WO2022067879A1 - 一种能够结合多种病毒的蛋白及其应用 - Google Patents

一种能够结合多种病毒的蛋白及其应用 Download PDF

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WO2022067879A1
WO2022067879A1 PCT/CN2020/120593 CN2020120593W WO2022067879A1 WO 2022067879 A1 WO2022067879 A1 WO 2022067879A1 CN 2020120593 W CN2020120593 W CN 2020120593W WO 2022067879 A1 WO2022067879 A1 WO 2022067879A1
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protein
virus
diarrhea
norovirus
composite material
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PCT/CN2020/120593
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French (fr)
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王大鹏
刘丹蕾
张子蕾
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上海交通大学
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    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61P1/12—Antidiarrhoeals
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • 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
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • 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 present application belongs to the field of biotechnology, and specifically relates to a protein capable of binding multiple viruses and applications thereof.
  • Binding of viruses to host cells is a key step for viruses to invade cells or infect the host. How to reduce the binding rate of virus to host cells has become an important way to treat viral infection. Therefore, mass production of specific molecules that can bind to viruses becomes the key to blocking virus (re)infection of the host. In addition, this specific molecule can also be used for the capture and concentration of target viruses.
  • HuNoVs human noroviruses
  • the specific molecules that can be combined with viruses in the prior art usually have the following disadvantages: long production cycle and high cost; poor ability to combine with viruses, narrow application range; large environmental pollution; strong dependence on experimental animals; poor safety. Therefore, there is still an urgent need for an easy-to-produce, low-cost, safe, environmentally friendly, efficient, and broad-spectrum product.
  • the present invention provides a protein synthesized by chemical means or genetic engineering, a composite material, a device and a method for detecting diarrhea-causing viruses, and for enriching diarrhea-causing viruses.
  • Viral composites and methods pharmaceutical compositions for preventing and/or treating diarrhea-causing viral infections, isolated DNA encoding the protein, expression vectors, host cells.
  • the present invention provides:
  • a protein synthesized by chemical means or genetic engineering characterized in that the protein has a functional domain capable of binding to the capsid protein of a diarrhea-causing virus, wherein the diarrhea-causing virus includes Calicivirus and/or Reovirus; and the amino acid sequence of said protein is as described in (a) or (b) below:
  • (b) has an amino acid sequence obtained by substituting, deleting, and/or adding one or more amino acids to the amino acid sequence of (a).
  • Said calicivirus is selected from Norovirus; said Norovirus includes GI-GX group; preferably, said Norovirus is selected from GI, GII, GIV, GVIII and/or GIX group; also preferably, said The norovirus is selected from groups GI and/or GII; and/or
  • Said reovirus is selected from rotavirus; said rotavirus includes group A-H; preferably, said rotavirus is selected from group A rotavirus; also preferably, said rotavirus is selected from group A Type G1P[8] and/or G9P[8].
  • a composite material for detecting a diarrhea-causing virus comprising the protein according to any one of (1) to (3), wherein the protein is bound to a solid-phase support and a label selected from the group consisting of at least one of them.
  • a device for detecting a diarrhea-causing virus comprising the composite material according to (4).
  • a method for detecting a virus that can cause diarrhea comprising:
  • Step A1 contacting the sample to be tested with the composite material according to (4);
  • step A2 the composite material obtained by the contact is detected for the virus that can cause diarrhea.
  • a composite material for enriching diarrhea-causing viruses comprising the protein according to any one of (1) to (3), wherein the protein is bound to a solid support.
  • a method for enriching a diarrhea-causing virus comprising:
  • Step B1 contacting the sample to be enriched containing the diarrhea-causing virus with the composite material according to (7);
  • step B2 the diarrhea-causing virus is eluted from the composite material obtained by the contact and collected.
  • a pharmaceutical composition for preventing and/or treating a viral infection that can cause diarrhea comprising: the protein according to any one of (1)-(3); and a pharmaceutically acceptable excipient.
  • An expression vector comprising the DNA according to (11) or (12) operably linked to a promoter.
  • a host cell for expression which contains the expression vector of (13); preferably, the host cell is selected from yeast cells or Escherichia coli cells.
  • the present invention has the following advantages and positive effects:
  • the invention obtains exogenously expressed target protein by artificially synthesizing nucleotide fragments, and carries out the screening of virus binding medium, thereby improving safety and targeting.
  • the present invention is based on the adsorption ligands obtained from oyster tissue that can specifically bind to the main capsid proteins of viruses that can cause diarrhea (such as Norovirus), artificially synthesize nucleic acid sequences, and use molecular cloning methods. Prokaryotic expression can shorten the production cycle, reduce production costs, and reduce the risk of environmental pollution.
  • the present invention adopts the microbial fermentation method to prepare the protein, and does not involve animal ethics.
  • the protein of the present invention can bind virus particles of different genotypes or multiple virus particles to form a complex, which increases the difficulty of combining the virus particles in the complex with the host cell receptor, thereby reducing the probability of the virus invading the host cell, and can be used to prevent or to treat viral infections.
  • the protein can recognize and bind to virus particles of different genotypes or multiple types of virus particles. Based on this, using the protein as a medium, an enrichment system of different virus particles can be established, and the detection efficiency of virus particles can be improved.
  • the protein of the present invention has the advantages of easy production, low cost, safety, environmental protection, high efficiency and broad spectrum.
  • FIG. 1 shows the results of SDS-PAGE analysis of the recombinant protein obtained in Preparation Example 1.
  • FIG. 2 shows the results of evaluating the binding efficiency (protein level) of the recombinant protein in Example 1 to norovirus particles of different genotypes.
  • FIG. 3 shows the results of evaluating the binding efficiency (nucleic acid level) of the recombinant protein in Example 1 to norovirus particles of different genotypes.
  • FIG. 4 shows the evaluation results (nucleic acid level) of the binding efficiency of the recombinant protein to rotavirus particles of different genotypes in Example 2.
  • Norovirus formerly known as Norwalk Viruses, is a virus belonging to the genus Norovirus in the family Human Calicivirus (HuCV).
  • Norovirus is a non-enveloped single-stranded positive-stranded RNA virus with a virion diameter of about 27-40 nm and a full genome length of about 7.5-7.7 kb.
  • the viral capsid consists of 180 major structural protein (VP1) and several minor structural protein (VP2) molecules.
  • VP1 major structural protein
  • VP2 major structural protein
  • the current in vitro culture system of human noroviruses (HuNoVs) is immature and cannot be identified by serotyping.
  • Noroviruses are divided into 10 genomes (Genogroups, GI-GX) according to their genetic characteristics. GI and GII are the two main genomes that cause acute gastroenteritis in humans. GIV, GIIIV, and GIX can also infect humans, but rarely was checked out.
  • Rotavirus is a double-stranded RNA virus belonging to the Reoviridae family. It is the main cause of diarrhea in infants and young children, and almost all infants and young children under the age of five in the world have been infected with rotavirus. At present, rotavirus has a total of 8 genomes, which are numbered A-H with English letters. Groups A, B, and C rotaviruses primarily infect humans, with the most common rotavirus infections being caused by group A strains.
  • Rotaviruses use a dual classification system, which is based on two structural proteins on the surface of the virion.
  • the glycoprotein VP7 defines the G-type
  • the protease-sensitive protein VP4 defines the P-type.
  • the P type is followed by a number to indicate the P serotype, and a number inside square brackets is used to indicate the corresponding P genotype.
  • the representation of G serotype is similar, but the numbers for the G genotype will be the same as for the G serotype.
  • Rotavirus particles are approximately 70 nm in diameter and have no Viral envelope.
  • the entire viral particle (virion) is composed of multiple viral structural proteins (viral protein, VP).
  • the VP4 protein is located on the surface of the virion to form spikes, which can bind to receptor molecules on the cell surface and assist the virus to enter the host cell.
  • the VP7 protein is a glycoprotein on the outer surface of the virus.
  • the VP7 protein like the VP4 protein, can be used as a target to block infection.
  • the inventors of the present application constructed an upgraded version of fake HuNoVs with a magnification of about 100 times by establishing a bacterial cell surface display system; using the fake virus as a bait, successfully isolated from food Proteins of HuNoVs.
  • this protein can bind to different virus particles (including different genotypes of norovirus and different genotypes of rotavirus), and its binding capacity is much greater than the currently recognized norovirus and rotavirus (helper). ) receptors (blood tissue antigens, histo-blood group antigens, HBGAs).
  • the protein is a virus infection (including: infection of a single genotype of norovirus, infection of a single genotype of rotavirus, mixed infection of different genotypes of norovirus, mixed infection of different genotypes of rotavirus, norovirus and The treatment of rotavirus mixed infection, etc.), the enrichment of different virus particles, etc., provide promising candidate materials.
  • the present invention provides a protein synthesized by chemical means or genetic engineering, characterized in that the protein has a functional domain capable of binding to the capsid protein of a virus that can cause diarrhea, wherein the protein can be Viruses that cause diarrhea include calicivirus and/or reovirus; and the amino acid sequence of the protein is as described in (a) or (b) below:
  • (b) has an amino acid sequence obtained by substituting, deleting, and/or adding one or more amino acids to the amino acid sequence of (a).
  • the protein of the present invention has a functional domain capable of binding to the capsid protein of the diarrhea-causing virus (eg, calicivirus or reovirus), which refers to the protein of the present invention.
  • the protein can bind to the capsid protein of the diarrhea-causing virus (such as calicivirus or reovirus) and can form a complex, which increases the difficulty of binding the virus particle in the complex to the host cell receptor, thereby reducing the The probability of a virus invading a cell.
  • Such cells include, for example, mammalian cells, such as human cell lines.
  • the protein of the invention has a capsid protein capable of interacting with the diarrhea-causing virus (eg, calicivirus (eg, norovirus) or reovirus (eg, rotavirus))
  • a capsid protein capable of interacting with the diarrhea-causing virus (eg, calicivirus (eg, norovirus) or reovirus (eg, rotavirus))
  • a functional domain that binds and/or has activity in inhibiting or blocking said diarrhea-causing viral infection.
  • the calicivirus is norovirus
  • its main capsid protein includes VP1
  • the reovirus is rotavirus
  • its main capsid protein includes VP4.
  • the amino acid sequence of the protein may be one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10...) amino acids, as long as it does not significantly affect the relationship between the protein and calicivirus or reovirus
  • the binding ability of the capsid protein of the virus may not significantly affect the activity of inhibiting or blocking the infection of the calicivirus or reovirus.
  • the amino acid sequence of the protein has at least 80.0%, preferably at least 90.0%, at least 95.0%, at least 99.0%, at least 99.5%, at least 99.7%, more At least 99.9% consistency is preferred.
  • the protein of the present invention is derived from oyster tissue.
  • the protein of the present invention is obtained by genetic engineering (eg, recombinant protein) or obtained by chemical synthesis.
  • the calicivirus is selected from Norovirus; the Norovirus includes GI-GX group (10 species); preferably, the Norovirus is selected from GI, GII, GIV , GVIII and/or GIX group; also preferably, the norovirus is selected from the GI and/or GII group; also preferably, the GI group norovirus is selected from the GI.1, GI.2, GI.3 , GI.4 and/or GI.5 type; also preferably, the GII group norovirus is selected from GII.2, GII.3, GII.4, GII.6, GII.12 and/or GII.17 type.
  • GI-GX group 10 species
  • the Norovirus is selected from GI, GII, GIV , GVIII and/or GIX group
  • the norovirus is selected from the GI and/or GII group
  • the GI group norovirus is selected from the GI.1, GI.2, GI
  • the reovirus is selected from rotavirus; the rotavirus includes groups A-H (8 kinds); preferably, the rotavirus is selected from the group A circulating strains; Also preferably, the group A rotavirus is selected from the G1P[8] and/or G9P[8] types.
  • the present invention also provides a composite material for the detection of diarrhea-causing viruses, such as calicivirus or reovirus, comprising a protein of the present invention, wherein the protein is bound to a solid support selected from the group consisting of: at least one of the markers.
  • a composite material for the detection of diarrhea-causing viruses such as calicivirus or reovirus
  • the solid support used for detection may be a support insoluble in the solvent used in the detection reaction system.
  • Shapes of solid supports include, but are not limited to: plates, beads, disks, tubes, filters, and membranes.
  • Materials for solid supports include, but are not limited to: polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene or polymethyl methacrylate; metals such as gold, silver or aluminium; or glass.
  • a method known in the art such as a physical adsorption method, a covalent bonding method, an ionic bonding method, or a cross-linking method can be used as a method for binding the protein of the present invention to a solid support.
  • the labels include, but are not limited to, fluorescent substances, luminescent substances, dyes, enzymes or radioactive substances.
  • a method known in the art such as a physical adsorption method, a covalent bonding method, an ionic bonding method or a cross-linking method can be used as a method for binding the protein of the present invention to the label.
  • the present invention also provides a device for detecting a diarrhea-causing virus (eg, calicivirus or reovirus), comprising the composite material for detection of the present invention.
  • a diarrhea-causing virus eg, calicivirus or reovirus
  • the present invention also provides a method of detecting a diarrhea-causing virus (such as calicivirus or reovirus), wherein the method comprises:
  • Step A1 contacting the sample to be tested with the composite material for detection of the present invention.
  • step A2 the composite material obtained by the contact is detected for a virus that can cause diarrhea.
  • the sample to be tested in order to detect a virus that can cause diarrhea, can be brought into contact with the composite material for detection of the present invention. Then, based on the reaction of the virus contained in the sample to be tested and the protein of the present invention contained in the composite material, changes in its physical quantity are detected. Examples of physical quantities may be luminous intensity, chromaticity, light transmittance, turbidity, absorbance, radiation dose, and the like.
  • detection method methods known in the art such as enzyme immunoassay, immunochromatography, latex agglutination, radioimmunoassay, fluorescence immunoassay, or surface plasmon resonance spectroscopy and the like can be used.
  • the present invention also provides a composite material for enriching diarrhea-causing viruses, such as calicivirus or reovirus, comprising a protein of the present invention, wherein the protein is bound to a solid support.
  • a composite material for enriching diarrhea-causing viruses such as calicivirus or reovirus
  • the solid support used for enrichment may be a support that is insoluble in the solvent used in the enrichment system.
  • Shapes of solid supports include, but are not limited to: plates, beads, disks, tubes, filters, and membranes.
  • Materials for solid supports include, but are not limited to: polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene or polymethyl methacrylate; metals such as gold, silver or aluminium; or glass.
  • a method known in the art such as a physical adsorption method, a covalent bonding method, an ionic bonding method, or a cross-linking method can be used as a method for binding the protein of the present invention to a solid support.
  • the present invention also provides a method for enriching diarrhea-causing viruses (eg, calicivirus or reovirus), wherein the method comprises:
  • Step B1 contacting the sample to be enriched containing the diarrhea-causing virus with the composite material for enrichment of the present invention.
  • step B2 the diarrhea-causing virus is eluted from the composite material obtained by the contact and collected.
  • the present invention also provides a pharmaceutical composition for preventing and/or treating a diarrhea-causing virus (eg, calicivirus or reovirus) infection, comprising: the protein of the present invention; and pharmaceutically acceptable excipients .
  • a diarrhea-causing virus eg, calicivirus or reovirus
  • the pharmaceutical composition comprises a therapeutically effective amount of a protein of the invention.
  • the pharmaceutically acceptable excipients that can be used in the present invention are those commonly used in the art. Appropriate excipients can be selected according to the needs of practical applications or the specific dosage forms used, for example, the reference “Li Baoqiu, editor-in-chief, “Peptide Drug Research and Development”, Chapter 4 "Research on Polypeptide Drug Preparations", pp. 82-100 , People's Health Publishing House, July 2011".
  • the pharmaceutically acceptable adjuvant is used in the composition of the present invention in its conventional dosage, and those skilled in the art can determine the appropriate dosage according to the needs of practical application.
  • the present invention also provides a use of the protein of the present invention in preparing a medicament for preventing and/or treating calicivirus or reovirus infection.
  • the present invention also provides an isolated DNA encoding the protein of the present invention.
  • nucleotide sequence of the DNA is shown in SEQ ID NO: 2 in the sequence listing.
  • the present invention also provides an expression vector containing the DNA of the present invention operably linked to a promoter.
  • the present invention also provides a host cell for expression, which contains the expression vector of the present invention; preferably, the host cell is selected from yeast cells (eg Pichia cells) or Escherichia coli cells (eg BL21(DE3) cell).
  • yeast cells eg Pichia cells
  • Escherichia coli cells eg BL21(DE3) cell.
  • the resulting recombinant protein can then be coated with an ELISA plate to evaluate the adsorption capacity of different viruses.
  • the related virus particles captured by the obtained recombinant protein can also be used as the object, and the thermally lysed viral nucleic acid can be used as the template to perform RT-qPCR detection to evaluate the adsorption capacity of the protein to different viruses.
  • the obtained recombinant protein can also be used as an adsorption ligand to capture relevant virus particles, so as to increase the virus titer and facilitate virus detection.
  • the resulting recombinant protein can also be incubated with clinical samples of viruses (eg GII.4 Norovirus) at various concentrations, and the incubated mixture can be infected with human cells (eg HIE cell line) to assess the reduction of viral reduction by the recombinant protein. (eg GII.4 type norovirus particles) infection efficiency of human derived cells (eg HIE cell line).
  • viruses eg GII.4 Norovirus
  • the experimental methods used in the following examples were performed using conventional experimental procedures, procedures, materials and conditions in the field of bioengineering. Unless otherwise specified, the same material is derived from the same source. Below, unless otherwise specified, the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v/v)) of the reagent.
  • nucleic acid coding sequence of the protein obtained from oyster tissue screening after nucleotide optimization was carried out according to the prokaryotic expression system, Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to artificially synthesize a nucleotide fragment of 1845 bp (that is, as shown in the sequence table SEQ Nucleotide sequence shown in ID NO: 2); in order to facilitate subsequent genetic engineering operations, Nco I and Xho I restriction enzyme sites were added at both ends of the above-mentioned nucleic acid fragment, a total of 1853bp, as shown in the sequence table SEQ ID The nucleotide sequence shown in NO:3.
  • the 1853bp target fragment was inserted into pUC57 cloning vector (available from Sangon Bioengineering (Shanghai) Co., Ltd.); using Nco I restriction endonuclease (available from ThermoFisher, FD0596) and Xho I restriction endonuclease (available from ThermoFisher, FD0695) was digested, and the recombinant expression vector pET-28a (available from Shanghai Yisheng Biotechnology Co., Ltd., 11905ES03) was subjected to the same treatment to construct a prokaryotic recombinant expression plasmid (wherein the recombinant protein was carried by pET-28a to carry His-tag ).
  • the target fragment was recovered using a DNA recovery kit (available from Nanjing Novizan Biotechnology Co., Ltd., DC301); under the action of DNA ligase (available from ThermoFisher, EL0014), The two target fragments are ligated to construct corresponding ligated products.
  • the ligation product was transformed into competent cells (cloning host, Escherichia coli DH5 ⁇ (available from Takara, 9057)), and coated with a solid plate containing Kan (100 ⁇ g/mL) (available from Sangon Bioengineering (Shanghai) Co., Ltd., A507003), incubated overnight at 37°C.
  • the plasmids of the correctly sequenced recombinant bacteria were extracted, transformed into competent cells (expression host, Escherichia coli BL21 (DE3) (available from Sangon Bioengineering (Shanghai) Co., Ltd., B528414)), coated with Kan (100 ⁇ g/mL) Solid LB plates were incubated overnight at 37°C. The next day, single clones of recombinant bacteria were picked from the plate and placed in 5 mL of liquid LB medium containing Kan (100 ⁇ g/mL), and cultured with shaking at 37° C. for 8-10 hours.
  • competent cells expression host, Escherichia coli BL21 (DE3) (available from Sangon Bioengineering (Shanghai) Co., Ltd., B528414)
  • Kan 100 ⁇ g/mL
  • Solid LB plates were incubated overnight at 37°C. The next day, single clones of recombinant bacteria were picked from the plate and placed in 5 mL of liquid
  • the recombinant bacterial monoclone that can express the target protein was picked again from the plate and placed in 5 mL of liquid LB medium containing Kan (100 ⁇ g/mL), and cultured at 37°C overnight. The next day, the cells were transferred to 400 ml of liquid LB medium containing Kan (100 ⁇ g/mL) at a ratio of 1:100, cultured at 37 °C until the OD600 was about 0.6, and IPTG with a final concentration of 0.5 mM was added for induction at 25 °C for 20 hours. .
  • Ni column available from Changzhou Tiandi Renren Biotechnology Co., Ltd.
  • ultrasonic buffer for 5 column volumes at a speed of 1 ml/min.
  • washing solution of 5 times the column volume (50mM NaH2PO4, 300mM NaCl, 20mM imidazole) to wash off the residual impurities on the Ni column (sample the liquid that flows out at the end of the wash as “washing solution”). "sample).
  • the target protein bound on the Ni column was eluted with an eluent (50mM NaH2PO4, 300mM NaCl, 100mM imidazole) at a rate of 1ml/min, using a computer nucleic acid protein detector (available from Shanghai Jia Peng Technology Co., Ltd., HD300) detects the elution process, collects the flow-through fluid from the peak section, and collects it as the target protein (sampling is taken as the "eluted protein" sample).
  • an eluent 50mM NaH2PO4, 300mM NaCl, 100mM imidazole
  • the target protein was analyzed by 10% (w/v) SDS-PAGE.
  • the results of recombinant protein expression detection are shown in Figure 1.
  • the first lane is "Marker” (standard protein) (available from Sangon Bioengineering (Shanghai) Co., Ltd., C610011)
  • the second lane is "Induced Bacterial Liquid”
  • the second lane is "Inducer”.
  • the third lane is "broken supernatant”
  • the fourth lane is "flow-through”
  • the fifth lane is "wash solution”
  • the sixth lane is "eluted protein".
  • the size of the main protein band in the lane is about 70kDa (compared with the expected The molecular weight of the protein composed of 615 amino acids is consistent). It can be seen from Figure 1 that the target recombinant protein with a molecular weight of about 70KDa has been successfully obtained.
  • Example 1 Evaluation of the binding ability of recombinant proteins to norovirus particles of different genotypes
  • ELISA plates available from Sangon Bioengineering (Shanghai) Co., Ltd. ) Inc., F605031
  • the coated wells were washed three times with Tris-HCl buffered saline solution (TBST, available from Sangon Bioengineering (Shanghai) Co., Ltd., B548105) containing 0.05% Tween-20, and 120 ⁇ L/well was added with 1% Serum albumin (BSA, available from Shanghai Yisheng Biotechnology Co., Ltd., 36101ES25), blocked at 37°C for 1 h.
  • BSA Serum albumin
  • 100 ⁇ L/well of norovirus samples of different genotypes (GI.1 and GII.4) (available from Beijing Center for Disease Control and Prevention) were added, and incubated at 37°C for 1 h.
  • the primary antibody of the corresponding viral capsid protein (the primary antibody can be prepared according to the method disclosed in the following reference: "Mengya Niu, et al. Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands.Frontiers in Microbiology.2015,6:1448.”), incubated at 37°C for 1h.
  • 100 ⁇ L/well of HRP-labeled goat anti-mouse secondary antibody available from Sangon Bioengineering (Shanghai) Co., Ltd., D110087) was added, and incubated at 37°C for 1 h.
  • the top legend indicates that the darker the color, the stronger the ability of the coating to bind to virus particles; the first row is GI.1 norovirus (shown as “HuNoV (GI.1)” in the figure ) and the difference in the binding ability of different coatings, the second row is the difference in the binding ability of GII.4 norovirus (shown as "HuNoV (GII.4)” in the figure) and different coatings; the first vertical Listed as the evaluation of the binding ability of the recombinant protein (20 ⁇ g/mL) coated ELISA plate of the present invention to noroviruses of different genotypes, the second column is the human-derived similar protein (20 ⁇ g/mL) similar to the recombinant protein of the present invention.
  • the third column is the binding ability of PGM (20 ⁇ g/mL, containing norovirus receptor) coated ELISA plate with different genotypes of norovirus
  • the fourth column is the evaluation of the binding ability of PGM (1 mg/mL, containing norovirus receptor) coated ELISA plate with different genotypes of norovirus. It can be seen from Figure 2 that the recombinant protein of the present invention can adsorb noroviruses of different genotypes, and the adsorption capacity is significantly better than similar proteins and PGM.
  • the recombinant protein (20 ⁇ g/mL) and PGM (1 mg/mL, containing human norovirus recognized receptor) purified according to the method of Preparation Example 1 were coated on the ELISA plate respectively, and incubated at 4°C overnight. The next day, after the coated wells were washed three times with TBST, 120 ⁇ L/well was added with 1% BSA, and the cells were blocked at 37°C for 1 h.
  • GI.1, GI.2, GI.3, GI.4, GI.5, GII.2, GII.3, Types GII.4, GII.6, GII.12 and GII.17 was added to clinical samples of different genotypes of norovirus (GI.1, GI.2, GI.3, GI.4, GI.5, GII.2, GII.3, Types GII.4, GII.6, GII.12 and GII.17) (available from Beijing Center for Disease Control and Prevention), incubated at 37°C for 1 h. After washing three times with TBS, 10 ⁇ L of diethyl pyrocarbonate (DEPC)-treated water was added to each well, sealed, incubated at 95°C for 5 min, and cooled at 4°C for use.
  • DEPC diethyl pyrocarbonate
  • the black bars represent the amplification signal intensity of the corresponding nucleic acid after capturing the norovirus particles with PGM (displayed as Ct value); the white bars represent the amplification signal of the corresponding nucleic acid after capturing the norovirus particles with the recombinant protein of the present invention Intensity (shown as Ct value); note: higher Ct value means lower amount of captured viral particles.
  • Ct value the recombinant protein of the present invention can adsorb at least 11 common genotype norovirus particles, and the adsorption capacity is significantly higher than that of PGM.
  • the RT-qPCR results showed that the Ct value of the recombinant protein-adsorbed virus particles of the present invention was 1-2 Ct values lower than that of the PGM-adsorbed virus.
  • Example 2 Evaluation of the binding ability of recombinant proteins to rotavirus particles of different genotypes
  • the recombinant protein (20 ⁇ g/mL) and PGM (1 mg/mL, also containing the receptor that can adsorb rotavirus) purified according to the method of Preparation Example 1 were coated on the ELISA plate respectively, and incubated at 4°C overnight. The next day, after the coated wells were washed three times with TBST, 120 ⁇ L/well was added with 1% BSA, and the cells were blocked at 37°C for 1 h.
  • the black bars represent the amplification signal intensity of the corresponding nucleic acid after capturing rotavirus particles with PGM (displayed as Ct value); the white bars represent the amplification signal of the corresponding nucleic acid after capturing rotavirus particles with the recombinant protein of the present invention Intensity (shown as Ct value); note: higher Ct value means lower amount of captured viral particles.
  • Ct value the recombinant protein of the present invention can adsorb at least two common genotype rotavirus particles, and the adsorption capacity is significantly higher than that of PGM.
  • RT-qPCR results showed that the Ct value of recombinant protein-adsorbed virus particles was 3-5 Ct values lower than that of PGM-adsorbed virus.

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Abstract

本发明提供了一种通过化学途径或基因工程合成的蛋白，其特征在于，所述蛋白具有能够与可引起腹泻的病毒的衣壳蛋白结合的功能性结构域，其中所述可引起腹泻的病毒包括杯状病毒和/或呼肠孤病毒；并且所述蛋白的氨基酸序列如以下(a)或(b)所述：(a)如序列表SEQ ID NO:1所示的氨基酸序列；或(b)具有在(a)所述氨基酸序列中替换、缺失、和/或添加一个或多个氨基酸而得到的氨基酸序列。本发明的蛋白具有易于生产、成本低、安全、环保、高效并且广谱的优点。

Description

一种能够结合多种病毒的蛋白及其应用 技术领域
本申请属于生物技术领域，具体涉及一种能够结合多种病毒的蛋白及其应用。
背景技术
病毒与宿主细胞结合是病毒入侵细胞或感染宿主的关键步骤。如何降低病毒与宿主细胞的结合率成为治疗病毒感染的一个重要途径。因此，大量制备可以与病毒结合的特异性分子成为阻断病毒(再)感染宿主的关键。另外，这种特异性分子还可用于目标病毒的捕获和浓缩。
以人源诺如病毒(Human noroviruses，HuNoVs)为例，近年，虽然有研究团队报道利用B细胞、HIE细胞以及斑马鱼陆续开展HuNoVs体外培养的研究工作，但是，所述细胞系和动物模型能够获得的病毒滴度较低且仅可培养几种特定基因型HuNoVs。迄今，已有报道称：HuNoVs至少有40种不同基因型，且因病毒体外增殖体系不成熟，以致无法真正开展病毒的生物学研究，以及病毒与宿主互作的相关研究工作，严重阻碍了治疗病毒感染药物和疫苗的研制等。那么，探寻可以与多种基因型HuNoVs结合的物质，阻断病毒与宿主细胞的结合成为降低病毒感染突破口。另外，食品或水体中HuNoVs的滴度较低，病毒检测依赖于分子检测方法且需进行病毒富集。但是，目前利用病毒抗体或非特异性的化学方法开展病毒富集，富集效率低且容易引入PCR抑制剂。探寻可广谱性结合HuNoVs的物质成为解决该技术瓶颈问题的关键。
现有技术中可以与病毒结合的特异性分子通常存在以下缺点：生产周期长、成本高；与病毒结合的能力差、应用范围窄；环境污染大；对实验动物依赖性强；安全性差。因此，仍然迫切需要一种易于生产、成本低、安全、环保、高效并且广谱的产品。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了通过化学途径或基因工程合成的蛋白，用于检测可引起腹泻的病毒的复合材料、装置和方法，用于富集可引起腹泻的病毒的复合材料和方法，用于预防和/或治疗可引起腹泻的病毒感染的药物组合物，分离的、编码所述蛋白的DNA，表达载体，宿主细胞。
具体而言，本发明提供了：
(1)一种通过化学途径或基因工程合成的蛋白，其特征在于，所述蛋白具有能够与可引起腹泻的病毒的衣壳蛋白结合的功能性结构域，其中所述可引起腹泻的病毒包括杯状病毒和/或呼肠孤病毒；并且所述蛋白的氨基酸序列如以下(a)或(b)所述：
(a)如序列表SEQ ID NO:1所示的氨基酸序列；或
(b)具有在(a)所述氨基酸序列中替换、缺失、和/或添加一个或多个氨基酸而得到的氨基酸序列。
(2)根据(1)所述的蛋白，其中所述的(b)具有与(a)所述氨基酸序列至少80.0％、优选至少90.0％、至少95.0％、至少99.0％、至少99.5％、至少99.7％、更优选至少99.9％的一致性。
(3)根据(1)所述的蛋白，其中：
所述杯状病毒选自诺如病毒；所述诺如病毒包括GI-GX组；优选地，所述诺如病毒选自GI、GII、GIV、GVIII和/或GIX组；还优选地，所述诺如病毒选自GI和/或GII组；并且/或者
所述呼肠孤病毒选自轮状病毒；所述轮状病毒包括A-H组；优选地，所述轮状病毒选自A组轮状病毒；还优选地，所述轮状病毒选自A组G1P[8]和/或G9P[8]型。
(4)一种用于检测可引起腹泻的病毒的复合材料，其包含根据(1)-(3)中任一项所述的蛋白，其中所述蛋白结合至选自固相支持物和标记物中的至少一种。
(5)一种用于检测可引起腹泻的病毒的装置，其包含根据(4)所述的复合材料。
(6)一种检测可引起腹泻的病毒的方法，其中，该方法包括：
步骤A1，将待测样品与根据(4)所述的复合材料接触；以及
步骤A2，将经所述接触得到的复合材料针对所述可引起腹泻的病毒进行检测。
(7)一种用于富集可引起腹泻的病毒的复合材料，其包含根据(1)-(3)中任一项所述的蛋白，其中所述蛋白结合至固相支持物。
(8)一种富集可引起腹泻的病毒的方法，其中，该方法包括：
步骤B1，将含有所述可引起腹泻的病毒的待富集样品与根据(7)所述的复合材料接触；以及
步骤B2，将所述可引起腹泻的病毒从经所述接触得到的复合材料上洗脱，并进行收集。
(9)一种用于预防和/或治疗可引起腹泻的病毒感染的药物组合物，其包含：根据(1)-(3)中任一项所述的蛋白；以及可药用的辅料。
(10)根据(1)-(3)中任一项所述的蛋白在制备用于预防和/或治疗可引起腹泻的病毒感染的药物中的用途。
(11)一种分离的、编码根据(1)-(3)中任一项所述的蛋白的DNA。
(12)根据(11)所述的DNA，所述DNA的核苷酸序列如序列表SEQ ID NO:2所示。
(13)一种表达载体，其含有与启动子有效连接的根据(11)或(12)所述的DNA。
(14)一种用于表达的宿主细胞，其含有(13)所述的表达载体；优选地，所述宿主细胞选自酵母细胞或大肠杆菌细胞。
本发明与现有技术相比具有以下优点和积极效果：
本发明通过人工合成核苷酸片段获得外源表达目标蛋白，并开展病毒结合介质的筛选，提高了安全性和靶向性。根据上述研究结果，本发明以从牡蛎组织中获得的可特异性结合可引起腹泻的病毒(例如诺如病毒)的主要衣壳蛋白的吸附配体为依据，人工合成核酸序列，利用分子克隆手段进行原核表达，能够缩短生产周期，降低生产成本，降低环境污染的风险。并且本发明采用微生物发酵法制备蛋白，不涉及动物伦理。
本发明的蛋白可以结合不同基因型病毒颗粒或多种病毒颗粒，形成复合物，增加复合物中病毒颗粒与宿主细胞受体结合的难度，从而降低病毒入侵宿主细胞的概率，进而能够用于预防或治疗病毒感染。另外，该蛋白可以识别并结合不同基因型病毒颗粒或多种病毒颗粒。基于此，以该蛋白为介质，能够建立不同病毒颗粒的富集体系，提高病毒颗粒的检出效率。
因此，本发明的蛋白具有易于生产、成本低、安全、环保、高效并且广谱的优点。
附图说明
图1示出制备例1中得到的重组蛋白的SDS-PAGE分析结果。
图2示出实施例1中重组蛋白与不同基因型诺如病毒颗粒的结合效率评价结果(蛋白质水平)。
图3示出实施例1中重组蛋白与不同基因型诺如病毒颗粒的结合效率评价结果(核酸水平)。
图4示出实施例2中重组蛋白与不同基因型轮状病毒颗粒的结合效率评价结果(核酸水平)。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
诺如病毒(Norovirus，简称NV)，曾称诺瓦克病毒(Norwalk Viruses)，是人类杯状病毒科(Human Calicivirus,HuCV)中诺如病毒属的一种病毒。诺如病毒为无包膜单股正链RNA病毒，病毒粒子直径约27-40nm，基因组全长约7.5-7.7kb。病毒衣壳由180个主要结构蛋白(VP1)和几个次要结构蛋白(VP2)分子构成。人源诺如病毒(human noroviruses，HuNoVs)目前体外培养体系不成熟，无法进行血清型分型鉴定。根据基因特征，诺如病毒被分为10个基因组(Genogroup，GI-GX)，GI和GII是引起人类急性胃肠炎的两个主要基因组，GIV、GIIIV和GIX也可感染人，但很少被检出。
轮状病毒(Rotavirus，简称RV)是一种双链RNA病毒，属于呼肠孤病毒科。它是婴幼儿腹泻的主因，几乎世界上五岁以下的婴幼儿都曾感染过轮状病毒。目前，轮状病毒共有8种基因组，分别以英文字母编号为A-H。A、B与C组轮状病毒主要感染人类，其中最常见的轮状病毒感染是A组毒株引起的。
在A组轮状病毒中有不同的病毒株，称之为血清变异株(serovar)。轮状病毒使用了双重的分类系统，这样分类法是依据这个病毒体表面的两个结构性蛋白质来作分类的。糖蛋白VP7定义了G型，而对于蛋白酶敏感的蛋白质VP4则定义了P型。P型跟一个数字来标示出P血清型，并用方括号内部的一个数字来标示所对应的P基因型。G血清型的表示方法很类似，但是G基因型的数字会与G血清型的数字相同。
轮状病毒颗粒直径大约70nm，且无包膜(Viral envelope)。由多个病毒结构蛋白(viral protein，VP)组成了整个病毒颗粒(病毒体)。其中VP4蛋白质位于病毒体的表面形成刺突(spike)，可与细胞表面的受体分子结合并协助病毒进入宿主细胞。VP7蛋白质是病毒外层表面的糖蛋白。VP7蛋白质跟VP4蛋白质一样，都可被用作阻断感染的靶标。
为了克服现有技术中存在的问题，本申请的发明人通过建立细菌细胞表面展示系统来构建约百倍放大的升级版假HuNoVs；以该假病毒为诱饵，从食品中成功分离到一种可以结合HuNoVs的蛋白质。令人惊奇的是，这种蛋白质可以结合不同病毒颗粒(包括不同基因型诺如病毒和不同基因型轮状病毒)，且其结合能力远大于目前公认的诺如病毒和轮状病毒的(辅助)受体(血组织抗原，histo-blood group antigens，HBGAs)。该蛋白为病毒感染(包括：单一基因型诺如病毒的感染，单一基因型轮状病毒的感染，不同基因型诺如病毒的混合感染，不同基因型轮状病毒的混合感染，诺如病毒和轮状病毒混合感染，等)的治疗，不同病毒颗粒的富集等提供了极具前景的候选材料。
具体而言，本发明提供了一种通过化学途径或基因工程合成的蛋白，其特征在于，所述蛋白具有能够与可引起腹泻的病毒的衣壳蛋白结合的功能性结构域，其中所述可引起腹泻的病毒包括杯状病毒和/或呼肠孤病毒；并且所述蛋白的氨基酸序列如以下(a)或(b)所述：
(a)如序列表SEQ ID NO:1所示的氨基酸序列；或
(b)具有在(a)所述氨基酸序列中替换、缺失、和/或添加一个或多个氨基酸而得到的氨基酸序列。
在本发明的一个实施方案中，本发明的蛋白具有能够与所述可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的衣壳蛋白结合的功能性结构域，是指本发明的蛋白能够与所述可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的衣壳蛋白结合，并能够形成复合物，增加复合物中病毒颗粒与宿主细胞受体结合的难度，从而降低病毒入侵细胞的概率。所述细胞包括(例如)：哺乳动物细胞，例如人类细胞系。
在本发明的一个实施方案中，本发明的蛋白具有能够与所述可引起腹泻的病毒(例 如杯状病毒(例如诺如病毒)或呼肠孤病毒(例如轮状病毒))的衣壳蛋白结合的功能性结构域，并且/或者具有抑制或阻断所述可引起腹泻的病毒感染的活性。其中，当杯状病毒为诺如病毒时，其主要衣壳蛋白包括VP1；当呼肠孤病毒为轮状病毒时，其主要衣壳蛋白包括VP4。
在本发明的一个实施方案中，所述蛋白的氨基酸序列可以是在如序列表SEQ ID NO:1所示的氨基酸序列中替换、缺失、和/或添加一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个…)氨基酸而得到的氨基酸序列，只要不显著影响所述蛋白与杯状病毒或呼肠孤病毒的衣壳蛋白的结合能力，或不显著影响抑制或阻断所述杯状病毒或呼肠孤病毒感染的活性即可。
在本发明的一个实施方案中，所述蛋白的氨基酸序列具有与(a)所述氨基酸序列至少80.0％、优选至少90.0％、至少95.0％、至少99.0％、至少99.5％、至少99.7％、更优选至少99.9％的一致性。
优选的是，本发明所述的蛋白来源于牡蛎组织。优选的是，本发明的蛋白是通过基因工程获得的(如重组蛋白)或者是通过化学合成方法获得的。
在本发明的一个实施方案中，所述杯状病毒选自诺如病毒；所述诺如病毒包括GI-GX组(10种)；优选地，所述诺如病毒选自GI、GII、GIV、GVIII和/或GIX组；还优选地，所述诺如病毒选自GI和/或GII组；还优选地，所述GI组诺如病毒选自GI.1、GI.2、GI.3、GI.4和/或GI.5型；还优选地，所述GII组诺如病毒选自GII.2、GII.3、GII.4、GII.6、GII.12和/或GII.17型。在本发明的一个实施方案中，所述呼肠孤病毒选自轮状病毒；所述轮状病毒包括A-H组(8种)；优选地，所述轮状病毒选自A组流行毒株；还优选地，所述A组轮状病毒选自G1P[8]和/或G9P[8]型。
本发明还提供了一种用于检测可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的复合材料，其包含本发明的蛋白，其中所述蛋白结合至选自固相支持物和标记物中的至少一种。
在本发明的一个实施方案中，用于检测的固相支持物可以是不溶于检测反应体系所用溶剂的支持物。固相支持物的形状包括但不限于：平板、珠、盘、管、滤器和膜。固相支持物的材料包括但不限于：聚合物，如聚对苯二甲酸乙二酯、醋酸纤维素、聚碳酸酯、聚苯乙烯或聚甲基丙烯酸甲酯；金属，如金、银或铝；或玻璃。可以使用本领域已知的方法如物理吸附法、共价结合法、离子键合法或交联法作为使本发明的蛋白结合于固相支持物的方法。所述标记物包括但不限于：荧光物质、发光物质、染料、酶或放射性物质。可以使用本领域已知的方法如物理吸附法、共价结合法、离子键合法或交联法作为使本发明的蛋白结合于标记物的方法。
本发明还提供了一种用于检测可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的装置，其包含本发明所述的用于检测的复合材料。
本发明还提供了一种检测可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的方 法，其中，该方法包括：
步骤A1，将待测样品与本发明所述的用于检测的复合材料接触；以及
步骤A2，将经所述接触得到的复合材料针对可引起腹泻的病毒进行检测。
在本发明的一个实施方案中，为检测可引起腹泻的病毒，可以使待测样品与本发明所述的用于检测的复合材料接触。随后，基于待测样品中含有的病毒与该复合材料中所含本发明的蛋白的反应，检测其物理量的变化。物理量的例子可以是发光强度、色度、透光性、浊度、吸光度和辐射剂量等。可以使用本领域已知的方法如酶免疫测定法、免疫色谱法、胶乳凝集反应法、放射免疫测定法、荧光免疫测定法或表面等离子体共振光谱法等作为检测方法的具体例子。
本发明还提供了一种用于富集可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的复合材料，其包含本发明的蛋白，其中所述蛋白结合至固相支持物。
在本发明的一个实施方案中，用于富集的固相支持物可以是不溶于富集体系所用溶剂的支持物。固相支持物的形状包括但不限于：平板、珠、盘、管、滤器和膜。固相支持物的材料包括但不限于：聚合物，如聚对苯二甲酸乙二酯、醋酸纤维素、聚碳酸酯、聚苯乙烯或聚甲基丙烯酸甲酯；金属，如金、银或铝；或玻璃。可以使用本领域已知的方法如物理吸附法、共价结合法、离子键合法或交联法作为使本发明的蛋白结合于固相支持物的方法。
本发明还提供了一种富集可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)的方法，其中，该方法包括：
步骤B1，将含有所述可引起腹泻的病毒的待富集样品与本发明所述的用于富集的复合材料接触；以及
步骤B2，将所述可引起腹泻的病毒从经所述接触得到的复合材料上洗脱，并进行收集。
本发明还提供了一种用于预防和/或治疗可引起腹泻的病毒(例如杯状病毒或呼肠孤病毒)感染的药物组合物，其包含：本发明的蛋白；以及可药用的辅料。
优选地，所述药物组合物包含治疗有效量的本发明的蛋白。
可用于本发明中的可药用的辅料为本领域通常所用的那些。可以根据实际应用的需要或所采用的具体剂型来选择合适的辅料，例如可以为参考文献“厉保秋主编，《多肽药物研究与开发》，第四章‘多肽药物制剂研究’，第82-100页，人民卫生出版社，2011年7月”中披露的那些。可药用的辅料以其常规用量用于本发明的组合物中，本领域技术人员可以根据实际应用的需要来确定合适的用量。
本发明还提供了一种本发明的蛋白在制备用于预防和/或治疗杯状病毒或呼肠孤病毒感染的药物中的用途。
本发明还提供了一种分离的、编码本发明的蛋白的DNA。
在本发明的一个实施方案中，所述DNA的核苷酸序列如序列表SEQ ID NO:2所 示。
本发明还提供了一种表达载体，其含有与启动子有效连接的本发明的DNA。
本发明还提供了一种用于表达的宿主细胞，其含有本发明的表达载体；优选地，所述宿主细胞选自酵母细胞(例如毕赤酵母细胞)或大肠杆菌细胞(例如BL21(DE3)细胞)。
本发明的一个实施方案中包括：
1、人工合成一段核苷酸序列(其中包含如序列表SEQ ID NO:2所示的核苷酸序列)，构建原核重组表达载体，并进行重组蛋白的表达。
2、利用Ni柱进行目标蛋白的纯化。
然后可以以所得到的重组蛋白包被酶标板，对不同种病毒的吸附能力进行评估。
也可以以所得到的重组蛋白捕获的相关病毒颗粒为对象，以热裂解的病毒核酸为模板，进行RT-qPCR检测，评估蛋白对不同种病毒的吸附能力。
也可以以所得到的重组蛋白为吸附配体捕获相关病毒颗粒，提高病毒滴度，便于病毒检测。
也可以以不同浓度的所得到的重组蛋白与病毒(例如GII.4型诺如病毒)临床样本进行孵育，将孵育后的混合物感染人源细胞(例如HIE细胞系)，以评估重组蛋白降低病毒(例如GII.4型诺如病毒颗粒)感染人源细胞(例如HIE细胞系)的效率。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
例子
除非特别说明，否则以下例子中所用实验方法均使用生物工程领域的常规实验流程、操作、材料和条件进行。除非特别说明，同样的材料来源相同。以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。
制备例1：重组蛋白的获得
A.重组菌的构建与验证：
参照从牡蛎组织筛选得到蛋白的核酸编码序列，根据原核表达系统进行核苷酸优化后，委托生工生物工程(上海)股份有限公司人工合成一段1845bp的核苷酸片段(即，如序列表SEQ ID NO:2所示的核苷酸序列)；为了便于后续基因工程操作，在上述核酸片段两端分别添加了Nco I和Xho I限制性酶切位点，共计1853bp，得到如序列表SEQ ID NO:3所示的核苷酸序列。1853bp目标片段嵌入pUC57克隆载体(可得自生工生物工程(上海)股份有限公司)；利用Nco I限制性内切酶(可得自ThermoFisher，FD0596)和Xho I限制性内切酶(可得自ThermoFisher，FD0695)进行消化处理，重 组表达载体pET-28a(可得自上海翊圣生物科技有限公司，11905ES03)做同样处理，构建原核重组表达质粒(其中通过pET-28a使重组蛋白携带His-tag)。经琼脂糖凝胶电泳后，利用DNA回收试剂盒(可得自南京诺唯赞生物科技股份有限公司，DC301)回收目标片段；在DNA连接酶(可得自ThermoFisher，EL0014)的作用下，将两条目标片段进行连接，构建相应连接产物。将连接产物转化感受态细胞(克隆宿主，大肠杆菌DH5α(可得自Takara，9057))，涂布含Kan(100μg/mL)的固体平板(可得自生工生物工程(上海)股份有限公司，A507003)，37℃过夜培养。次日，从平板上挑取重组菌单克隆于5mL含有Kan(100μg/mL)的液体LB培养基(可得自青岛海博生物，HB0128)中，37℃震荡培养8-10小时。将重组菌送上海桑尼生物科技有限公司测序。
B.重组蛋白诱导表达：
提取测序正确重组菌的质粒，转化感受态细胞(表达宿主，大肠杆菌BL21(DE3)(可得自生工生物工程(上海)股份有限公司，B528414))，涂布含有Kan(100μg/mL)的固体LB平板，37℃过夜培养。次日，从平板上挑取重组菌单克隆于5mL含有Kan(100μg/mL)的液体LB培养基中，37℃震荡培养8-10小时。以1:100的比例转接至400mL含有Kan(100μg/mL)的液体LB培养基中，37℃培养至OD600为0.6左右，加入终浓度为0.5mM的异丙基硫代半乳糖苷(IPTG)(可得自Takara，9030)于37℃诱导3小时。取样作为“诱导菌液”用于后续10％SDS-PAGE电泳针对重组蛋白进行的检测。
重新从平板上挑取可以表达目的蛋白的重组菌单克隆于5mL含有Kan(100μg/mL)的液体LB培养基中，37℃培养过夜。次日，以1:100的比例转接至400ml含有Kan(100μg/mL)的液体LB培养基中，37℃培养至OD600为0.6左右，加入终浓度为0.5mM的IPTG于25℃诱导20小时。
C.重组蛋白纯化：
1、5000×g离心培养液5min，收集诱导后菌体。
2、向收集的菌体中加入至少3倍体积的超声缓冲液(50mM NaH2PO4(可得自上海沪试)，300mM NaCl(可得自上海沪试)，10mM咪唑(可得自生工生物工程(上海)股份有限公司)，并加入终浓度为1mM苯甲基磺酰氟(PMSF)(可得自上海碧云天生物技术有限公司，ST505)，1mg/mL溶菌酶(可得自上海碧云天生物技术有限公司，ST206)，混匀后置于冰上30min。
3、冰上用超声破碎仪(可得自宁波新芝，SCIENT2-IID)破碎，直到菌液澄清。
4、超声后的菌液10000×g离心5min，收集上清(取样作为“破碎上清”样品)，并以0.45μm膜(可得自天津市津腾实验设备有限公司)过滤；滤液置于4℃备用。
5、Ni柱(可得自常州天地人和生物科技有限公司)先使用超声缓冲液以1ml/min 速度活化5个柱体积。
6、以0.5mL/min速率，将经过滤的菌液加入活化后的Ni柱(将此时直接流出的液体取样作为“流穿液”样品)。
7、以0.5mL/min速率，以3个柱体积超声缓冲液清洗Ni柱。
8、以1ml/min速率用，以5倍柱体积的洗涤液(50mM NaH2PO4，300mM NaCl，20mM咪唑)洗除Ni柱上残留的杂蛋白(将洗到最后时流出的液体取样作为“洗涤液”样品)。
9、以1ml/min速率，以5倍柱体积的洗脱液(50mM NaH2PO4，300mM NaCl，100mM咪唑)将Ni柱上结合的目标蛋白洗脱，使用电脑核酸蛋白检测仪(可得自上海嘉鹏科技有限公司，HD300)检测洗脱过程，收集起峰段的流穿液，作为目标蛋白收集(取样作为“洗脱蛋白”样品)。
将目标蛋白进行10％(w/v)的SDS-PAGE分析。重组蛋白表达检测结果如图1所示，第一泳道为“Marker”(标准蛋白)(可得自生工生物工程(上海)股份有限公司，C610011)，第二泳道为“诱导菌液”，第三泳道为“破碎上清”，第四泳道为“流穿液”，第五泳道为“洗涤液”，第六泳道为“洗脱蛋白”。第六泳道与第三泳道相比，诱导菌液上清中的大部分杂蛋白已经被除去；与第一泳道的蛋白Marker相比，泳道中主蛋白条带大小约为70kDa(与预期的由615个氨基酸组成的蛋白质分子量相符)。由图1可知，已成功获得分子量约70KDa的目标重组蛋白。
实施例1：重组蛋白与不同基因型诺如病毒颗粒的结合能力评价
(1)酶联免疫吸附试验：
将按照制备例1的方法纯化的重组蛋白(20μg/mL)、类似蛋白(重组人热休克蛋白70(可得自北京义翘神州科技股份有限公司，11660-H07H))(20μg/mL)、猪胃粘膜提取物(pig stomach mucin,PGM,可得自Sigma，20μg/mL和1mg/mL，含人源诺如病毒公认受体)分别包被酶标板(可得自生工生物工程(上海)股份有限公司，F605031)，4℃过夜孵育。次日，包被孔以含0.05％吐温-20的Tris-HCl缓冲盐溶液(TBST，可得自生工生物工程(上海)股份有限公司，B548105)洗涤三次后，120μL/孔加入1％牛血清白蛋白(BSA，可得自上海翊圣生物科技有限公司，36101ES25)，37℃封闭1h。包被孔以TBST洗涤三次后，100μL/孔加入不同基因型诺如病毒样本(GI.1和GII.4型)(可得自北京市疾病预防控制中心)，37℃孵育1h。以TBST洗涤三次后，100μL/孔加入相应病毒衣壳蛋白的一抗(该一抗可按如下参考文献中披露的方法制备：“Mengya Niu,et al.Engineering bacterial surface displayed human norovirus capsid proteins:A novel system to explore interaction between norovirus and ligands.Frontiers in Microbiology.2015,6:1448.”)，37℃孵育1h。以TBST洗涤三次后，100μL/孔加入HRP标记的羊抗鼠二抗(可得自生工生物工程(上海)股份有限公司，D110087)，37℃孵育 1h。以TBST洗涤三次后，100μL/孔加入3,3',5,5'-四甲基联苯胺(可得自上海碧云天生物技术有限公司，P0209)，室温避光孵育10min，每孔加入50μL 2.0mol/L H2SO4。酶标仪(可得自Tecan Sunrise)测定450nm吸光度。结果如图2所示，最上图例表示颜色越深包被物与病毒颗粒结合的能力越强；第一行是GI.1型诺如病毒(图中示出为“HuNoV(GI.1)”)与不同包被物结合能力的差异，第二行是GII.4型诺如病毒(图中示出为“HuNoV(GII.4)”)与不同包被物结合能力的差异；第一纵列为本发明的重组蛋白(20μg/mL)包被的酶标板与不同基因型诺如病毒结合能力的评价，第二纵列为与本发明的重组蛋白相似的人源类似蛋白(20μg/mL)包被酶标板与不同基因型诺如病毒结合能力的评价，第三列为PGM(20μg/mL，含诺如病毒受体)包被酶标板与不同基因型诺如病毒结合能力的评价，第四列为PGM(1mg/mL，含诺如病毒受体)包被酶标板与不同基因型诺如病毒结合能力的评价。由图2可知本发明的重组蛋白可以吸附不同基因型诺如病毒，且吸附能力显著优于类似蛋白和PGM。
(2)原位捕获RT-qPCR方法：
将按照制备例1的方法纯化的重组蛋白(20μg/mL)、PGM(1mg/mL，含人源诺如病毒公认受体)分别包被酶标板，4℃过夜孵育。次日，包被孔以TBST洗涤三次后，120μL/孔加入1％BSA，37℃封闭1h。包被孔以TBST洗涤三次后，100μL/孔加入不同基因型诺如病毒临床样本(GI.1、GI.2、GI.3、GI.4、GI.5、GII.2、GII.3、GII.4、GII.6、GII.12和GII.17型)(可得自北京市疾病预防控制中心)，37℃孵育1h。以TBS洗涤三次后，每孔分别加入10μL焦碳酸二乙酯(DEPC)处理的水，封口后，95℃孵育5min，4℃冷却备用。以原位捕获RT-qPCR方法(该方法可参见参考文献“Dapeng Wang and Peng Tian.Inactivation conditions for human norovirus measured by an in situ capture-qRT-PCR method.International Journal of Food Microbiology.2014,172:76-82.”)检测上述热裂解病毒的核酸。结果如图3所示，其中横轴表示不同基因型诺如病毒临床样本，纵轴表示Ct值。黑色柱条表示以PGM捕获诺如病毒颗粒后，相应核酸的扩增信号强度(以Ct值显示)；白色柱条表示以本发明的重组蛋白捕获诺如病毒颗粒后，相应核酸的扩增信号强度(以Ct值显示)；注意：Ct值越大意味着所捕获病毒颗粒的量越少。由图3可知本发明的重组蛋白至少可以吸附11种常见基因型诺如病毒颗粒，且明显高于PGM的吸附能力。RT-qPCR结果显示：本发明的重组蛋白吸附病毒颗粒的Ct值较PGM吸附病毒低1-2个Ct值。
实施例2：重组蛋白与不同基因型轮状病毒颗粒的结合能力评价
将按照制备例1的方法纯化的重组蛋白(20μg/mL)和PGM(1mg/mL，也含可吸附轮状病毒的受体)分别包被酶标板，4℃过夜孵育。次日，包被孔以TBST洗涤三次后，120μL/孔加入1％BSA，37℃封闭1h。包被孔以TBST洗涤三次后，100μL/ 孔加入不同基因型轮状病毒样本(G1P[8]、G9P[8]型)(可得自北京市疾病预防控制中心)，37℃孵育1h。以TBS洗涤三次后，每孔分别加入10μL DEPC处理的水，封口后，95℃孵育5min，4℃冷却备用。以原位捕获RT-qPCR方法检测上述热裂解病毒的核酸。结果如图4所示，其中横轴表示不同基因型轮状病毒样本，纵轴表示Ct值。黑色柱条表示以PGM捕获轮状病毒颗粒后，相应核酸的扩增信号强度(以Ct值显示)；白色柱条表示以本发明的重组蛋白捕获轮状病毒颗粒后，相应核酸的扩增信号强度(以Ct值显示)；注意：Ct值越大意味着所捕获病毒颗粒的量越少。由图4可知本发明的重组蛋白至少可以吸附2种常见基因型轮状病毒颗粒，且明显高于PGM的吸附能力。RT-qPCR结果显示：重组蛋白吸附病毒颗粒的Ct值较PGM吸附病毒低3-5个Ct值。

Claims (14)

  1. 一种通过化学途径或基因工程合成的蛋白，其特征在于，所述蛋白具有能够与可引起腹泻的病毒的衣壳蛋白结合的功能性结构域，其中所述可引起腹泻的病毒包括杯状病毒和/或呼肠孤病毒；并且所述蛋白的氨基酸序列如以下(a)或(b)所述：
    (a)如序列表SEQ ID NO:1所示的氨基酸序列；或
    (b)具有在(a)所述氨基酸序列中替换、缺失、和/或添加一个或多个氨基酸而得到的氨基酸序列。
  2. 根据权利要求1所述的蛋白，其中所述的(b)具有与(a)所述氨基酸序列至少80.0％、优选至少90.0％、至少95.0％、至少99.0％、至少99.5％、至少99.7％、更优选至少99.9％的一致性。
  3. 根据权利要求1所述的蛋白，其中：
    所述杯状病毒选自诺如病毒；所述诺如病毒包括GI-GX组；优选地，所述诺如病毒选自GI、GII、GIV、GVIII和/或GIX组；还优选地，所述诺如病毒选自GI和/或GII组；并且/或者
    所述呼肠孤病毒选自轮状病毒；所述轮状病毒包括A-H组；优选地，所述轮状病毒选自A组轮状病毒；还优选地，所述轮状病毒选自A组G1P[8]和/或G9P[8]型。
  4. 一种用于检测可引起腹泻的病毒的复合材料，其包含根据权利要求1-3中任一项所述的蛋白，其中所述蛋白结合至选自固相支持物和标记物中的至少一种。
  5. 一种用于检测可引起腹泻的病毒的装置，其包含根据权利要求4所述的复合材料。
  6. 一种检测可引起腹泻的病毒的方法，其中，该方法包括：
    步骤A1，将待测样品与根据权利要求4所述的复合材料接触；以及
    步骤A2，将经所述接触得到的复合材料针对所述可引起腹泻的病毒进行检测。
  7. 一种用于富集可引起腹泻的病毒的复合材料，其包含根据权利要求1-3中任一项所述的蛋白，其中所述蛋白结合至固相支持物。
  8. 一种富集可引起腹泻的病毒的方法，其中，该方法包括：
    步骤B1，将含有所述可引起腹泻的病毒的待富集样品与根据权利要求7所述的复合材料接触；以及
    步骤B2，将所述可引起腹泻的病毒从经所述接触得到的复合材料上洗脱，并进行收集。
  9. 一种用于预防和/或治疗可引起腹泻的病毒感染的药物组合物，其包含：根据权利要求1-3中任一项所述的蛋白；以及可药用的辅料。
  10. 根据权利要求1-3中任一项所述的蛋白在制备用于预防和/或治疗可引起腹泻的病毒感染的药物中的用途。
  11. 一种分离的、编码根据权利要求1-3中任一项所述的蛋白的DNA。
  12. 根据权利要求11所述的DNA，所述DNA的核苷酸序列如序列表SEQ ID NO:2所示。
  13. 一种表达载体，其含有与启动子有效连接的根据权利要求11或12所述的DNA。
  14. 一种用于表达的宿主细胞，其含有权利要求13所述的表达载体；优选地，所述宿主细胞选自酵母细胞或大肠杆菌细胞。
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