WO2024093910A1 - 一种基于病毒阻断剂的洗手液及其应用 - Google Patents

一种基于病毒阻断剂的洗手液及其应用 Download PDF

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WO2024093910A1
WO2024093910A1 PCT/CN2023/127755 CN2023127755W WO2024093910A1 WO 2024093910 A1 WO2024093910 A1 WO 2024093910A1 CN 2023127755 W CN2023127755 W CN 2023127755W WO 2024093910 A1 WO2024093910 A1 WO 2024093910A1
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hand sanitizer
virus
blocker
protein
amino acid
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PCT/CN2023/127755
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English (en)
French (fr)
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郭敏
伍志
刘章
徐丽琼
于雪
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康码（上海）生物科技有限公司
康码(上海)海洋科技有限公司
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Publication of WO2024093910A1 publication Critical patent/WO2024093910A1/zh

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    • 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
    • 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
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/345—Alcohols containing more than one hydroxy group
    • 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
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/41—Amines
    • 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
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/41—Amines
    • A61K8/416—Quaternary ammonium compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • 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
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations

Definitions

  • the invention relates to the technical field of hand sanitizer, and in particular to a hand sanitizer based on a virus blocking agent and application thereof.
  • Hand washing products are a type of skin cleanser.
  • hand sanitizer has gradually become a substitute for traditional hand washing products.
  • Hand sanitizer has the characteristics of mild performance, no skin irritation, easy to use, hard water resistance, easy to clean, rich foam and good skin lubrication after washing. It is clean, hygienic and easy to store.
  • the bottle pump head is squeezed to take the amount, which avoids cross infection when used by different people, and is therefore widely welcomed by consumers.
  • the ingredients of hand sanitizer include surfactants, emollients, thickeners, pH adjusters, pigments, pearlescent agents, natural active ingredients, etc.
  • Hand sanitizer is a skin care cleaning liquid used to clean hands, which can remove dirt on the hands and attached microorganisms such as bacteria and viruses.
  • Hand sanitizer is a skin care cleansing liquid used to clean hands. Hand sanitizer is used to clean the user's hands. It can clean and remove dirt from the user's hands, care for the skin, and thoroughly clean the hands. Most existing hand sanitizers have a disinfection function, which can disinfect the hands while cleaning, thereby making the hands cleaner.
  • hand sanitizer Today, when water conservation is advocated, the application environment of hand sanitizer is more extensive. It only needs to be applied to the hands, and the hands are rubbed together continuously to spread the hand sanitizer evenly. The hand sanitizer will automatically take away the dirt on the hands as it evaporates, and disinfect the hands.
  • the hand sanitizer is not restricted by the use environment, and can also be used to clean and disinfect the hands in a waterless environment. It should usually be equipped with disinfectant ingredients and volatile solvents. In order to avoid excessive foam, the content of surfactant components that play a washing role in the hand sanitizer should not be too much, generally 2%-8%, and some low-boiling point alcohol solvents are selected to be added.
  • hand sanitizers are also added with certain moisturizing ingredients.
  • Commonly used moisturizers include glycerin and polyethylene glycol series products.
  • the simplest formula of hand sanitizer on the market is similar to disinfectant wipes, which are prepared with volatile components.
  • patent CN113081881A discloses an antibacterial and antiviral disposable hand sanitizer with catechins as antibacterial and antiviral active components
  • patent CN110478293A discloses a disposable disinfectant hand sanitizer, which uses Zanthoxylum bungeanum fruit extract and Forsythia suspensa extract as antibacterial ingredients, but requires acrylic acid (ester)/C10-30 alkyl acrylate cross-linked polymer as a thickener and chemical components such as triethanolamine and ethanol
  • patent CN101766552B discloses a disposable antibacterial hand sanitizer, and its raw material formula includes The invention discloses a terpene-based antibacterial water-free hand sanitizer, which is prepared from the following raw materials: 0.1%
  • the existing hand sanitizer products on the market do not contain active protein components that have the effect of blocking the new coronavirus (SARS-CoV-2).
  • the present invention provides a hand sanitizer based on a virus blocker and its application.
  • the hand sanitizer is added with a protein with a novel coronavirus (SARS-CoV-2) blocking effect, so that the hand sanitizer can play a good protective role against the novel coronavirus.
  • SARS-CoV-2 novel coronavirus
  • the protein will not irritate the user's hands and play an effect of protecting the hand skin. Therefore, this kind of hand sanitizer with a novel coronavirus (SARS-CoV-2) blocking effect is prepared.
  • the present invention provides a hand sanitizer based on a virus blocker, characterized in that it comprises an effective amount of a virus blocker, wherein the virus blocker is a virus blocking protein having a spike binding domain that can specifically bind to the S protein of a coronavirus to inactivate the virus, preferably a (SARS-CoV-2) virus.
  • the virus blocker is a virus blocking protein having a spike binding domain that can specifically bind to the S protein of a coronavirus to inactivate the virus, preferably a (SARS-CoV-2) virus.
  • the "effective amount of virus blocker" of the present invention refers to the amount of virus blocker in the hand sanitizer that can effectively make the virus lose the function and pathway of infecting the body when the hand sanitizer is used, and finally achieve the effect of blocking the spread and infection of the new coronavirus. It can be regarded as neutralization ability, using high affinity to capture the characteristics of adhesion to the new coronavirus from multiple angles, and then make the virus lose the function and pathway of infecting the body.
  • the effective amount of the virus blocker is 0.1nM or more, such as 1-100nM, preferably 5-50nM, and more preferably 10-20nM.
  • the hand sanitizer based on virus blocking agents provided by the present invention further comprises one or more of the following components:
  • Moisturizer used for skin moisturizing.
  • Moisturizers can leave a protective film on the skin surface after the water evaporates, preventing or slowing down the loss of water inside the skin and maintaining the skin's moisturizing properties.
  • Thickeners are used to improve the viscosity of hand sanitizer products.
  • hand sanitizer if the viscosity is too low and it is not easy to stay on the hands, the cleaning ability will be affected. Generally, the viscosity is 0.5Pa, which is ideal. However, when the total surfactant content is 10%, the solution viscosity regulator, the hand sanitizer has a certain viscosity, which can not only improve the product appearance, make the product have a certain stability, but also make it convenient to use.
  • Antibacterial agents are preparations that can be used to effectively control or kill microorganisms on the skin surface, such as bacteria, fungi, and algae.
  • Metal ion chelating agent wherein the metal chelating agent is selected from EDTA, 8-hydroxyquinoline or a combination thereof.
  • Surfactant whose basic function is to remove grease and dirt on hands and generate certain foam.
  • the general principle of selecting surfactant is to enhance the detergency and safety protection.
  • the surfactant of the present invention is selected from anionic surfactant, amphoteric surfactant and nonionic surfactant, and anionic surfactant is preferably used as the main surfactant.
  • Fat-liquor In order to prevent the degreasing effect of hand soap from making the skin feel dry, a certain amount of fat-liquor can be added to the hand soap.
  • Appearance regulators In order to make the product pleasing to the eye, appropriate fragrances and pigments can be added to the hand sanitizer.
  • the appearance regulator is selected from a fragrance, a pigment or a combination thereof.
  • the mass proportion of the water solvent in the hand sanitizer of the present invention is greater than or equal to 90%, preferably, greater than or equal to 95%, and more preferably, greater than or equal to 99%; preferably, the water solvent is a combination of one or more of water, distilled water, sterile water, ultrapure water and deionized water.
  • the moisturizing agent is selected from one or more of 1,2-propylene glycol, high molecular weight sodium hyaluronate, medium molecular weight sodium hyaluronate, low molecular weight sodium hyaluronate, oligomeric sodium hyaluronate, sodium lactate, allantoin, trimethylglycine, liquid paraffin, glycerol, ethylene glycol, sorbitol, propylene glycol and alkyl glycosides, preferably, the moisturizing agent is selected from glycerol.
  • the content of the moisturizing agent is 0-100 mg/mL, preferably 0.1-50 mg/mL, and more preferably 1-20 mg/mL.
  • the thickener is an inorganic salt selected from one or more of sodium chloride, ammonium chloride and potassium chloride. They are cheap, have good thickening effect and are easy to use.
  • the content of the thickener is 0-10 mg/mL, Preferably it is 0.05-5 mg/mL, more preferably 0.1-1 mg/mL.
  • the antibacterial agent can be selected from one or more selected from benzalkonium chloride, chlorhexidine acetate, chlorhexidine hydrochloride, chlorhexidine sulfate, chlorhexidine nitrate, chlorhexidine hydrobromide, chlorhexidine hydroiodide and chlorhexidine gluconate.
  • the hand sanitizer contains an effective amount of antibacterial agent.
  • the amount is 0.01-10 mg/mL, preferably 0.05-5 mg/mL, more preferably 0.1-1 mg/mL.
  • the thickener is one or more of hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose.
  • the metal chelator is selected from one of EDTA and 8-hydroxyquinoline or a combination thereof.
  • the content of the metal chelator in the hand sanitizer is 0-10 mg/mL, preferably 0.05-5 mg/mL, and more preferably 0.1-1 mg/mL.
  • the surfactant is one or more of lauroamidopropyl betaine, cocamidopropyl hydroxybetaine, lauroamidopropyl hydroxysulfobetaine, lauroamidopropyl hydroxysulfobetaine and sodium laurylamphoacetate.
  • the content of the surfactant is 0-10 mg/mL, preferably 0.05-5 mg/mL, and more preferably 0.1-1 mg/mL.
  • the fatting agent is selected from one or more of glycerol fatty acid ester, propylene glycol fatty acid ester, fatty acid ester, alkoxy carboxylic acid, alkoxy alcohol, fatty alcohol and fatty acid.
  • the fatty acid ester is selected from one or more of stearic acid hexadecyl alcohol fat, cetyl hexadecyl fat, stearic acid cetyl alcohol ester, lauric acid isopropyl alcohol ester, myristic acid isopropyl alcohol ester, palmitic acid isopropyl alcohol ester.
  • the fatty alcohol may include but is not limited to octyldodecanol, lauryl alcohol, tetradecyl alcohol, hexadecanol, octadecyl alcohol, behenyl alcohol and their combination.
  • Suitable ethers used for emollients can also be used, such as eucalyptol, cetearyl glucoside, dimethyl isosorbide polyglyceryl 3-3 hexadecyl ether, polyglyceryl-3-decyl tetradecyl alcohol, propylene glycol tetradecyl ether.
  • the fatty acid is selected from one or more of lauric acid, palmitic acid, stearic acid, linoleic acid, hexadecanoic acid.
  • the content of the fatliquor is 0-100 mg/mL, preferably 0.5-50 mg/mL, more preferably 1-30 mg/mL.
  • the flavoring agent is preferably polyols, peppermint oil and/or essence, etc.
  • the content of the flavoring agent in the hand sanitizer is 0-20 ng/mL, preferably 0.1-10 ng/mL, and more preferably 1-5 ng/mL.
  • the fragrance of the essence can be selected from cool fragrance, leaf fragrance, citrus fragrance, fruity fragrance, floral fragrance, spicy fragrance, grass fragrance, resin fragrance, cream fragrance, woody fragrance, soil fragrance, moss fragrance, animal fragrance, leather fragrance, aldehyde fragrance and the like.
  • the pigments are natural colorants mainly including red yeast rice, sorghum red, Gardenia yellow, paprika, beetroot red, turmeric, beta-carotene, chlorophyll, cochineal red, etc.
  • virus inhibitor also has a combination of one or more of the following features:
  • virus blocking agent The various components of the virus blocking agent are connected in the order of N-terminus to C-terminus.
  • the polymeric molecule is a single-chain protein or polypeptide having a polymerization function, and the polymerization function means that the single-chain protein has the function of naturally polymerizing into a polymer.
  • the polymeric molecule comprises streptavidin, and further preferably, comprises an amino acid sequence that is consistent with SEQ ID NO: 1 or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% consistency therewith;
  • the linking molecule of the present invention can maintain the three-dimensional structure and improve the biological activity of the recombinant protein.
  • the linking molecule comprises any one or more of fluorescent protein, human immunoglobulin G4, Fc and HSA.
  • the linking molecule is eGFP fluorescent protein or is obtained by its modification, preferably, by deleting some amino acids from eGFP; or: the linking molecule comprises an amino acid sequence that is consistent with SEQ ID NO: 2 or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% consistency with it, or is consistent with SEQ ID NO: 3 or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% consistency with it.
  • the acidic structure is a short-chain polymer of amino acid residues with negative charge. Further, the acidic structure has a combination of one or more of the following characteristics:
  • the acidic structure is set at the C-terminus
  • the number of amino acid residues 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 contains at least two consecutive D-aspartic acid amino acids, and/or at least Two consecutive E glutamic acid amino acids, and/or a combination of at least one group of D aspartic acid amino acids and E amino acids.
  • the acidic structure contains 10 consecutive aspartic acids (SEQ ID NO: 11), 10 consecutive E glutamic acids (SEQ ID NO: 12), 8 consecutive D aspartic acids and 8 consecutive E glutamic acids (SEQ ID NO: 14), and 5 DE (SEQ ID NO: 13).
  • the leader peptide comprises an amino acid sequence that is identical to SEQ ID NO: 10 or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity thereto.
  • the tag protein comprises an amino acid sequence that is identical to SEQ ID NO: 15 or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity thereto.
  • the tag protein is connected to the N-terminus of the blocking binding molecule unit via the C-terminus, that is, the C-terminus of the tag protein is on the N-terminal side of the blocking binding molecule unit.
  • the spike binding domain contains at least one blocking molecule that binds to the receptor binding region of the virus.
  • the blocking molecule contains an amino acid sequence that is consistent with any one of SEQ ID NO: 7-9 or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% consistency therewith.
  • the spike binding domain comprises a sequence having one or several amino acid substitutions, deletions or additions compared to the polypeptide shown in any one of the amino acid sequences of SEQ ID NO.7-9, and the number of the substituted, deleted or added amino acids may be 1-20, preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the blocker comprises at least one spike binding domain, preferably more than two spike binding domains, such as 1-10, preferably 2-8, and most preferably 4-8.
  • virus blocking agent of the present invention is produced by protein engineering technology, such as expression in host cells, cell secretory expression or in vitro cell-free synthetic expression.
  • the virus blocker is preferably produced by an in vitro cell-free protein synthesis system produced by Kangma (Shanghai) Biotechnology Co., Ltd., such as a D2P system, such as comprising the following steps: the gene is optimized by coding and cloned into a pD2P vector.
  • the plasmid is amplified using the Ampi system and then added to the protein factory rapid reaction system (Kangma (Shanghai) Biotechnology Co., Ltd.) at a volume ratio of 1:30.
  • the reaction mixture is incubated at 30°C for 4 hours and then collected and purified by centrifugation.
  • the supernatant of the cell-free mixture is rotated with magnetic His Monster Beads (Kangma (Shanghai) Biotechnology Co., Ltd.) at 4°C for 1 hour. Wash with a washing buffer (50mM Tris-HCl, pH 8.0, 500mM NaCl, 10mM imidazole) and an elution buffer (50mM Tris-HCl, pH 8.0, 500mM NaCl, 250mM imidazole).
  • a washing buffer 50mM Tris-HCl, pH 8.0, 500mM NaCl, 10mM imidazole
  • an elution buffer 50mM Tris-HCl, pH 8.0, 500mM NaCl, 250mM imidazole.
  • the Ampi system is: random primers with a final concentration of 20-30 ⁇ M, 0.05-0.15 ⁇ g/mL of plasmid template, 0.5-1 mM dNTP, 2 ⁇ BSA, 0.05-0.1 mg/mL of phi29 DNA polymerase, and 1 ⁇ phi29 reaction buffer (components are 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH7.5).
  • the virus blocking agent is Kansetin protein (commercially available from Shanghai Biotechnology Co., Ltd.).
  • the present invention also provides the use of the hand sanitizer based on virus blocking agents in hand cleaning and/or disinfection, preferably in the protection against the novel coronavirus during hand cleaning and/or disinfection.
  • the hand sanitizer of the present invention you only need to put an appropriate amount of hand sanitizer in your hands, wash your hands thoroughly, and dry them. After use, your hands will feel comfortable and non-irritating.
  • the third aspect of the present invention provides the use of the virus blocking agent in the preparation of an antibacterial and antiviral hand sanitizer, and the hand sanitizer is preferably a wash-free hand sanitizer.
  • the present invention has the following beneficial effects:
  • this product contains a new type of virus blocker, which has a strong affinity for the new coronavirus and other viruses, and can effectively block the virus from infecting human cells by adhering to the virus. It has an effective blocking effect on the new coronavirus and its different variants ( ⁇ , ⁇ , ⁇ , etc.), and has the advantages of being safer, having a higher antiviral effect, and being more durable. At the same time, protein will not irritate the user's hands, and can better protect the hand skin than other antiviral preparations.
  • the hand sanitizer of the present invention contains an effective amount of an environmentally friendly bactericide, so that it has both antiviral and bactericidal effects, and has a good killing effect on common bacteria and fungi such as Escherichia coli, Staphylococcus aureus, and Candida albicans.
  • the present invention adds glycerin as a moisturizer and fat-liquor, thereby achieving better skin care effects while killing bacteria.
  • the hand sanitizer product of the present invention has high stability and excellent bactericidal and antiviral effects.
  • the preparation process of the hand sanitizer of the present invention is simple, low-cost, and can be industrialized for production.
  • the present invention provides a method for improving the efficiency of protein expression in vitro.
  • the present invention is further described in detail by examples listed below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
  • the "antibacterial agent” in the present invention is generally referred to as an agent that can effectively control or kill microorganisms - bacteria, fungi, Preparations of bacteria and algae.
  • coronavirus in the present invention refers to a coronavirus that belongs to the Nidovirales order, Coronaviridae family, and the genus Coronavirus in the systematic classification and uses ACE2 as a binding receptor, including but not limited to SARS-CoV, MERS-CoV, SARS-CoV-2, etc.
  • ACE2 The ACE2 described in the present invention is also called ACEH, which is called angiotensin converting enzyme 2.
  • ACE2 is composed of 805 amino acids and is a type I transmembrane glycoprotein with a single extracellular catalytic domain.
  • ACE2 is a receptor protein for coronaviruses such as SARS-Cov-2 to infect human cells.
  • spike protein in the present invention refers to a protein on the surface of the coronavirus that can mediate viral attachment to target host cells directly (e.g., by interacting with a viral receptor (e.g., Ace2)) or indirectly (e.g., by mediating the interaction of one or more other proteins or molecules with a viral receptor (e.g., Ace2)).
  • a viral receptor e.g., Ace2
  • a viral receptor e.g., Ace2
  • the "D2P" system includes but is not limited to IVTT reaction (in vitro transcription and translation reaction).
  • IVTT reaction is preferred.
  • IVTT reaction corresponding to IVTT system, is the process of transcribing and translating DNA into protein in vitro.
  • in vitro protein synthesis system D-to-P system, D_to_P system, DNAto-Protein system
  • the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method, which has the same meaning as "in vitro cell-free protein synthesis system", “in vitro expression system”, “in vitro protein synthesis system”, “in vitro protein synthesis reaction system”, “cell-free protein synthesis system” and other expressions.
  • Protein in vitro 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 descriptions.
  • CFS system cell-free system
  • CFPS system cell-free protein synthesis system
  • the Kansetin protein described in the present invention is a new type of blocking protein independently developed and synthesized by Kangma (Shanghai) Biotechnology Co., Ltd. (hereinafter referred to as "Kangma Bio”).
  • the protein has a strong affinity for viruses such as the new coronavirus and achieves the effect of efficiently blocking the virus from infecting human cells by adhering to the virus.
  • the Kansetin protein has an effective blocking effect on different variants of the new coronavirus ( ⁇ , ⁇ , ⁇ , etc.), and the IC50 detection values are all at the pM level (the IC50 detection index refers to the concentration of the new protein when the new coronavirus is inhibited by 50%. In layman's terms, the lower the IC50 value, the better the blocking effect of the new protein).
  • the blocking efficiency of "Erta Delta" is the strongest, more than 10,000 times. Constantin protein has extremely high thermal stability and can be stably placed at room temperature for more than one year.
  • the protein components (such as 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 reference can be made to existing 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" and the gene modification methods provided by the references thereof, specifically including but not limited to: inserting the coding sequence into an intracellular free plasmid, integrating the coding gene into the cell genome, and combinations thereof.
  • exogenous means the dosage can be controlled and adjusted according to the needs of the system.
  • the host cell host cells are well known in the art, including but not limited to E. coli, CHO cells, Chinese hamster ovary, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, HEK-293 cells and many other cell lines. Fall armyworm (Spodoptera fr ⁇ giperda or Trichoplusiani)), amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataeaminuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae.
  • Saccharomyces sp. Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum), Physcomitrella patens, and Neurospora crassa.
  • the in vitro cell-free protein synthesis system includes, but is not limited to, an in vitro protein synthesis system of Escherichia coli, an in vitro protein synthesis system of bacteria, an in vitro protein synthesis system of mammalian cells (such as HF9, Hela, CHO, HEK293), an in vitro protein synthesis system of plant cells, an in vitro protein synthesis system of yeast cells, and an in vitro protein synthesis system of insect cells.
  • it is an in vitro protein synthesis system of yeast, more preferably, an in vitro protein synthesis system of Kluyveromyces, and most preferably, an in vitro protein synthesis system of Kluyveromyces lactis.
  • the in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, various detection methods and other technical elements of the present invention can also be independently selected from the following documents to select appropriate implementation methods or implementation methods, including but not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN10942 3497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A,
  • the optimized gene sequence of the target protein (such as Constin protein) was inserted into the pD2P plasmid and then added into the homemade Kluyveromyces lactis in vitro cell-free protein synthesis system to prepare the in vitro cell-free synthesis system using Kluyveromyces lactis NRRL Y-1140.
  • the in vitro cell-free protein synthesis system used in this example includes 50% (v/v) Kluyveromyces lactis cell extract, 22 mM tris (pH 8), 90 mM potassium acetate, 4.0 mM magnesium acetate, 3.0 mM nucleoside tris 19 phosphate mixture, 0.16 mM amino acid mixture, 22 mM potassium phosphate, 0.003 mg/mL amylase, 3% (w/v) polyethylene glycol (PEG-8000), 340 mM maltodextrin (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.
  • the above reaction system is placed in an environment of 22-30°C and incubated for about 20 hours.
  • the mixture was prepared according to the following formula: 2 mg/ml glycerol, 0.111 mg/ml disodium ethylenediaminetetraacetic acid dihydrate, 0.2 mg/ml benzalkonium chloride, 10 nM virus blocking protein prepared in Example 1, and deionized water as the solvent.
  • the mixture was prepared according to the following formula: 5 mg/ml glycerol, 0.111 mg/ml disodium ethylenediaminetetraacetic acid dihydrate, 0.2 mg/ml benzalkonium chloride, 10 nM virus blocking protein prepared in Example 1; and the solvent was deionized water.
  • the mixture was prepared according to the following formula: 10 mg/ml glycerol, 0.111 mg/ml disodium ethylenediaminetetraacetic acid dihydrate, 0.2 mg/ml benzalkonium chloride, 10 nM virus blocking protein prepared in Example 1; the solvent was Deionized water.
  • the mixture was prepared according to the following formula: 2 mg/ml glycerol, 0.111 mg/ml disodium ethylenediaminetetraacetic acid dihydrate, 4 mg/ml sodium chloride, 0.5 mg/ml chlorhexidine acetate, 10 nM virus blocking protein prepared in Example 1, and distilled water as solvent.
  • the mixture was prepared according to the following formula: 5 mg/ml glycerol, 0.111 mg/ml disodium ethylenediaminetetraacetic acid dihydrate, 4 mg/ml sodium chloride, 0.5 mg/ml chlorhexidine acetate, 10 nM virus blocking protein prepared in Example 1, and distilled water as solvent.
  • the mixture was prepared according to the following formula: 10 mg/ml glycerol, 0.111 mg/ml disodium ethylenediaminetetraacetic acid dihydrate, 4 mg/ml sodium chloride, 0.5 mg/ml chlorhexidine acetate, 10 nM virus blocking protein prepared in Example 1, and distilled water as solvent.
  • FBS Fetal Bovine Serum
  • Penicillin streptomycin (Pen-Strep dual antibody)
  • the HEK293T-ACE2 cells to be infected were inoculated in a 48-well cell culture plate.
  • the inoculation amount was 1.5 ⁇ 104 cells per well; when the pseudovirus infection experiment was performed the next day, the cell density was about 30%.
  • the SARS-CoV-2 (Omicron) S protein pseudovirus was taken out from -80°C and placed on ice to thaw naturally.
  • test sample was mixed with the pseudovirus dilution solution at a ratio of 1:1 and incubated at room temperature for 1 h.
  • hand sanitizers A-F can effectively block the infection of the new coronavirus pseudovirus (Omicron strain) on HEK293T-ACE2 cells.
  • the inhibition rate of hand sanitizers A, B, and C on the new coronavirus was 99.9%, and the inhibition rate of prescriptions D, E, and F on the new coronavirus was 95.5%.
  • Staphylococcus aureus (ATCC6538), provided by Beina Chuanglian Biotechnology Co., Ltd., cultured at 3 generation
  • Candida albicans (ATCC10231), provided by Beina Chuanglian Biotechnology Co., Ltd., cultured at the 3rd generation
  • Gender female; Weight: 2.0kg ⁇ 3.0kg.
  • Rearing environment ordinary animal room, room number: rabbit room 321, temperature of the rearing room 16°C ⁇ 26°C, relative humidity 40% ⁇ 70%,
  • the hand sanitizer based on virus blockers provided by the present invention has the dual effects of antibacterial and antiviral, and has a good killing effect on common bacteria and fungi such as Escherichia coli, Staphylococcus aureus, Candida albicans, etc., and has a good protective effect on coronaviruses such as the new coronavirus.
  • the protein will not irritate the user's hands, and has the effect of protecting the hand skin.

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Abstract

提供一种基于病毒阻断剂的洗手液及其应用，该洗手液具备抗菌抗病毒的作用，对常见的大肠杆菌、金黄色葡萄球菌、白色念珠菌等细菌和真菌都具有良好的杀灭效果，且通过添加了病毒阻断剂，使得洗手液能对新冠病毒等冠状病毒起到很好防护作用，同时蛋白质不会刺激使用者手部，起到保护手部皮肤的效果。

Description

一种基于病毒阻断剂的洗手液及其应用 技术领域
本发明涉及洗手液技术领域，尤其涉及一种基于病毒阻断剂的洗手液及其应用。
背景技术
洗手用品属于皮肤清洁剂中的一种，近年来洗手液逐渐成为传统洗手用品的替代品。洗手液具有性能温和、不刺激皮肤、使用方便、抗硬水、易清洗，泡沫丰富和洗后皮肤润滑感好等特点，且清洁卫生，易于存放。尤其采用瓶体泵头挤压式取量，避免了不同人员使用时的交叉感染，因而受到了广大消费者的普遍欢迎。洗手液的成分包括表面活性、润肤保湿剂、增稠剂、pH调节剂、色素、珠光剂、天然活性成分等。洗手液是一种用于清洁手部的护肤清洁液，可以清除手上的污垢和附着的细菌和病毒等微生物。
洗手液是一种用来清洁手部的护肤清洁液，使用洗手液用来对使用者手部进行清洗，能够对使用者手部进行清洁去污、护理皮肤，并对手部进行彻底清洁，且现有大多数洗手液都具备消毒功能，能够在清洁的同时对手部进行消毒，进而使得手部更加洁净。
在倡导节约水资源、节约用水的今天，免洗洗手液的应用环境更加广泛，它仅需涂抹在手部，并持续使双手相互揉搓，进而将免洗洗手液抹匀，免洗洗手液会随着自身的挥发进而自动带走手上的污垢，并对手部进行消毒，且免洗洗手液不受制于使用环境，无水环境下亦可进行手部的清洁消毒。通常应配有消毒成分和挥发性的溶剂。为了避免过多的泡沫产生，免洗型洗手液中起洗涤作用的表面活性剂成分的含量不宜过多，一般在2％-8％，并选则一些低沸点的醇类溶剂加入，在清洗手上的污垢后，溶剂会自动挥发，无需再用水冲洗。为了保护手部皮肤，许多免洗型洗手液中还添加了一定的保湿成分，常用的保湿剂有甘油和聚乙二醇系列产品等。现在市面上最简单的免洗型洗手液配方类似消毒湿巾，它采用具有挥发性的组分制备而成。
现有技术中制备免洗消毒洗手液时，会向免洗消毒洗手液中添加大量化学成 分，故会使得洗手液较为伤手，不够柔和，如专利CN113081881A公开了一种抗菌抗病毒的免洗洗手液以儿茶素类物质作为抗菌抗病毒活性组分；专利CN110478293A公开了一种免洗消毒洗手液，其采用花椒果提取物和连翘提取物作为抗菌成分，但需要丙烯酸(酯)类/C10-30烷醇丙烯酸酯交联聚合物作为增稠剂和三乙醇胺和乙醇等化学成分；专利CN101766552B公开了一种免洗抗菌洗手液，其原料配方包括氯己定盐1g-10g、硝酸银5μg-1000μg、醇类物质30-90ml、护肤剂2g-50g、增粘剂0.5g-3g、表面活性剂0.5g-3g、pH调节剂0.5g-5g、去离子水15ml-50ml；专利CN104173236B公开了一种萜类方向抗菌免水洗手液，由97％天然芳樟醇0.1％～0.3％、无患子精油0.2％～1.0％、白樟油2.0％～5.0％、芳樟水5.0％～40.0％、卡波姆-940 0.15％～0.3％、三乙醇胺0.15％～0.30％和天然色素0.005％～0.015％的原料配制而成。
同时，市面上现有的免洗洗手液产品均不含具有新冠病毒(SARS-CoV-2)阻断效果的活性蛋白组分。
发明内容
本发明针对现有技术存在的上述问题，提供一种基于病毒阻断剂的洗手液及其应用，该洗手液添加了一种带有新冠病毒(SARS-CoV-2)阻断效果的蛋白质，使得洗手液能对新冠病毒起到很好防护作用，同时蛋白质不会刺激使用者手部，起到保护手部皮肤的效果，故制备此种带有新冠病毒(SARS-CoV-2)阻断效果的免洗洗手液。
本发明提供一种基于病毒阻断剂的洗手液，其特征在于：包含有效量的病毒阻断剂，所述病毒阻断剂为一种病毒阻断蛋白具有能与冠状病毒的S蛋白特异性结合而使得病毒失活的刺突结合域，优选为(SARS-CoV-2)病毒。
本发明“有效量的病毒阻断剂”是指洗手液在使用时洗手液中的病毒阻断剂含量能够有效使病毒丧失侵染机体的功能和途径，最终达到阻断新冠病毒传播及感染的效果的量，其可视为中和能力，利用高亲和力多角度捕获黏连新冠病毒的特性，进而使病毒丧失侵染机体的功能和途径的用量。在一种可行的实施方案中，所述病毒阻断剂的有效量为0.1nM以上，比如为1-100nM，优选为5-50nM，更优选为10-20nM。
进一步地，本发明提供的基于病毒阻断剂的洗手液还包含如下组分中一种或多种：
(1)保湿剂，用于皮润肤保湿。
保湿剂可在皮肤表面水分挥发后留下一层保护膜，阻止或减缓皮肤内部水分的流失，保持皮肤的润湿性。
(2)增稠剂，用于改善洗手液产品的粘度。在使用洗手液时，如果粘度太小，不易在手中停留，则去污能力会受到影响。一般情况下，粘度为0.5Pa，较为理想。但当总表面活性剂含量为10％时，溶液粘度调节剂，洗手液具有一定的粘稠度既可以改善产品外观、使产品具有一定的稳定性，也可方便使用。
(3)抑菌剂，可用于有效地控制或杀死皮肤表面的微生物，如细菌、真菌和藻类等的制剂。
(4)金属离子螯合剂，所述的金属螯合剂选自EDTA、8-羟基喹啉中的一种或其组合。
(5)表面活性剂，其基本功能是用于祛除手上的油垢和污垢，并产生一定的泡沫。选择表面活性剂总的原则是加强去污活性与安全保护性。本发明所述的表面活性剂选自阴离子表面活性剂、两性表面活性剂和非离子表面活性，优选阴离子表面活性剂作为主表面活性剂。
(6)加脂剂，为了避免洗手液的脱脂作用使皮肤感到干燥，可在洗手液中加入一定的加脂剂。
(7)外观调节剂，为了使产品令人感到愉悦，可在洗手液中加入适当的香味剂和颜料。
进一步地，所述的外观调节剂选自香味剂、颜料或其组合。
进一步地，本发明的洗手液水溶剂的质量占比范围为大于等于90％，优选地，大于等于95％，更优选地，大于等于99％；优选地，所述水溶剂为水、蒸馏水、无菌水、超纯水以及去离子水中的一种或多种的组合。
进一步地，所述的保湿剂选自1,2-丙二醇、大分子量透明质酸钠、中分子量透明质酸钠、小分子量透明质酸钠、寡聚透明质酸钠、乳酸钠、尿囊素、三甲基甘氨酸、液体石蜡、甘油、乙二醇、山梨醇、丙二醇和烷基糖苷中的一种或多种，优选地，所述保湿剂选自甘油。优选地，所述保湿剂的含量为0-100mg/mL，优选为0.1-50mg/mL，更优选为1-20mg/mL。
进一步，所述增稠剂是无机盐，选自氯化钠、氯化铵和氯化钾中的一种或多种，他们价格便宜，增稠效果好，使用方便。所述增稠剂的含量为0-10mg/mL， 优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
进一步地，所述抑菌剂可选自选自选自苯扎氯铵、醋酸氯己定、盐酸氯己定、硫酸氯己定、硝酸氯己定、氢溴酸氯己定、氢碘酸氯己定和葡萄糖酸氯已定中的一种或多种。优选地，所述的洗手液含有有效含量的抑菌剂。优选地，所量为0.01-10mg/mL，优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
进一步地，所述的增稠剂为羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素中的一种或几种。
进一步地，所述的金属螯合剂选自EDTA、8-羟基喹啉中的一种或其组合。所述洗手液中的金属螯合剂的含量为0-10mg/mL，优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
进一步地，所述的表面活性剂为月桂酰胺丙基甜菜碱、椰油酰胺丙羟基甜菜碱、月桂酰胺丙基羟磺基甜菜碱、月桂酰胺丙基羟磺基甜菜碱和月桂基两性醋酸钠中一种或多种。优选地，所述表面活性剂的含量为0-10mg/mL，优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
进一步地，所述的加脂剂选自甘油脂肪酸酯、丙二醇脂肪酸酯、脂肪酸酯、烷氧基羧酸、烷氧基醇、脂肪醇和脂肪酸中的一种或多种。优选地，所述的脂肪酸脂选自硬脂酸十六烷醇脂、鲸蜡醇十六酸脂，硬脂酸鲸蜡醇酯、月桂酸异丙醇酯、豆蔻酸异丙醇酯、棕榈酸异丙醇酯中的一种或多种。优选地，所述的脂肪醇可以包括但不限于辛基十二烷醇、月桂醇、十四烷醇、十六烷醇、十八烷醇、二十二醇以及它们的组合。还可以为润肤剂使用的合适的醚，例如桉叶脑、鲸蜡硬脂酸酯基葡萄苷、二甲基异山梨醇聚甘油基3-3十六烷基醚、聚甘油基-3-癸基十四醇、丙二醇十四烷基醚中的一中或多种。进一步优选地，所述的脂肪酸选自月桂酸、棕榈酸、硬脂酸、亚油酸、十六烷酸中的一种或多种。优选地，所述加脂剂的含量为0-100mg/mL，优选为0.5-50mg/mL，更优选为1-30mg/mL。
进一步地，所述的香味剂优选为多醇类、薄荷油和/或香精等。优选地，所述洗手液中的香味剂的含量为0-20ng/mL，优选为0.1-10ng/mL，更优选为1-5ng/mL。
优选地，所述的香精的香型可选自清凉香、叶香、柑橘香、果香、花香、辛香、草香、树脂香、膏香、木香、壤香、苔香、动物香、皮革香、醛香等。
在一种可行的实施方案中，所诉的颜料为天然着色剂主要有红曲红、高粱红、 栀子黄、辣椒红、甜菜红、姜黄、β-胡萝卜素、叶绿素、胭脂虫红等。
进一步地，所述病毒阻剂还具有以下特征中的一个或多个的组合：
(1)聚合分子；
(2)连接分子；
(3)酸性结构域；
(4)先导肽；
(5)标签蛋白；
(6)病毒阻断剂中具有的各个组成部分之间按N-C端的顺序连接。
进一步地，所述聚合分子为具有聚合功能的单链蛋白、多肽，所述聚合功能是指所述单链蛋白具有天然聚合为多聚体的功能，优选地，聚合分子包含链霉亲和素，进一步优选地，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；
本发明所述的连接分子可以保持三维结构并提高重组蛋白的生物活性。所述的连接分子包含有荧光蛋白、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，通过对eGFP删除部分氨基酸得到；或：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
进一步，所述酸性结构为带有负电的氨基酸残基短链聚合物，进一步地，酸性结构具有以下特征中的一个或多个的组合：
(1)酸性结构设置在C末端；
(2)所述的短链聚合物的氨基酸残基的个数为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；
进一步地，所述酸性结构含有至少两个连续的D天冬氨酸氨基酸，和/或至少 两个连续的E谷氨酸氨基酸，和/或至少一组D天冬氨酸氨基酸和E氨基酸的组合。例如，酸性结构含有10个连续的天冬氨酸(SEQIDNO:11)、10个连续的E谷氨酸(SEQIDNO:12)，8个连续的D天冬氨酸和8个连续的E谷氨酸(SEQIDNO:14)，5个D-E(SEQIDNO:13)。
进一步，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。
进一步地，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，也即标签蛋白的C端在阻断结合分子单元N端一侧。
进一步地，所述刺突结合域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，优选地，阻断分子含有与SEQIDNO:7-9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。
进一步地，所述的刺突结合域包含与如SEQ ID NO.7-9任一氨基酸序列所示的多肽具有一个或几个氨基酸的置换、缺失或添加的序列，所述置换、缺失或添加的氨基酸的数量可以为1-20个，优选为1-10个，如1、2、3、4、5、6、7、8、9、10。
优选地，为了增加重组蛋白与刺突蛋白的结合亲和力，所述的阻断剂包含至少一个刺突结合域，优选包含两个以上的刺突结合域，如1-10个，优选2-8个，最优选4-8个。
进一步地，本发明的的病毒阻断剂通过蛋白质工程技术进行生产，如利用宿主细胞内表达、细胞分泌性表达或体外无细胞合成表达获得。
进一步地，所述的病毒阻断剂优选通过康码(上海)生物科技有限公司生产的体外无细胞蛋白合成体系进行生产，如D2P系统，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(康码(上海)生物科技有限公司)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)。
所述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)。
作为本发明最优选的技术方案，所述的病毒阻断剂为康码(上海生物科技有限公司市售康斯汀(Kansetin)蛋白。
本发明还提供所述的基于病毒阻断剂的洗手液在手部清洁和/或消毒中的应用，优选在手部清洁和/或消毒时防护新冠病毒中的应用。使用本发明的洗手液时，只需将适量洗手液置于手中，将手部全面清洗，晾干即可，使用后，手感舒适，无刺激，
本发明第三个方面提供所述病毒阻断剂在制备抗菌抗病毒的洗手液中的应用，优选所所述的洗手液为免洗洗手液。
本发明与现有技术相比，具有如下有益效果：
(1)与传统含有酒精的洗手液相比，本产品含新型病毒阻断剂，该阻断剂对新冠病毒新冠病毒等病毒具有极强亲和力，并通过对病毒的粘连达到高效阻断病毒侵染人体细胞的效果，对新冠病毒及其不同变种(α,β,δ等)都具有效的阻断作用，具有更安全、抗病毒效果更高、更持久等优点；同时蛋白质不会刺激使用者手部，相比其它抗病毒制剂更加能够起到保护手部皮肤的效果。
(2)本发明的洗手液含有有效含量的环保杀菌剂，使其在具有抗病毒功效同时兼具杀菌功效，对常见的大肠杆菌、金黄色葡萄球菌、白色念珠菌等细菌和真菌都具有良好的杀灭效果。
(3)本发明通过添加甘油的保湿剂、加脂剂，杀菌的同时具有更好的护肤效果。
(4)本发明的洗手液产品稳定性高，杀菌和抗病毒效果优。
(5)本发明的洗手液制备工艺简单、成本低，可实现工业化生产。
具体实施方式
本发明提供了一种提高蛋白体外表达效率的方法，为使本发明的目的、技术方案及效果更加清楚、明确，以下列举举实例对本发明作进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
术语
本发明中的“抑菌剂”又称通常是指能有效地控制或杀死微生物——细菌、真 菌和藻类的制剂。
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。
本发明所述的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。
本发明中的“刺突蛋白”是指在冠状病毒表面上，并且能够直接(例如通过与病毒受体(例如Ace2)相互作用)或间接(例如通过介导一种或多种其它蛋白或分子与病毒受体(例如Ace2)的相互作用)介导病毒附着于靶宿主细胞。
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。
本发明所述康斯汀(Kansetin)蛋白是康码(上海)生物科技有限公司(以下简称“康码生物”自主研发并合成的一款新型阻断蛋白，该蛋白对新冠病毒新冠病毒等病毒具有极强亲和力，并通过对病毒的粘连达到高效阻断病毒侵染人体细胞的效果。经湖北省疾控中心(CDC)卫生检验检测研究所权威鉴定，康斯汀蛋白对新冠病毒新冠病毒不同变种(α,β,δ等)都具有效阻断作用，IC50检测值均在pM级(IC50检测指标是指新冠病毒被新型蛋白抑制到50％时新型蛋白的浓度。通俗来说，IC50值越低，表明新型蛋白的阻断效果越好)。对新冠病毒变异株“德 尔塔Delta”的阻断效率最强超过1万倍以上。康斯汀蛋白具有极高的热稳定性，室温下可稳定放置1年以上。多家第三方检测机构平行安全性测试显示，康斯汀蛋白在100倍使用剂量喷涂、口服、注射的情况下，对实验动物均无任何急性、毒性反应，表现安全(参考康码公司官网：https://www.ourproteinfactory.com/)。
本发明所述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。
所述宿主细胞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 frμgiperda)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，所述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，所述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。优选为酵母体外蛋白合成体系，更优选即为克鲁维酵母体外蛋白合成体系，最优选地为乳酸克鲁维酵母体外蛋白合成体系。
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。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于阐述说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如“Sambrook等人，分子克隆：实验室手册(New York:Cold SpringHarbor LaboratoryPress,1989)”、《无细胞蛋白合成实验手册》“Edited by AlexanderS.Spirin and James R.Swartz.Cell-free protein ynthesis:methods and protocols[M].2008”等文献中所述的实验条件，或者按照制造厂商所建议的条件，或者按照、参考上文所述的具体实施方式指引的条件。除非另外说明，否则本发明中提及的百分比和份数是重量百分比和重量份数。
本文前述提及的序列，汇总如表1所示.
表1 序列表


实施例1 病毒阻断蛋白的制备
将优化后的目标蛋白(如康斯汀蛋白)的基因序列插入到pD2P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)制备体外无细胞合成体系。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。将上述反应体系置于22-30℃的环境中，静置孵育约20h。
反应后的溶液，4000rpm 44℃离心10min。上清液加入5％(w/v)的硫酸铵粉末，充分搅拌溶解，4000rpm 44℃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 44℃离心10min。弃上清，沉淀用30％(w/v)的硫酸铵溶液漂洗1次。12000rpm 4℃离心10min。沉淀用PBS重悬，充分溶解。12000rpm，4℃离心10min，用0.22μm的针筒滤器过滤后，即获得病毒阻断蛋白，KMds4℃保存待用，BCA法测定蛋白浓度，要求不低于1mg/ml。
实施例2 洗手液A：
按如下配方进行配置：甘油2mg/ml，二水合乙二胺四乙酸二钠0.111mg/ml，苯扎氯铵0.2mg/ml，实施例1所制备的病毒阻断蛋白10nM，溶剂为去离子水。
实施例3 洗手液B：
按如下配方进行配置：甘油5mg/ml，二水合乙二胺四乙酸二钠0.111mg/ml，苯扎氯铵0.2mg/ml，实施例1所制备的病毒阻断蛋白10nM；溶剂为去离子水。
实施例4 洗手液C：
按如下配方进行配置：甘油10mg/ml，二水合乙二胺四乙酸二钠0.111mg/ml，苯扎氯铵0.2mg/ml，实施例1所制备的病毒阻断蛋白10nM；溶剂为 去离子水。
实施例5：洗手液D：
按如下配方进行配置：甘油2mg/ml，二水合乙二胺四乙酸二钠0.111mg/ml，氯化钠4mg/ml，醋酸氯己定0.5mg/ml，实施例1所制备的病毒阻断蛋白10nM，溶剂为蒸馏水。
实施例6：洗手液E：
按如下配方进行配置：甘油5mg/ml，二水合乙二胺四乙酸二钠0.111mg/ml，氯化钠4mg/ml，醋酸氯己定0.5mg/ml，实施例1所制备的病毒阻断蛋白10nM，溶剂为蒸馏水。
实施例7：洗手液F：
按如下配方进行配置：甘油10mg/ml，二水合乙二胺四乙酸二钠0.111mg/ml，氯化钠4mg/ml，醋酸氯己定0.5mg/ml，实施例1所制备的病毒阻断蛋白10nM，溶剂为蒸馏水。
实施例8：对新冠病毒抑制活性检测
1.实验材料
T-25flask:surface area 25cm2
DMEM培养基
Fetal Bovine Serum(FBS胎牛血清)
Penicillin streptomycin(Pen-Strep双抗)
0.25％Trypsin-EDTA胰酶
Puromycin
1x PBS
DMSO(Tissue Culture Grade)二甲亚砜
75％Ethanol乙醇(清洁用)
1.仪器
电子天平
电动移液器
生物安全柜
2.实验步骤
2.1.样品信息
取新配制的6个洗手液样品，检测细胞学活性。
2.2.细胞铺板
将待感染HEK293T-ACE2细胞接种于48孔细胞培养板中。接种量为1.5×104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。
血细胞计数板计算方法：
1)在细胞60-70％汇合度时传代，不让细胞长到多于80％。
2)在生物安全柜内，倒掉培养基，并加入1-2mL预热的1x PBS。在生物安全柜内静置1-2min之后倒掉PBS。
3)在T-25内加入1mL预热的trypsin-EDTA胰酶，放入37°培养箱消化1-2min，不多于2min。
4)在T-25内加入2-3mL完全培养基中和胰酶，用移液器吹打细胞，使细胞分散。转移至15mL离心管；1,000×rpm,4℃离心5分钟。
5)用5mL完全培养基重悬细胞，轻轻吹打使细胞分散。取出100-200微升细胞悬液至1个1.5毫升离心管。
6)在血细胞计数板上盖上玻璃片(注意小心不要打碎玻璃片)。从细胞悬液里取出10微升并加入血细胞计数板和玻璃片之间的缝隙中。
7)数网格1-5内细胞个数，并取平均值。
8)混合完全培养基及细胞悬液，并进行铺板。
2.3.感染(样品检测)
从-80℃取出SARS-CoV-2(Omicron)S蛋白假病毒，置于冰上自然融化。
待完全融化后，将假病毒用完全培养基进行270倍稀释。取40ul假病毒，加入到10.8ml完全培养基中，混合均匀。没用完的假病毒稀释液存放于-80℃冰箱。
将检测样品与假病毒稀释液1:1混合，室温孵育1h。
取样品220ul于灭过菌的1.5mL离心管中，加入假病毒稀释液220ul， 混合均匀。放置于室温，孵育1h，同时，设置阳性对照(100nm实施例1所制备的重组蛋白)和阴性对照品PBS(0nM)。
2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，每个样品加2个复孔。将48孔板放入培养箱中培养6小时。
2 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。
2.4.细胞裂解
v用水稀释5x细胞裂解液至1x，细胞裂解液总体积＝65μL/孔x孔总数x 1.1。多余的1x裂解液可放在4°冰箱。
v感染的细胞在孔板中培养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。
v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。v 15-20分钟后，立即检测荧光素酶的活性，或置于-20℃保存。
2.5.检测荧光
v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，将混合液加入白色384孔板中；
v立即用Perkin Elmer EnVision 2102多功能酶标仪上检测荧光素酶的活性，并判定洗手液样品的中和效率。
3.实验结果
取新配制的洗手液A-F，检测细胞学活性。
结果分析：相比较于PBS溶液(阴性对照)，洗手液A-F均能有效阻断新冠假病毒(Omicron株)对HEK293T-ACE2细胞的感染作用。经计算，洗手液A、B、C对新冠病毒的抑制率为99.9％，处方D、E、F对新冠病毒的抑制率为95.5％。
实施例9：抗(抑)菌产品抑菌性能试验
1.实验器材
1.1试验菌株：大肠杆菌(8099)，由广东省食品微生物安全工程技术研究开发中心提供，培养第3代
金黄色葡萄球菌(ATCC6538)，由北纳创联生物科技有限公司提供，培养第3 代
白色念珠菌(ATCC10231)，由北纳创联生物科技有限公司提供，培养第3代
1.2培养基：NA/20220808、SDA/20220808
1.3仪器设备：电热恒温培养箱LRH-250F(WPE-RH0342)、生物安全柜BSC-1604ⅡB2(WPE-RH0311)
2.实验方法
2.1检验依据：GB 15979-2002《一次性使用卫生用品卫生标准》
2.2检测环境：温度：24.8℃，湿度：49％RH
2.3抑菌性能试验：受试物作用2min，5min，10min，20min，试验重复3次
3实验结果
3.1经3次重复试验，洗手液A分别作用2min，5min，10min，20min对大肠杆菌、金黄色葡萄球菌、白色念珠菌抑菌率均≥90％，产品有较强抑菌作用，符合GB 15979-2002《一次性使用卫生用品卫生标准》附录C4.2的要求，结果见表2、3和4：
表2 对金黄色葡萄球菌的抑菌效果的试验结果
表3 对金黄色葡萄球菌的抑菌效果的试验结果

表4 对白色念珠菌的抑菌效果的试验结果
由此可见，洗手液A在试验温度20～25℃，湿度50～70％RH条件下，受试物消毒作用60min后，对1m3密闭房间空气中人工污染的白色葡萄球菌的平均杀灭率均＞99.91％，符合《消毒技术规范》(2002年版)2.1.3.4的要求
3.2重复上述实验，实验证实洗手液B-F在试验温度20～25℃，湿度50～70％RH条件下，受试物消毒作用60min后，对1m3密闭房间空气中人工污染的白色葡萄球菌的平均杀灭率均＞99.9％，符合《消毒技术规范》(2002年版)2.1.3.4的要求。
实施例10：多次完整皮肤刺激试验
1.实验器材
1.1实验动物和饲养环境
动物品种：新西兰白兔；等级：普通级；
来源：上海市松江区车墩实验动物良种场有限公司，生产许可证号：SCXK(沪)2022-0001，质量合格证号：20220001000062；
性别：雌性；体重：2.0kg～3.0kg。
饲料来源：江苏省协同医药生物工程有限责任公司，
生产许可证号：苏饲证(2019)01008。
饲养环境：普通级动物房，房间号：兔房321，饲养室温度16℃～26℃，相对湿度40％～70％，
实验动物使用许可证号：SYXK(沪)2021-0023。
1.3仪器设备：电子计数秤ACS-30(WPE-TL0055)
2实验方法
2.1.检验依据：《消毒技术规范》(2002年版)2.3.3
2.2操作程序：
1)试验前约24h，将实验动物背部脊柱两侧被毛剃掉，去毛范围左右各约3cm×3cm不得损伤皮肤。
2)次日称重后将受试物0.5ml涂抹于面积为2.5cm×2.5cm左侧去毛皮肤上，右侧作为空白对照。在涂抹后4h后用温水除去残留物。每天涂抹1次，连续涂抹14d。
3)临床观察：在每次涂抹24h后观察结果，同时为了便于受试物的涂抹和结果观察，需要时可剪毛。记录每天每只动物红斑和水肿的积分。
4)判断标准和评分：按《消毒技术规范》(2002年版)2.3.3.4的评价规定进行多次完整皮肤刺激反应积分评分，对样品和对照的红斑和水肿进行观察并评分，计算出每天每只动物皮肤刺激反应积分均值(刺激指数)，按照皮肤刺激强度分级来判断皮肤刺激强度
3实验结果
经检验，该样品对新西兰白兔多次完整皮肤刺激后，每天每只动物皮肤刺激反应积分均值(刺激指数)为0.00，属无刺激性，符合《消毒技术规范》(2002年版)标准要求，结果见表5：
表5 新西兰白兔多次完整皮肤刺激性试验结果

由此可见，洗手液A对新西兰白兔多次完整皮肤刺激后，每天每只动物皮肤刺激反应积分均值(刺激指数)为0.00，属无刺激性，符合《消毒技术规范》(2002年版)标准要求。
3.2重复上述实验，实验证实洗手液B-F在32m3房间里作用60min后，对空气自然菌的消亡率3次试验结果均>90％，符合《消毒技术规范》(2002年版)2.1.3.5的要求。
通过以上实验证明，本发明提供的基于病毒阻断剂的洗手液，具备抗菌抗病毒的双重功效，对常见的大肠杆菌、金黄色葡萄球菌、白色念珠菌等细菌和真菌都具有良好的杀灭效果，且对新冠病毒等冠状病毒起到很好防护作用，同时蛋白质不会刺激使用者手部，起到保护手部皮肤的效果。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (20)

  1. 一种基于病毒阻断剂的洗手液，其特征在于：包含有效量的病毒阻断剂，所述病毒阻断剂具有能与冠状病毒的S蛋白特异性结合而使得病毒失活的刺突结合域，优选地，所述病毒阻断剂的有效量为0.1nM以上，优选为1-50nM，更优选为10-20nM。
  2. 根据权利要求1所述的基于病毒阻断剂的洗手液，其特征在于：所述洗手液还包含如下(1)～(7)组份中的一种或多种：
    (1)保湿剂；
    (2)增稠剂；
    (3)抑菌剂；
    (4)金属离子螯合剂；
    (5)表面活性剂；
    (6)加脂剂；
    (7)外观调节剂。
  3. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于，所述增稠剂是无机盐，选自氯化钠、氯化铵和氯化钾中的一种或多种；所述增稠剂的含量为0.01-50mg/mL，优选为0.1-40mg/mL，更优选为1-10mg/mL。
  4. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述抑菌剂可选自苯扎氯铵、醋酸氯己定、盐酸氯己定、硫酸氯己定、硝酸氯己定、氢溴酸氯己定、氢碘酸氯己定和葡萄糖酸氯已定中的一种或多种；优选地，所述的洗手液含有有效含量的抑菌剂；优选地，所述抑菌剂的含量为0.01-10mg/mL，优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
  5. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述的保湿剂选自1,2-丙二醇、大分子量透明质酸钠、中分子量透明质酸钠、小分子量透明质酸钠、寡聚透明质酸钠、乳酸钠、尿囊素、三甲基甘氨酸、液体石蜡、甘油、乙二醇、山梨醇、丙二醇和烷基糖苷中的一种或多种，优选地，所述保湿剂选自甘油；优选地，所述保湿剂的含量为0-100mg/mL，优选为0.1-50mg/mL，更优选为1-20mg/mL。
  6. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述的金 属螯合剂选自EDTA、8-羟基喹啉中的一种或其组合；所述洗手液中的金属螯合剂的含量为0-10mg/mL，优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
  7. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述的表面活性剂为月桂酰胺丙基甜菜碱、椰油酰胺丙羟基甜菜碱、月桂酰胺丙基羟磺基甜菜碱、月桂酰胺丙基羟磺基甜菜碱和月桂基两性醋酸钠中一种或多种；优选地，所述表面活性剂的含量为0-10mg/mL，优选为0.05-5mg/mL，更优选为0.1-1mg/mL。
  8. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述的加脂剂选自甘油脂肪酸酯、丙二醇脂肪酸酯、脂肪酸酯、烷氧基羧酸、烷氧基醇、脂肪醇和脂肪酸中的一种或多种；优选地，所述加脂剂的含量为0.1-100mg/mL，优选为0.5-50mg/mL，更优选为1-30mg/mL。
  9. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述的外观调节剂包含香味剂和/或颜料；所述的香味剂优选为多醇类、薄荷油和/或香精；优选地，所述外观调节剂的含量为0-20ng/mL，优选为0.1-10ng/mL，更优选为1-5ng/mL。
  10. 根据权利要求2所述的基于病毒阻断剂的洗手液，其特征在于：所述病毒阻断剂还具有以下特征中的一个或多个的组合：
    (1)聚合分子；
    (2)连接分子；
    (3)酸性结构域；
    (4)先导肽；
    (5)标签蛋白；
    (6)病毒阻断剂中具有的各个组成部分之间按N-C端的顺序连接。
  11. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：所述聚合分子为具有聚合功能的单链蛋白、多肽，所述聚合功能是指所述单链蛋白具有天然聚合为多聚体的功能，优选地，所述聚合分子包含链霉亲和素，进一步优选地，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。
  12. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：所述的连接分子包含有荧光蛋白、人免疫球蛋白G4、Fc以及HSA中的任意一种或多种，优选地，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。
  13. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：所述酸性结构域为带有负电的氨基酸短链聚合物，进一步地，酸性结构具有以下特征中的一个或多个的组合：
    (1)酸性结构设置在C末端；
    (2)所述的短链聚合物的氨基酸残基的个数为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。
  14. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：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。
  15. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：所述标签蛋白包含与SEQ ID NO: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；和/或，
    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。
  16. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：所述刺突结合域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，优选地，阻断分子含有与SEQID NO:7-9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。
  17. 根据权利要求10所述的基于病毒阻断剂的洗手液，其特征在于：所述的病毒阻断剂通过体外无细胞蛋白合成体系制备。
  18. 根据权利要求1-17任一项所述的基于病毒阻断剂的洗手液，其特征在于： 所述洗手液水溶剂的质量占比范围为大于等于90％，优选地，大于等于95％，更优选地，大于等于99％；优选地，所述水溶剂为水、蒸馏水、无菌水、超纯水以及去离子水中的一种或多种的组合。
  19. 根据权利要求1-18任一项所述的基于病毒阻断剂的洗手液在手部清洁和/或消毒中的应用，优选在手部清洁和/或消毒时防护新冠病毒中的应用。
  20. 根据权利要求1-18任一项所述的病毒阻断剂在制备抗菌抗病毒的洗手液中的应用，优选所述的洗手液为免洗洗手液。
PCT/CN2023/127755 2022-10-31 2023-10-30 一种基于病毒阻断剂的洗手液及其应用 WO2024093910A1 (zh)

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