任意所述的聚合分子，其特征在于：
    其中，所述细胞受体为ACE2。
  10. 根据权利要求8或9所述的聚合分子，其特征在于：
    其中，所述阻断结合分子单元含有至少一个第一阻断分子和/或至少一个第二阻断分子，所述第一阻断分子针包含与SEQIDNO:7具有一致性或与其具有至少50％、60％、70、80％、85％、90％、 95％或99％一致性的氨基酸序列，所述第二阻断分子包含与SEQIDNO:8以及16-19中任意一个具有一致性或与与SEQIDNO:8以及16-19中任意一个具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，
    优选地，所述阻断结合分子单元含有两个所述第一阻断分子或两个所述第二阻断分子更优选地，两个所述第一阻断分子之间或两个所述第二阻断分子之间通过一个的N端和另一个的C端连接；
    再一优选地，所述阻断结合分子单元含有所述第一阻断分子和所述第二阻断分子所述第一阻断分子与所述第二阻断肽段之间通过一个的N端和另一个的C端连接，更优选地，所述第二阻断分子的N端和所述第一阻断分子的C端连接。
  11. 根据权利要求1-10任意一项所述的聚合分子，其特征在于：其中，所述单一结构还含有先导肽，
    所述先导肽包含与SEQIDNO:10具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序，列优选地，所述先导肽通过C端任意一个所述阻断肽段的N端连接。
  12. 根据权利要求1-11任意一项所述的聚合分子，其特征在于：
    其中，所述单一结构还含有所述酸性结构，所述酸性结构为带有负电的氨基酸短链聚合物，进一步地，所述酸性结构具有以下 特征中的一个或多个的组合：
    (1)所述酸性结构设置在C末端；
    (2)所述的短链聚合物的氨基酸的个数为0-50、2-40、3-30、2-20或2-10；
    (3)所述的带有负电的氨基酸为天冬氨酸和/或谷氨酸，
    优选地，所述酸性结构与所述聚合分子的C端连接。
  13. 根据权利要求1-12任意一项所述的聚合分子，其特征在于：
    其中，所述单一结构还含有标签蛋白，
    所述标签蛋白包含与SEQIDNO:15具有一致性或与其具有至少50％、60％、70、80％、85％、90％、95％或99％一致性的氨基酸序列，
    优选地，所述标签蛋白通过C端与所述阻断结合分子单元的N端之间连接，当所述阻断结合分子单元为阻断肽段时，所述标签蛋白的C端与任意一个所述阻断肽段的N端连接；
    更优选地，当所述单一结构还含有所述先导肽时，所述先导肽通过所述标签蛋白与所述阻断结合分子单元之间连接。
  14. 据权利要求1-13任意一项所述的聚合分子，其特征在于：
    其中，所述病毒为乙肝病毒、狂犬病毒、HVP以及新冠病毒中的一种或多种。
  15. 一种单一结构，其特征在于，含有：
    权利要求1-14任意一项所述的聚合分子，
    优选地，所述单一结构的大小为30-80KD，
    再一优选地，所述单一结构为权利要求2-14中任意一项涉及到的单一结构。
  16. 一种聚合结构，其特征在于：
    由多个权利要求15所述的单一结构聚合而成。
  17. 根据权利要求16所述的聚合结构，其特征在于：
    其中，所述聚合结构由数量为2-10中任意一种的所述单一结构聚合而成，优选地，所述聚合结构为4个所述单一结构聚合而成。
  18. 根据权利要求16或17所述的聚合结构，其特征在于：
    其中，所述阻断结构单元的结合力为纳米抗体的1000-1M倍。
  19. 根据权利要求16-18任意一项所述的聚合结构，其特征在于：
    其中，所述聚合结构是可溶性地。
  20. 一种核酸，其编码聚合分子、单一结构或聚合结构，其特征在于：
    其中，所述聚合分子为权利要求1-15中任意一项所述的聚合分子；
    所述单一结构为权利要求15所述的单一结构；
    所述聚合结构为权利要求16-19中任意一项所述的聚合结构。
  21. 一种载体，其含有权利要求20所述的核酸。
  22. 一种真核宿主细胞，其包含根据权利要求20所述的核酸或21所述的载体，用于细胞内表达、细胞分泌性或体外无细胞合成表达单一结构或聚合结构，
    其中，所述单一结构为权利要求15所述的单一结构；
    所述聚合结构为权利要求16-19中任意一项所述的聚合结构。
  23. 一种权利要求20所述的核酸或21载体在制备权利要求15所述的单一结构和/或权利要求16-19中任意一项所述多聚结构的方法中的应用。
  24. 聚合分子、单一结构以及聚合结构中的任意一项在以下中的应用：
    在治疗病毒的药品、病毒检测诊断、医学用途、用于针对病毒的消毒产品、化妆品、护肤品、护理品、食品、清洁产品，
    其中，所述聚合分子为权利要求1-14中任意一项所述的聚合分子；
    所述单一结构为权利要求15所述的单一结构；
    所述聚合结构为权利要求16-19中任意一项所述的聚合结构，
    优选地，直接采用经无细胞体外合成所述聚合结构得到的未纯化的产物进行所述应用。
  25. 一种消毒产产品、化妆品、化妆品、护肤品、护理品、食品或清洁产品，其特征在于，包括：
    聚合分子、单一结构以及聚合结构中的任意一项的一种或多种，
    其中，所述聚合分子为权利要求1-14中任意一项所述的聚合分子；
    所述单一结构为权利要求15所述的单一结构；
    所述聚合结构为权利要求16-19中任意一项所述的聚合结构。
  26. 一种药品，其特征在于，包括：
    聚合分子、单一结构以及聚合结构中的一种或多种和医药学上可接受的载剂、稀释剂或赋形剂，
    其中，所述聚合分子为权利要求1-14中任意一项所述的聚合分子；
    所述单一结构为权利要求15所述的单一结构；
    所述聚合结构为权利要求16-19中任意一项所述的聚合结构。
PCT/CN2023/070015 2021-12-31 2023-01-03 聚合分子、包括其的单一结构和多聚结构 WO2023126009A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020247025893A KR20240128089A (ko) 2021-12-31 2023-01-03 중합체 분자 및 이를 포함한 단량체 구조 및 중합체 구조

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN202111668011 2021-12-31
CN202111668011.3 2021-12-31
CN202210114482.8 2022-01-30
CN202210114482 2022-01-30
CN202210773393 2022-07-01
CN202210773393.4 2022-07-01
CN202210968998.9 2022-08-12
CN202210968998 2022-08-12

Publications (1)

Publication Number Publication Date
WO2023126009A1 true WO2023126009A1 (zh) 2023-07-06

Family

ID=86968175

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/070015 WO2023126009A1 (zh) 2021-12-31 2023-01-03 聚合分子、包括其的单一结构和多聚结构

Country Status (3)

Country Link
KR (1) KR20240128089A (zh)
CN (3) CN116370335A (zh)
WO (1) WO2023126009A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117942275A (zh) * 2022-10-31 2024-04-30 康码（上海）生物科技有限公司 一种基于病毒阻断剂的洗手液及其应用

Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1798770A (zh) * 2003-04-04 2006-07-05 洛桑大学 用于治疗癌症的多聚肽体
CN101094688A (zh) * 2004-09-13 2007-12-26 建新公司 多聚构建体
CN106978439A (zh) 2010-12-30 2017-07-25 陶氏益农公司 赋予对鞘翅目有害生物的抗性的核酸分子
CN106978349A (zh) 2016-09-30 2017-07-25 康码（上海）生物科技有限公司 一种体外蛋白质合成的试剂盒及其制备方法
CN107592865A (zh) * 2015-05-11 2018-01-16 扬森疫苗与预防公司 中和流感病毒的拟肽化合物
CN108535489A (zh) 2017-03-04 2018-09-14 康码（上海）生物科技有限公司 一种用于体外蛋白质合成的蛋白合成体系、试剂盒及其制备方法
CN108642076A (zh) 2017-03-23 2018-10-12 康码（上海）生物科技有限公司 一种体外DNA-to-Protein(D2P)的合成体系、制剂、试剂盒及制备方法
CN108690139A (zh) 2017-07-31 2018-10-23 康码（上海）生物科技有限公司 新型融合蛋白的制备及其在提高蛋白质合成的应用
CN108949801A (zh) 2017-11-24 2018-12-07 康码（上海）生物科技有限公司 一种通过对核酸酶系统敲除以调控体外生物合成活性的方法
CN109022478A (zh) 2017-06-09 2018-12-18 康码（上海）生物科技有限公司 一种用于高通量体外蛋白质合成的dna元件
CN109423496A (zh) 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 一种细胞中内源性表达rna聚合酶的核酸构建物
CN109423497A (zh) 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 增强蛋白质合成效率的rna元件
CN109423509A (zh) 2017-08-27 2019-03-05 康码（上海）生物科技有限公司 一种葡萄糖检测方法、试剂盒及其应用
CN109837293A (zh) 2017-11-24 2019-06-04 康码（上海）生物科技有限公司 一种能够增强蛋白质合成效率的串联dna元件
CN109971783A (zh) 2017-12-28 2019-07-05 康码（上海）生物科技有限公司 一种体外蛋白合成体系、试剂盒及其制备方法
CN109971775A (zh) 2017-12-27 2019-07-05 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法
CN109988801A (zh) 2017-12-29 2019-07-09 康码（上海）生物科技有限公司 一种用于体外生物反应体系的新型的高效的能源再生体系(bes)、试剂盒及制备方法
CN110093284A (zh) 2018-01-31 2019-08-06 康码（上海）生物科技有限公司 一种在细胞中提高蛋白合成效率的方法
CN110408635A (zh) 2018-04-28 2019-11-05 康码（上海）生物科技有限公司 一种含有链霉亲和素元件的核酸构建物在蛋白质表达、纯化中的应用
CN110408636A (zh) 2018-04-28 2019-11-05 康码（上海）生物科技有限公司 多重标签串联的dna序列及其在蛋白质表达纯化系统的应用
CN110551745A (zh) 2018-05-31 2019-12-10 康码（上海）生物科技有限公司 一种多重组氨酸序列标签及其在蛋白质表达、纯化中的应用
CN110551785A (zh) 2018-06-01 2019-12-10 康码（上海）生物科技有限公司 一种用于体外蛋白质合成的无细胞冻干制剂、其制法和用途
CN110551700A (zh) 2018-05-31 2019-12-10 康码（上海）生物科技有限公司 Adh蛋白家族突变体及其应用
CN110819647A (zh) 2018-08-07 2020-02-21 康码（上海）生物科技有限公司 信号肽相关序列及其在蛋白质合成中的应用
CN110845622A (zh) 2018-08-21 2020-02-28 康码（上海）生物科技有限公司 不同结构域缺失融合蛋白的制备及其在提高蛋白质合成的应用
CN110938649A (zh) 2018-09-25 2020-03-31 康码（上海）生物科技有限公司 一种提高外源蛋白表达量的蛋白合成体系及其应用方法
CN110964736A (zh) 2018-09-28 2020-04-07 康码（上海）生物科技有限公司 一种体外蛋白合成体系及其用于提高蛋白合成效率的方法、试剂盒
CN111378708A (zh) 2018-12-28 2020-07-07 康码（上海）生物科技有限公司 一种优化的体外无细胞蛋白合成体系及其应用
CN111484998A (zh) 2019-05-30 2020-08-04 康码（上海）生物科技有限公司 体外定量共表达多种蛋白的方法及其应用
WO2021219121A1 (zh) * 2020-04-30 2021-11-04 养生堂有限公司 用于筛选冠状病毒感染阻断剂的检测试剂及检测方法

Patent Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1798770A (zh) * 2003-04-04 2006-07-05 洛桑大学 用于治疗癌症的多聚肽体
CN101094688A (zh) * 2004-09-13 2007-12-26 建新公司 多聚构建体
CN106978439A (zh) 2010-12-30 2017-07-25 陶氏益农公司 赋予对鞘翅目有害生物的抗性的核酸分子
CN107592865A (zh) * 2015-05-11 2018-01-16 扬森疫苗与预防公司 中和流感病毒的拟肽化合物
CN106978349A (zh) 2016-09-30 2017-07-25 康码（上海）生物科技有限公司 一种体外蛋白质合成的试剂盒及其制备方法
CN108535489A (zh) 2017-03-04 2018-09-14 康码（上海）生物科技有限公司 一种用于体外蛋白质合成的蛋白合成体系、试剂盒及其制备方法
CN108642076A (zh) 2017-03-23 2018-10-12 康码（上海）生物科技有限公司 一种体外DNA-to-Protein(D2P)的合成体系、制剂、试剂盒及制备方法
CN109022478A (zh) 2017-06-09 2018-12-18 康码（上海）生物科技有限公司 一种用于高通量体外蛋白质合成的dna元件
CN108690139A (zh) 2017-07-31 2018-10-23 康码（上海）生物科技有限公司 新型融合蛋白的制备及其在提高蛋白质合成的应用
CN109423509A (zh) 2017-08-27 2019-03-05 康码（上海）生物科技有限公司 一种葡萄糖检测方法、试剂盒及其应用
CN109423496A (zh) 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 一种细胞中内源性表达rna聚合酶的核酸构建物
CN109423497A (zh) 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 增强蛋白质合成效率的rna元件
CN108949801A (zh) 2017-11-24 2018-12-07 康码（上海）生物科技有限公司 一种通过对核酸酶系统敲除以调控体外生物合成活性的方法
CN109837293A (zh) 2017-11-24 2019-06-04 康码（上海）生物科技有限公司 一种能够增强蛋白质合成效率的串联dna元件
CN109971775A (zh) 2017-12-27 2019-07-05 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法
CN109971783A (zh) 2017-12-28 2019-07-05 康码（上海）生物科技有限公司 一种体外蛋白合成体系、试剂盒及其制备方法
CN109988801A (zh) 2017-12-29 2019-07-09 康码（上海）生物科技有限公司 一种用于体外生物反应体系的新型的高效的能源再生体系(bes)、试剂盒及制备方法
CN110093284A (zh) 2018-01-31 2019-08-06 康码（上海）生物科技有限公司 一种在细胞中提高蛋白合成效率的方法
CN110408635A (zh) 2018-04-28 2019-11-05 康码（上海）生物科技有限公司 一种含有链霉亲和素元件的核酸构建物在蛋白质表达、纯化中的应用
CN110408636A (zh) 2018-04-28 2019-11-05 康码（上海）生物科技有限公司 多重标签串联的dna序列及其在蛋白质表达纯化系统的应用
CN110551700A (zh) 2018-05-31 2019-12-10 康码（上海）生物科技有限公司 Adh蛋白家族突变体及其应用
CN110551745A (zh) 2018-05-31 2019-12-10 康码（上海）生物科技有限公司 一种多重组氨酸序列标签及其在蛋白质表达、纯化中的应用
CN110551785A (zh) 2018-06-01 2019-12-10 康码（上海）生物科技有限公司 一种用于体外蛋白质合成的无细胞冻干制剂、其制法和用途
CN110819647A (zh) 2018-08-07 2020-02-21 康码（上海）生物科技有限公司 信号肽相关序列及其在蛋白质合成中的应用
CN110845622A (zh) 2018-08-21 2020-02-28 康码（上海）生物科技有限公司 不同结构域缺失融合蛋白的制备及其在提高蛋白质合成的应用
CN110938649A (zh) 2018-09-25 2020-03-31 康码（上海）生物科技有限公司 一种提高外源蛋白表达量的蛋白合成体系及其应用方法
CN110964736A (zh) 2018-09-28 2020-04-07 康码（上海）生物科技有限公司 一种体外蛋白合成体系及其用于提高蛋白合成效率的方法、试剂盒
CN111378708A (zh) 2018-12-28 2020-07-07 康码（上海）生物科技有限公司 一种优化的体外无细胞蛋白合成体系及其应用
CN111484998A (zh) 2019-05-30 2020-08-04 康码（上海）生物科技有限公司 体外定量共表达多种蛋白的方法及其应用
WO2021219121A1 (zh) * 2020-04-30 2021-11-04 养生堂有限公司 用于筛选冠状病毒感染阻断剂的检测试剂及检测方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Disinfection Technical Standard", 2002, article "Acute Oral Toxicity Test, Standard of Inspection Technology for Disinfection Product (Part 2"
"Pharmacopoeia of the People's Republic of China", 2020, article "Principles and Inspection Methods for Biological Products, General Principles"
"Structural insights into hepatitis C virus receptor binding and entry", NATURE
MOLECULAR ANDCELLULAR BIOLOGY, vol. 10, no. 1, 1990, pages 353 - 360
YAHI N, SABATIER J M, BAGHDIGUIAN S, GONZALEZ-SCARANO F, FANTINI J: "Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 69, no. 1, 1 January 1995 (1995-01-01), US , pages 320 - 325, XP093075365, ISSN: 0022-538X, DOI: 10.1128/jvi.69.1.320-325.1995 *

Also Published As

Publication number Publication date
CN116370335A (zh) 2023-07-04
KR20240128089A (ko) 2024-08-23
CN116370399A (zh) 2023-07-04
CN116769048A (zh) 2023-09-19

Similar Documents

Publication Publication Date Title
WO2022262142A1 (zh) 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
WO2022022445A1 (zh) 一种特异性结合冠状病毒的抗体或其抗原结合片段
CN113817029B (zh) 一种新型冠状病毒s-rbd三聚体蛋白疫苗、其制备方法和应用
CN111620952A (zh) 基于嵌合型病毒样颗粒的新型冠状病毒疫苗
JP7554837B2 (ja) 可溶性ａｃｅ２及び融合タンパク質、並びにその適用
WO2023126009A1 (zh) 聚合分子、包括其的单一结构和多聚结构
CN116693673B (zh) 广谱抗SARS和SARS-CoV-2的纳米抗体、多价纳米抗体及其应用
TW200902043A (en) Novel formulations for delivery of antiviral peptide therapeutics
CN114805560B (zh) 纳米抗体r14的构建体及其应用
WO2023179546A1 (zh) 纳米抗体s43的构建体及其应用
WO2023138334A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
JPH0768267B2 (ja) フラビウイルス抗原
CN110467672B (zh) 一种针对sftsv的全人源单克隆中和抗体及其应用
CN106480070B (zh) 一种用于展示目的多肽的多肽载体及其用途
US20240067706A1 (en) Fully human broad-spectrum neutralizing antibody 76e1 against coronavirus, and use thereof
WO2021164576A1 (zh) 用于抗冠状病毒感染的药物及其应用
EP3892298A1 (en) Epitopes having sequence homology to coronavirus spike protein subunit and uses thereof
KR20150135231A (ko) 인간 메타뉴모바이러스에 특이적인 인간 항체 또는 그의 항원 결합성 단편
CN106892979B (zh) 一种全人源抗h7n9病毒的中和抗体
CN113813375B (zh) 一种新型抗新冠病毒复合物的组成及其在防治冠状病毒感染疾病药物中的应用
US20030148265A1 (en) Anti-viral conjugate comprising a factor allowing the translocation of a protein across a cell membrane and comprising a single-chain antibody fragment directed against a viral protein
CN101851289B (zh) H5亚型禽流感病毒血凝素蛋白的人源化抗体及其用途
AU2018323504B2 (en) Modified HSV gB protein and HSV vaccine including same
WO2023143619A1 (zh) 一种阻断病毒侵染细胞的中和阻断剂及其制备方法和应用
CN103450351B (zh) 一种对抗新城疫病毒感染的长效多肽及其修饰物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23735114

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 20247025893

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2023735114

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023735114

Country of ref document: EP

Effective date: 20240731