WO2023013618A1 - Récepteur de l'infection par l'astrovirus humain et son application - Google Patents

Récepteur de l'infection par l'astrovirus humain et son application Download PDF

Info

Publication number
WO2023013618A1
WO2023013618A1 PCT/JP2022/029580 JP2022029580W WO2023013618A1 WO 2023013618 A1 WO2023013618 A1 WO 2023013618A1 JP 2022029580 W JP2022029580 W JP 2022029580W WO 2023013618 A1 WO2023013618 A1 WO 2023013618A1
Authority
WO
WIPO (PCT)
Prior art keywords
hastv
fcgrt
gene
genetically modified
cells
Prior art date
Application number
PCT/JP2022/029580
Other languages
English (en)
Japanese (ja)
Inventor
章 中西
和彦 片山
慧 芳賀
玲子 戸高
紫文 小池
栞 工藤
Original Assignee
国立研究開発法人国立長寿医療研究センター
学校法人北里研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立研究開発法人国立長寿医療研究センター, 学校法人北里研究所 filed Critical 国立研究開発法人国立長寿医療研究センター
Priority to JP2023540351A priority Critical patent/JPWO2023013618A1/ja
Publication of WO2023013618A1 publication Critical patent/WO2023013618A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • 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
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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

Definitions

  • the present invention relates to the cell infection mechanism of viruses and its application. More specifically, the invention relates to an infection receptor for human astrovirus, one of major gastroenteritis viruses, and its application.
  • HstV Human Astrovirus
  • HAstV Human Astrovirus
  • Infants under the age of 5 are mainly infected after 5 to 6 months of age, causing acute gastroenteritis. It can also infect adults and cause large-scale viral food poisoning incidents.
  • HAstV is the third most frequently reported pathogenic virus for viral gastroenteritis in the epidemic prediction project of the Ministry of Health, Labor and Welfare, next to norovirus and rotavirus, along with sapovirus.
  • HAstV is transmitted from person to person via the fecal-oral route, but outbreaks can also occur with contaminated food and drink as the source of infection. Infection through food is thought to be caused by drinking water and bivalves such as oysters and green goats carrying HAstV, and the number of viral particles required for HAstV infection is said to be 100 or more.
  • Astrovirus particles are stable at pH 3 and are resistant to various detergents (nonionic, anionic, zwitterionic) and lipid solvents including chloroform.
  • HAstV remains infective by heat treatment at 60° C. for 5 minutes, but is inactivated by heat treatment for 10 minutes.
  • HAstV develops with symptoms such as diarrhea, vomiting, and abdominal pain 2-3 days after infection, and the symptoms last for about 4 days. There are also subclinical infections in which no symptoms are observed even if infected, but more serious symptoms are observed in immunocompromised patients. Shedding of HAstV in healthy infant patients can persist for several weeks even when the condition improves and becomes asymptomatic. On the other hand, persistent viral shedding may occur in immunosuppressed individuals.
  • Non-Patent Document 1 Human astrovirus (also referred to herein as HAstV) can be grown and cultured in Caco2, a continuous cell line of human colon adenocarcinoma (Non-Patent Document 1), research on the replication mechanism of the virus has progressed. Nakanishi, one of the present inventors, has also constructed a plasmid-based reverse genetics system. However, the HAstV receptor molecule has not yet been discovered, and the initial steps of replication, such as cell adhesion, invasion, and enucleation, have not been elucidated. Furthermore, HAstV vaccines, therapeutic agents, efficient HAstV detection means, etc. have not yet been developed, and the identification of receptor molecules is extremely important for the development of HAstV infection control methods.
  • the present inventors conducted intensive studies with the aim of elucidating the nature of the HAstV receptor molecule.
  • the FCGRT receptor protein and B2M protein were found to be HAstV receptor molecules. More specifically, on the cell surface of a mammal, the FCGRT receptor protein may be alone, but the FCGRT receptor protein and the B2M protein form a heterodimer as a pair with each other, and the form of the heterodimer is It was found that this is the form that best exhibits the receptor function of HAstV. They also found that the B2M protein plays a role in assisting the HAstV receptor function in the FCGRT receptor protein.
  • the FCGRT gene encodes a fetal Fc receptor (neonatal Fc receptor: FcRn).
  • the FCGRT receptor has a similar structure to the MHC1 molecule and, like the MHC1 molecule, forms a heterodimer with ⁇ 2 microglobulin (B2M) on the cell surface.
  • B2M microglobulin
  • FcRn fetal Fc receptor
  • FcRn is expressed in various tissues even in adults, and its main function is to extend the half-life of IgG in plasma by returning IgG that has been taken up into cells back into the plasma. It turns out.
  • B2M forms a heterodimer with FCGRT receptor on the cell surface and constitutes a part of Fc receptor. That is, Fc receptors are composed of two polypeptide chains, the ⁇ chain and ⁇ 2 microglobulin (B2M), which are non-covalently bound by interactions between the B2M and ⁇ 3 domains. known to be connected.
  • B2M microglobulin
  • the nucleotide sequence of the FCGRT receptor gene (NM_001136019: SEQ ID NO: 1) is and The amino acid sequence of the FCGRT receptor protein (NM_001136019: SEQ ID NO: 2) is ⁇ mgvprpqpwalglllfllpgslgaeshlsllyhltavsspapgtpafwvsgwlgpqqylsynslrgeaepcgawvwenqvswywekettdlrikeklfleafkalggkgpytlqgllgcelgpdntsvptakfalngeefmnfdlkqgtwggdwpealaisqrwqqqdkaankeltfllfscphrlrehlergrgnlewkeppsmrlkarpsspgfsvltcsafsfyppelqlrflrnglaagt
  • the base sequence of the B2M gene (NM_004048: SEQ ID NO: 3) is ⁇ atgtctcgctccgtggccttagctgtgctcgcgctactctctctttctggcctggaggctatccagcgtactccaaagattcaggtttactcacgtcatccagcagagaatggaaagtcaaattttcctgaattgtctgggtttcatccatccgacattgaagttgacttactgaagaatggagagagaattgaaaaagtggagcattcagacttgtcttttcagcaaggactggtctttctatctcttgtactacactgaattcacccccactgaaaaagatgagtatgcctgccgtgtga
  • nucleotide sequences SEQ ID NOS: 1 and 3 and amino acid sequences (SEQ ID NOS: 2 and 4) are sequences related to the human genome, and naturally there are sequences that are modified by natural occurrences and by genetic polymorphisms. .
  • the genetic polymorphism contains a single nucleotide substitution (SNP).
  • SNP single nucleotide substitution
  • the present invention has an aspect using a gene encoding an FCGRT receptor (FCGRT receptor gene or FCGRT gene) and/or a gene encoding a B2M protein (B2M gene) (first aspect); Secondly, there is an aspect (second aspect) using FCGRT receptor protein (FCGRT receptor protein or FCGRT protein) and/or B2M protein (B2M protein).
  • the general structure of the astrovirus genome has ORF1a and ORF1b encoding nonstructural proteins at the 5' end and ORF2 encoding the structural protein at the 3' end.
  • This arrangement resembles the genomic structure of caliciviruses.
  • features such as polyprotein size, number and processing, RNA helicase domains, and ribosomal frameshift mechanisms. Therefore, astroviruses are classified into the Astroviridae family, which is a virus family different from calciviruses and the like.
  • Viruses of the Astroviridae family have been isolated from humans and many other animal species and have been identified by the International Committee on Taxonomy of Viruses (ICTV) using the full-length polypeptide sequence of the viral capsid. According to phylogenetic analysis, they are classified into two genera: mammalian astroviruses (MAstV) that infect mammals including humans, and astroviruses isolated from birds (Avastrovirus). MAstV was further subdivided into two gene groups, Mamastrovirus genogroups I and II, and Avastrovirus was similarly subdivided into two gene groups. Furthermore, these gene groups were subdivided into multiple genotypes.
  • MAstV mammalian astroviruses
  • Avastrovirus astroviruses isolated from birds
  • the Astroviridae family consists of two virus genera (Astrovirus: Mamastrovirus, Avastrovirus), but the above Mamastrovirus includes 19 virus species (mamastrovirus 1-19), and Avastrovirus includes 3 viruses. It was assumed to contain species (avastrovirus 1-3). In other words, by integrating the gene groups and genotypes that had been established under the virus genus, the virus species were unified and organized.
  • HAstV Astroviruses that infect humans
  • MAstV a viral species included in MAstV (because the antigenicity of these eight genotypes is different from each other). synonymous with serotype).
  • HAstV genotype 1 HAstV1
  • mastroviruses 6, 8, and 9 which are genetically distant from mastrovirus 1, also infect humans like other genotypes. It has become clear since 2008 that
  • the human astrovirus (HAstV) to be the object of the present invention is an astrovirus having the ability to infect humans, and includes all of the above HAstV types 1 to 8 (genotype) and mastroviruses 6 and 8. ⁇ 9 is included.
  • a permissible B2M gene or an FCGRT receptor gene in which partial modification of the nucleotide sequence represented by SEQ ID NO: 1 is permissible and which is infected with HAstV
  • proliferative genetically modified mammalian cultured cells hereinafter also referred to as cultured cells of the present invention.
  • the present invention provides an FCGRT receptor gene that permits partial modification of the base sequence represented by SEQ ID NO: 1 and a B2M gene that permits partial modification of the base sequence represented by SEQ ID NO: 3, or sequences A genetically modified mammal (excluding humans) in which the FCGRT receptor gene, which permits partial modification of the base sequence represented by No. 1, is integrated, and the gene has the ability to be infected with HAstV or proliferate.
  • a recombinant mammal hereinafter also referred to as the recombinant animal of the present invention.
  • the partial modification of the base sequence above refers to the replacement, deletion, or insertion of part of the reference base sequence.
  • the modification is permissible and not particularly limited as long as the genetically modified mammalian cultured cells or genetically modified mammals into which the above-mentioned receptor gene of the modified base sequence is integrated have the ability to be infected with HAstV or proliferate. not something.
  • Genetic modifications according to natural occurrences, genetic modifications according to genetic polymorphisms, etc. based on information obtained by human gene analysis are included in the partial modifications of the base sequences described above.
  • the genetic polymorphism includes a single nucleotide substitution (SNP).
  • SNP single nucleotide substitution
  • the gene to be incorporated into cultured mammalian cells or mammals can be the FCGRT receptor gene alone, but it is preferable that both the FCGRT receptor gene and the B2M gene are incorporated.
  • the FCGRT receptor gene and the B2M gene, or the FCGRT receptor gene are introduced into cultured mammalian cells or mammals, thereby introducing HAstV into the cultured mammalian cells or mammals.
  • the point is that the ability to be infected or the ability to proliferate is conferred.
  • the origin of the mammalian cultured cells to be the subject of the above genetic recombination is not limited, and includes cells derived from mammals such as humans, rats, hamsters, guinea pigs, mice, rabbits, cats, dogs, pigs, and monkeys.
  • human cultured cells include HEK293T cells, Caco2 cells, Intestine407, macrophage-based cultured cells 15310-LN cells, NALM-6 cells, etc.
  • Mouse cultured cells include RAW264.7 cells, NIH3T3 cells, Examples include M1 cells.
  • BHK cells derived from hamsters
  • CHO cells derived from hamsters
  • CRFK cells derived from cats
  • MDCK cells derived from dogs
  • PK-15 cells derived from pigs
  • VERO cells derived from monkeys
  • COS7 cells derived from monkeys origin
  • cultured mammalian cells in addition to the above-mentioned established cultured cells, organoids derived from biopsy samples, immortalization-inducing cells, iPS cells, and the like can also be used.
  • FCGRT receptor gene and/or the B2M gene can be improved.
  • the method of introducing the FCGRT receptor gene and B2M gene, or the FCGRT receptor gene, into cultured mammalian cells is not particularly limited, and is carried out according to a conventional method.
  • a vector for gene transfer that is, a mouse leukemia virus vector incorporating an FCGRT receptor gene and a B2M gene, or an FCGRT receptor gene, a retroviral vector such as a lentiviral vector, an adenoviral vector , adeno-associated virus vector, herpes simplex type I vector, modified viral vector such as HVJ-liposome, plasmid vector having CMV promoter, GAC promoter, EF-1 ⁇ promoter, SV40 promoter that functions in cultured mammalian cells, etc. introduction method.
  • introduction method for introduction of genes, in addition to using recombinant viral vectors, calcium phosphate method, lipofection method, commercially available transfection reagent, microinjection method, stamping method, particle gun method and the like can also be used.
  • the mammals to be genetically modified above are not particularly limited, and include non-human mammals such as rats, hamsters, guinea pigs, mice, rabbits, cats, dogs, pigs, and monkeys.
  • Gene integration methods for these mammals include, for example, the FCGRT receptor gene and the B2M gene, or the FCGRT receptor gene, retroviral vectors such as mouse leukemia virus vectors, lentiviral vectors, adenovirus vectors, adenoviral vectors, adenovirus vectors, etc.
  • Associated virus vectors herpes simplex type I vectors, modified viral vectors such as HVJ-liposomes, CMV promoters that function in mammalian cells, GAC promoters, EF-1 ⁇ promoters, plasmid vectors having SV40 promoters, etc.
  • a gene can be introduced into a mammal of interest and expressed.
  • the FCGRT receptor gene and B2M gene can be secured by standard methods. That is, the above genes can be easily obtained by amplifying the gene region by a gene amplification method such as PCR using cDNA containing these genes as a template. It is possible to do this at home, but it is also possible to outsource it, and if it is available on the market, it is also possible to use a commercially available product.
  • the present invention provides the following method.
  • the present invention provides an FCGRT receptor gene that permits partial modification of the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide represented by SEQ ID NO: 3 for cultured mammalian cells or mammals (excluding humans).
  • the above-mentioned mammals genetically modified by introducing the B2M gene, which permits partial modification of the sequence, or the FCGRT receptor gene, which permits partial modification of the base sequence represented by SEQ ID NO: 1.
  • a method for imparting or enhancing the ability to be infected with HAstV in cultured cells or mammals (hereinafter also referred to as the method for imparting/enhancing the present invention) is provided.
  • the cultured cells of the present invention and the recombinant animals of the present invention are recombinant mammalian cultured cells and recombinant mammals to which HAstV infectivity has been imparted or enhanced by the imparting method of the present invention.
  • imparting the ability to be infected" by HAstV means to newly impart the ability to be infected by HAstV to cultured mammalian cells or mammals, which initially lacked the ability to be infected by HAstV.
  • the term "enhancement of the ability to be infected” means enhancement of the ability to be infected by HAstV in cultured mammalian cells or mammals in which the ability to be infected by HAstV has already been confirmed.
  • the present invention relates to the production of HAstV by infecting the cultured cells of the present invention or the recombinant animals (excluding humans) of the present invention with HAstV and proliferating the HAstV in the cultured cells or recombinant animals.
  • a method hereinafter also referred to as the production method of the present invention.
  • the present invention provides that subculturing of HAstV in the cultured cells of the present invention (including human-derived cultured cells) or the recombinant animals of the present invention (excluding humans) is permitted as a live vaccine for pathogenicity.
  • a method for producing a live vaccine against HAstV hereinafter also referred to as a method for producing a vaccine of the present invention, in which HAstV, which has lost the ability to proliferate, is obtained as a live vaccine by continuing subculturing until it has decreased to a certain level.
  • the reduction in pathogenicity of HAstV subcultured as described above is brought about by changes in temperature sensitivity, changes in pH sensitivity, or reduction in susceptibility to original infected target cells in HAstV. be.
  • the vaccine production method of the present invention is a method aimed at producing a live HAstV vaccine.
  • the pathogenicity of HAstV is recognized because the virus possesses both the ability to infect and the ability to grow in cells to be infected.
  • a live vaccine against HAstV is obtained by eliminating or reducing the ability to proliferate in human cells while leaving only the ability to infect humans from HAstV, which originally has the ability to infect and proliferate in humans. be able to.
  • the live vaccine against HAstV obtained by the vaccine production method of the present invention eliminates or reduces the pathogenicity in humans of HAstV subcultured in the cultured cells or recombinant animals of the present invention.
  • HAstV The reduction in virulence in production of HAstV is required to be reduced compared to the wild-type strain to the extent that it is acceptable as a live vaccine.
  • This "permissible degree as a live vaccine” means that when administered to humans, symptoms such as diarrhea, vomiting, and abdominal pain when infected with wild-type HAstV are not observed, and in vivo immunity to HAstV is constructed.
  • aspects in which the pathogenicity of HAstV in humans disappears or decreases during subculturing include changes in temperature sensitivity, changes in pH sensitivity, or decreased susceptibility to the same original infected target cells in HAstV. is mentioned.
  • HAstV confirmedation of reduction or disappearance of pathogenicity in subcultured HAstV
  • HAstV to be tested is administered to the recombinant animal of the present invention, and the presence or absence of HAstV infection symptoms is examined. If the infection symptoms are not observed and the desired immune response to HAstV is observed, the above It can be recognized that the virulence of HAstV is reduced (including eliminated) to an "acceptable level for a live vaccine".
  • the pathogenicity of the passaged HAstV is strongly reduced, or whether or not it has disappeared can be confirmed by using the test HAstV It can be done by conducting an infection test of That is, when the human-derived cultured cells are infected with the HAstV to be tested and no proliferation is observed, or when the ability to proliferate is remarkably reduced, the completion of the live vaccine can be determined.
  • the above-mentioned "human-derived cultured cells that are infected and proliferate with HAstV” can be cultured cells of the present invention using human-derived cells.
  • human-derived cultured cells having
  • human-derived cultured cells such as Caco2 cells, which originally have the ability to be infected and proliferate with HAstV, are enhanced by the introduction of the FCGRT receptor gene and / or the B2M gene.
  • the cultured cells of the present invention infected with HAstV or the recombinant animals of the present invention are brought into contact with a substance to be screened, and the cultured cells or recombinant animals of the present invention are infected with HAstV.
  • a screening method for acquiring information on the action of the substance to be screened for HAstV by detecting proliferation By contacting the cultured cells of the present invention or the recombinant animals (excluding humans) of the present invention with a substance to be screened and HAstV, and detecting the growth of HAstV in the cultured cells or recombinant animals, A screening method (the above two methods are hereinafter also referred to as the screening method of the present invention) for obtaining information on the action of the screening target substance on HAstV is provided.
  • the action to be screened is preferably HAstV inactivation action or growth inhibitory action.
  • the present invention immunizes the cultured cells of the present invention or the recombinant animals (excluding humans) of the present invention with a vaccine to be screened, and detects the protective action against HAstV in the cultured cells or recombinant animals.
  • a screening method (hereinafter, also referred to as a vaccine screening method of the present invention) for obtaining information on the action of the screening target vaccine is provided.
  • the present invention provides a genetic set obtained by subjecting the cultured cells of the present invention or the recombinant animals of the present invention (excluding humans) to wild-type HAstV and predetermined genetic modifications using techniques such as reverse genetics. Recombinant HAstV is attempted to infect each individual group, and the Astrovirus infection mode in the genetically modified mammalian cultured cell group or the genetically modified mammalian group before and after the infection is associated with the genetic modification.
  • an information acquisition method also referred to as an information acquisition method of the present invention for acquiring information received.
  • the "aspect of astrovirus infection” that constitutes the information acquired in the information acquisition method of the present invention refers to attempts to infect the cultured cells or recombinant animals of the present invention with HAstV, which has undergone a predetermined genetic modification,
  • HAstV which has undergone a predetermined genetic modification
  • the ratio of the number of infected cells to the number of test cells, the number of HAstV introduced into each infected cell, the growth rate of HAstV introduced, the degree of cells, etc. are exemplified.
  • the presence/absence, content, and degree of symptoms of HAstV infection are exemplified.
  • a solid-phase carrier carrying an acceptable B2M protein or an FCGRT receptor protein in which a partial modification of the amino acid sequence represented by SEQ ID NO: 2 is allowed, wherein the solid-phase carrier has binding ability to HAstV. (hereinafter also referred to as the solid phase carrier of the present invention).
  • Partial modification of the amino acid sequence refers to substitution, deletion, or insertion of part of the reference amino acid sequence. The modification is permissible as long as the receptor protein with the modified amino acid sequence has the ability to bind to HAstV, and is not particularly limited. Modifications of amino acid sequences according to natural occurrences, modifications of amino acid sequences according to genetic polymorphisms, etc. based on information obtained by human genetic analysis are included in the above partial modifications of amino acid sequences. ing.
  • the genetic polymorphism includes a single nucleotide substitution (SNP).
  • SNP single nucleotide substitution
  • the above-mentioned receptor protein supported on a solid phase carrier can be FCGRT receptor protein alone, but the fact that the FCGRT receptor protein and B2M protein are supported as a set in a heterodimeric state indicates that the binding to HAstV Moreover, the presence of both receptors on actual human cells is reproduced, making it suitable for screening substances that act on the cell surface binding ability of HAstV.
  • FCGRT receptor protein and B2M protein can be produced by known methods, specifically genetic engineering methods or chemical synthesis methods. It is also possible to produce all of these receptor proteins together, or to produce each part separately and then combine the parts with each other by a chemical modification method. Binding between polypeptides via a linker can be achieved by binding lysine residues or cysteine residues in each polypeptide with a linker having a succinimide group or a maleimide group.
  • a nucleic acid encoding all or part of the FCGRT receptor protein or B2M protein to be produced is transformed into host cells such as E. coli, yeast, insect cells, animal cells, or E. coli. It can be expressed in a cell-free expression system such as an extract, rabbit reticulocyte extract, or wheat germ extract. Expression vectors incorporating these nucleic acids can be used according to each expression system. For example, pET for E. coli expression, pAUR for yeast expression, pIEx-1 for insect cell expression, animal cell Examples include pBApo-CMV for expression and pF3A for wheat germ extract expression.
  • the chemical synthesis method it is possible to use a known peptide chemical synthesis method. That is, it is possible to produce all or part of the conjugated protein of the present invention using liquid-phase peptide synthesis or solid-phase peptide synthesis, which have been established as conventional methods. Further, the solid-phase peptide synthesis method, which is generally recognized as a suitable chemical synthesis method, can also use the Boc solid-phase method or the Fmoc solid-phase method. It is also possible to use In addition, individual amino acids can be produced by known methods, and commercially available products can also be used.
  • the shape, size, material, and form of the solid phase carrier are not particularly limited as long as they can support all or part of the FCGRT receptor protein or B2M protein.
  • Materials include, but are not limited to, nonwoven fabric, filter paper, glass, glass fiber, nitrocellulose filter, polyethersulfone filter, nylon filter, polyvinylidene fluoride filter, and porous materials (silica, etc.). , may be singular or compounded.
  • the shape includes, but is not limited to, plate-like, perforated plate, spherical, amorphous, rod-like, elongated, and the like.
  • Insoluble carrier particles such as latex particles, silica particles, and metal colloids can also be used as solid phase carriers.
  • the FCGRT receptor protein and the B2M protein, or the FCGRT receptor protein are supported on a solid phase carrier, so that HAstV binds to the receptor protein, and the binding is
  • the main point is that it serves as a detection index for HAstV in the detection target.
  • the present invention involves contacting an object to be detected for HAstV with the solid-phase carrier of the present invention, and detecting HAstV in the object using the binding of HAstV to the receptor supported on the solid-phase carrier as a signal.
  • a detection method (hereinafter also referred to as the detection method of the present invention) is provided.
  • the object to be detected for HAstV in the detection method of the present invention is not particularly limited as long as the presence of HAstV poses a problem. exemplified. Examples include preparations such as feces, vomit, food and drink, river water, and sewage, as well as washing water for clothes, furniture surfaces, and the like.
  • the binding signal of HAstV to the receptor is not particularly limited as long as it is a means capable of detecting HAstV captured by the receptor supported by the solid phase carrier of the present invention.
  • a highly sensitive ELISA-based measurement method such as a competitive method, a method of measuring binding of two molecules such as Alphascreen, a method of detecting a change in mass due to binding of two molecules such as Biacore, a method of detecting a change in mass due to binding of two molecules such as Biacore, Examples include a method of detecting change.
  • the solid-phase carrier of the present invention, HAstV, and a substance to be screened are allowed to coexist in a liquid phase, and the binding of HAstV to the receptor supported on the solid-phase carrier is used as a signal, HAstV is quantified, and the quantified value is compared with the quantified value obtained when the screening target substance is excluded in the quantification system, thereby screening the binding of HAstV to the receptor in the screening target substance.
  • Screening methods also referred to as binding screening methods of the invention are provided.
  • the substance to be screened in the binding screening method of the present invention is preferably a substance that inhibits the binding of HAstV to the FCGRT receptor protein and B2M protein, or to the FCGRT receptor protein on the solid phase carrier of the present invention.
  • Methods for quantifying binding signals include ELISA, sandwich method, competitive method, etc., ELISA-based highly sensitive measurement methods, methods for measuring binding of two molecules such as Alphascreen, and two-molecule binding methods such as Biacore. Examples include a method of detecting a change in mass due to molecular binding, a method of detecting a change in charge, and the like.
  • recombinant mammalian cultured cells and recombinant mammals endowed with human astrovirus (HAstV) infectivity and proliferation ability are provided, and the recombinant mammalian cultured cells and recombinant mammals are used.
  • Methods of producing HAstV and live vaccines of HAstV are provided.
  • a method of screening test substances and vaccines for HAstV action using the above recombinant mammalian cultured cells and recombinant mammals Also provided is a method for obtaining information that associates gene modification of HAstV with pathogenicity.
  • the present invention provides a solid-phase carrier carrying a receptor protein that binds to HAstV by a mechanism similar to that on the cell surface. Also provided are a method of detecting HAstV in a test subject and a method of screening for a substance having an effect on the binding of HAstV to the above-mentioned receptor, using the solid-phase carrier.
  • FIG. 1-A is a control with actin.
  • 1 is a drawing showing the results of examination of HAstV-1 infection susceptibility in Caco-2 cells by knocking out the FCGRT gene and B2M gene.
  • FIG. 2 is a drawing showing the results of examination of susceptibility to HAstV-4 infection in Caco-2 cells by knocking out the FCGRT gene and B2M gene.
  • FIG. 2 shows the results of changes in HAstV-1 RNA levels in Caco-2 cells in which the FCGRT gene and B2M gene were knocked out.
  • Fig. 3 shows the results of changes in the amount of HAstV-4 RNA in Caco-2 cells in which the FCGRT gene and B2M gene were knocked out.
  • Knockout of FCGRT protein expression in human intestinal organoids (SI1) in which the FCGRT gene was knocked out (Fig. 4-B)
  • FIG. 4-C knockout of B2M protein expression in human intestinal organoids (SI1) in which the B2M gene was knocked out
  • FIG. 2 is a drawing showing the results of examination of susceptibility to HAstV infection in human intestinal organoids by knocking out the FCGRT gene and B2M gene.
  • FIG. Fig. 3 shows the results of changes in the amount of HAstV-1 RNA in human intestinal organoids in which the FCGRT gene and B2M gene were knocked out.
  • Fig. 3 shows the results of changes in the amount of HAstV-4 RNA in human intestinal organoids in which the FCGRT gene and the B2M gene were knocked out.
  • Schematic diagram showing steps for introduction of FCGRT gene and B2M gene into MDCK cells by lentivirus.
  • FIG. 2 is a drawing showing the results of Western blotting examination of the expression of the corresponding proteins in MDCK cells transfected with the FCGRT gene, the B2M gene, and the FCGRT gene and the B2M gene.
  • Fig. 9 shows the results of examining HAstV-1 (Fig. 9-A) and HAstV-4 (Fig. 9-B) for the amount of HAstV-RNA in the culture supernatant of candidate gene-introduced MDCK cells.
  • FIG. 2 is a drawing showing the steps of analyzing the binding between HAstV particles and heterodimers of FCGRT protein and B2M protein by ELISA.
  • HAstV receptors were identified using Caco-2 cells, which are susceptible to HAstV infection, HAstV-1, which has a large number of cases among MAstV1 types, and HAstV-4.
  • EMEM Minimum Essential Medium Eagle
  • Anti-Rabbit antibody (abcam, ab75853, derived from Rabbit) and anti-Goat antibody (Santa Cruz Biotechnology, Inc., sc-2020, derived from Goat) were used as secondary antibodies and incubated at room temperature for 1 hour.
  • Chemi-Lumi One L (nacalai tesque, 07880-54) was used as a coloring solution.
  • Virus strain HAstV-1 (VR-1936 TM ) purchased from ATCC (the American Type Culture Collection) and stored at the Kitasato Research Institute Omura Satoshi Memorial Institute Viral Infection Control Laboratory (Katayama Laboratory) HAstV-4 was used. These were infected with Caco-2/Cas9, 1% NEAA, 1% Na pyruvate, 1% Anti-Anti, 20 mM Hepes (1 mol / l-HEPES Buffer Solution: nacalai tesque, 17557-94) was added. The cells were cultured under the conditions of 37° C. and 5% CO 2 using EMEM (to which the above was added, hereinafter referred to as EMEM( ⁇ )).
  • HAstV indicates both HAstV-1 and HAstV-4 above.
  • Virus Infection and Culture Caco-2 cells cultured in EMEM(+) and made to be 80% confluent in a collagen-coated flask or well plate were infected with HAstV.
  • HAstV was suspended in EMEM(-) to which Trypsin IX-S (Sigma, T0303-1G) was added at 10 ⁇ g/ml for virus activation treatment, and incubated at 37° C. under 5% CO 2 conditions for 1 hour.
  • 10% FBS was added before use for infection.
  • FBS was similarly added to a virus solution that had not been subjected to activation treatment in order to prepare the conditions.
  • Trypsin IX-S was added to EMEM(-) at 5 ⁇ g/ml.
  • HAstV Infection After Activation Treatment and Cultivation under Addition of Trypsin Caco-2 cells were washed with PBS( ⁇ ) to remove serum, and cultured in EMEM( ⁇ ) at 37° C. under 5% CO 2 conditions for 1 hour. .
  • the activated virus solution was added to the cells and infected for 1 hour at 37° C., 5% CO 2 conditions. After 1 hour, the cells were washed with PBS(-) and cultured in trypsin-added EMEM(-) for 6 days.
  • HAstV Infection After Activation Treatment and Cultivation Without Addition of Trypsin Viruses were infected in the same manner as in (4-1) above, and after washing Caco-2 cells, they were cultured in EMEM(-) for 6 days.
  • Non-activated HAstV infection and culture without Trypsin addition Serum was removed from Caco-2 cells, HAstV was suspended in non-activated virus solution (EMEM (-) without Trypsin added, 37 ° C., 5 % CO2 for 1 hour) was added to the cells and infected for 1 hour at 37°C, 5% CO2 . After 1 hour, the cells were washed with PBS(-) and cultured in EMEM(-) for 6 days.
  • EMEM (-) non-activated virus solution
  • HAstV Infection with Inactivated HAstV and Culture under Addition of Trypsin Prior to activation, HAstV was inactivated by heat treatment. In the preliminary test, three types of viruses were infected: untreated, 60°C for 5 minutes, and 10 minutes, but virus RNA replication was confirmed even at 60°C for 10 minutes, so in this test, 60°C for 15 minutes. , inactivated by heat treatment for 30 min. Inactivated HAstV and untreated HAstV were infected in the same manner as in (4-1) above, and cultured for 6 days.
  • HAstV Infection and Culture of Human Intestinal Organoid Human intestinal organoids (SI1) cultured in ENRA (*1) and confluent in a 96-well plate were infected with HAstV.
  • SI1 was used as a control against SI1/FCGRT KO and SI1/B2M KO in which the FCGRT gene and B2M gene were knocked out, respectively.
  • Cells were infected with activated HAstV for 1 hour at 37° C., 5% CO 2 . After 1 hour, cells were washed with 3+ (*2), fresh ENRA was added and cultured for 3 days.
  • EGF EGF
  • B27 Invitrogen, #17504-044
  • 10 nM Gastrin Sigma-Aldrich, G9145-1MG
  • 10% Noggin Peprotech, 250 -38
  • 3+ with 10% R-spondine R&D, #4645-RS
  • 1 mM nAc Sigma-Aldrich, A9165-5G
  • 0.5 ⁇ M A83 Tocris, #2939)
  • NucleoSpin (registered trademark) 8 virus (MACHEREY-NAGEL, 740643.5) was used to extract RNA from 100 ⁇ l of culture supernatant with 100 ⁇ l of Elution Buffer.
  • HAstV genomic RNA Extraction of HAstV genomic RNA in cells
  • Cells on day 0 after infection (day 0), culture day 3 (day 3), culture day 6 (day 6) were used as samples, and HAstV genomic RNA in infected cells was extracted at n 3.
  • NucleoSpin® RNA (MACHEREY-NAGEL, 740955.50) was used to extract 60 ⁇ l of RNA from 1 well of a 24-well plate.
  • cDNA from HAstV Genomic RNA 10 ⁇ l of cDNA was prepared from 5 ⁇ l of the RNA extracted in (5, 6) above using SuperScript TM III Reverse Transcriptase (Invitrogen, 18080-085). The HAstV-specific primer described in (9) below was used as the reverse primer.
  • HAstV genomic RNA was quantified by qPCR using THUNDERBIRD® Next SYBR® qPCR Mix (TOYOBO, QPX-201). For each sample, 10 ⁇ l of THUNDERBIRD (registered trademark) Next SYBR (registered trademark) qPCR Mix, 6 pmol of Forward Primer (below: 9), 6 pmol of Reverse Primer (below: 9), and 2 ⁇ l of cDNA were added to make a total of 20 ⁇ l. was added with sterile water. A PCR fragment was used as a standard for quantifying HAstV genomic RNA.
  • cDNA derived from HAstV genomic RNA was amplified by PCR, and the genome copy number in the solution was calculated based on the concentration measured with Qubit TM dsDNA BR Assay Kit (Invitrogen, Q32850).
  • HAstV Primers In this test, the following primers common to HAstV-1 and HAstV-4 were used.
  • the nucleotide sequence of the forward primer shown in Table 1 below is SEQ ID NO: 5
  • the nucleotide sequence of the reverse primer is SEQ ID NO: 6.
  • FCGRT gene and the B2M gene are known to form heterodimers of expressed receptor proteins and are expressed on the cell surface, and function as receptors for HAstV. Because of this possibility, we conducted a more detailed verification. That is, the FCGRT gene and gRNA of the B2M gene were introduced into Caco-2/Cas9, single cell cloning was performed, and knockout cells of the FCGRT gene and B2M gene were established.
  • Fig. 1-A shows the results of reacting with an anti-actin antibody as a primary antibody and then reacting with an anti-Goat antibody as a secondary antibody.
  • Figure 1-B shows the results of reacting with an anti-FCGRT protein antibody as a primary antibody and then reacting with an anti-Rabbit antibody as a secondary antibody.
  • Figure 1-C shows the results of reacting with an anti-B2M protein antibody as a primary antibody and then reacting with an anti-rabbit antibody as a secondary antibody.
  • HAstV Changes in susceptibility to HAstV infection by knocking out the FCGRT gene and B2M gene
  • HAstV (as described above, The cells were infected with HAstV-1 and HAstV-4), and the cell morphology and the amount of viral RNA replication were observed. Since HAstV is said to become infectious particles when activated by Trypsin, Caco-2 was infected with Trypsin-activated virus and cultured for 6 days in a medium with or without Trypsin. The results are shown in Figures 2-1 and 2-2.
  • FIG. 2-1 relates to HAstV-1
  • FIG. 2-2 relates to HAstV-4. Both figures show micrographs of cells before infection and cells on days 3 and 6 after culture of infection.
  • HAstV RNA levels in FCGRT and B2M gene-knocked out cells After HAstV is replicated in cells and viral particles are assembled, it is released outside the cells and then activated by Trypsin to become mature viruses. ing. Therefore, in this study, the virus was activated with Trypsin prior to infection in order to efficiently infect HAstV. In addition, Trypsin was added to the culture medium and cultured so that trypsin could activate the new virus released extracellularly during the 6-day culture.
  • RNA is extracted from the collected culture supernatant, cDNA is prepared, RT - The quantified value by PCR was used as the desired amount of RNA.
  • Caco-2/Cas9, Caco-2/Cas9/FCGRT KO, and Caco-2/Cas9/B2M KO were seeded on a collagen-coated 24-well plate to construct a HAstV infection system.
  • This HAstV infection system was infected with HAstV-1 at a concentration of 6.84 ⁇ 10 4 / ⁇ l of RNA and with HAstV-4 at a concentration of 4.29 ⁇ 10 3 / ⁇ l.
  • the culture solution the new medium after infection and washing was cultured on day 0 (day0), and then cultured for 6 days. They were harvested and their RNA content was quantified.
  • Table 2-1 and Figure 3-1 for HAstV-1 and Table 2-2 and Figure 3-2 for HAstV-4 The results are shown in Table 2-1 and Figure 3-1 for HAstV-1 and Table 2-2 and Figure 3-2 for HAstV-4.
  • FIGS. 3-1 and 3-2 the common logarithms were calculated from the amounts of RNA shown in Tables 2-1 and 2-2, respectively, and their averages and standard deviations were obtained.
  • the vertical axis of the graph represents the HAstV RNA copy number present per 1 ⁇ l of the culture supernatant.
  • day 0 the average values on day 0 of infection
  • day 3 day 3
  • day 6 day 6 of culture
  • Error bars are calculated standard deviations.
  • “A” as a knockout (KO) target indicates the FCGRT gene
  • "B” indicates the B2M gene.
  • RNA copy number of Caco-2/Cas9 was amplified to about 1000-fold on day 0 after 3 days of culture, reaching a peak. After 6 days, no significant difference was observed from that after 3 days.
  • Caco-2/Cas9/FCGRT KO after 3 days of culture, the amount of RNA was amplified to about 100-fold after 0 days, but did not reach the peak. After 6 days of culture, RNA was further amplified from the 3rd day and reached about 1000-fold compared to the 0th day, similar to Caco-2/Cas9.
  • Caco-2/Cas9/B2M KO also gave generally similar results to Caco-2/Cas9/FCGRT KO.
  • Caco-2/Cas9 peaked after 3 days of culture, with the amount of RNA amplified to 100,000-fold or more on day 0. No significant difference was observed after 6 days of culture and after 3 days.
  • Caco-2/Cas9/FCGRT KO after 3 days of culture, the amount of RNA was amplified to about 100-fold on day 0, but did not reach its peak. After 6 days of culture, RNA was further amplified from the 3 day time point, reaching approximately 10000-fold compared to day 0, but not after 6 days of Caco-2/Cas9.
  • Caco-2/Cas9/B2M KO after 3 days the amount of RNA was amplified to about 10-fold on day 0, but did not reach the peak. After 6 days, the amount of RNA reached about 10000-fold compared to day 0, but as with Caco-2/Cas9/FCGRT KO, it was less than after 6 days of Caco-2/Cas9.
  • HAstV infection of human intestinal organoids and quantification of genomic RNA 3-1 Establishment of Knockout Cells It is known that HAstV infects and proliferates in human intestinal organoids (SI1). SI1 is a cell established from intestinal stem cells, and can be differentiated into epithelial or goblet cells by inducing differentiation in vitro. Also in this human intestinal organoid (SI1), knockout cells were established to evaluate whether the FCGRT gene and B2M gene are involved in HAstV infection susceptibility. For knockout, SI1/FCGRT KO in which the FCGRT gene was knocked out and SI1/B2M KO in which the B2M gene was knocked out were obtained using the same method as for Caco2 cells described above.
  • FIGS. 4-A and B were originally one membrane, cleaved at about 30 kDa after protein transcription, and reacted with different antibodies.
  • Fig. 4-A shows the results of reacting with an anti-Goat antibody as a secondary antibody after reacting with an anti-actin antibody as a primary antibody.
  • Figure 4-B shows the results of reacting with an anti-FCGRT protein antibody as a primary antibody and then reacting with an anti-Rabbit antibody as a secondary antibody.
  • Fig. 4-C shows the results of reacting with an anti-B2M protein antibody as a primary antibody and then reacting with an anti-rabbit antibody as a secondary antibody.
  • FIG. 5 shows micrographs of HAstV-infected SI1, SI1/FCGRT KO, and SI1/B2M KO on day 0 of infection (day 0) and day 3 of culture (day 3).
  • HAstV infection system was infected with HAstV-1 at a concentration of 4.32 ⁇ 10 5 / ⁇ l of RNA and with HAstV-4 at a concentration of 5.24 ⁇ 10 6 / ⁇ l.
  • SI1 infected with HAstV-1 the copy number of HAstV RNA after 3 days of culture was amplified about 1000-fold on day 0 of infection.
  • SI1/FCGRT KO the amount of RNA was amplified about 100-fold on the third day of culture.
  • the SI1/B2M KO was also amplified about 100-fold like the SI1/FCGRT KO.
  • SI1 infected with HAstV-4 the copy number of HAstV RNA on day 3 of culture was amplified to about 100-fold on day 0 of infection.
  • SI1/FCGRT KO the amount of RNA was amplified about 10-fold on the third day of culture.
  • the SI1/B2M KO was also amplified about 10-fold like the SI1/FCGRT KO.
  • FCGRT receptor and B2M may be HAstV infection receptors, and since these receptors form heterodimers, they may function as the infection receptors in conjunction with each other. was taken.
  • MDCK cells purchased from ATCC
  • MEM medium at 37° C. and 5% CO 2 until confluent.
  • seeded on a 6-well plate cultured until confluent again, added with the above lentiviral vector, and recombined lentivirus using the drug resistance of the introduced lentivirus at 37 ° C., 5% CO 2 , That is, cells into which the gene of interest was introduced were selected (step 4-5).
  • Gene transfer was carried out in three patterns of FCGRT gene only, B2M gene only, and both FCGRT gene and B2M gene to obtain respective cells.
  • step 5 The cells selected in step 5 were scaled up and cultured under the condition that a drug for cell selection was added, and the proliferated cells were stocked (step 6).
  • the blot images in FIG. 8 are, from the left, MDCK cells, MDCK cells introduced with the FCGRT gene, MDCK cells introduced with the B2M gene, MDCK cells introduced with both the FCGRT gene and the B2M gene, Caco-2/Cas9 cells ( control). From FIG. 8, it became clear that each target gene was expressed in each gene-introduced cell.
  • each recombinant MDCK cell was seeded in 24 wells, serum-free medium was added and cultured overnight at 37°C and 5% CO 2 , then the cells were washed with PBS (-), A serum-free medium was added and the cells were cultured at 37° C. and 5% CO 2 for 1 hour.
  • HAstV HAstV-1, HAstV-4
  • trypsin at 37° C. for 1 hour
  • the serum-free medium was removed, the activated HAstV solution was added, culture was carried out at 37° C., 5% CO 2 , the virus solution was removed, and the cells were washed with PBS( ⁇ ).
  • HAstV-1 was infected at a concentration of 6.84 ⁇ 10 4 / ⁇ l of RNA, and HAstV-4 was infected at a concentration of 4.29 ⁇ 10 3 / ⁇ l.
  • FIG. 9-A shows the results for HAstV-1
  • FIG. 9-B shows the results for HAstV-9.
  • "A” indicates introduction of the FCGRT gene
  • "B” indicates introduction of the B2M gene.
  • MDCK cells MDCK cells introduced with the FCGRT gene
  • MDCK cells introduced with the B2M gene MDCK cells introduced with both the FCGRT gene and the B2M gene
  • Caco-2 / Cas9 cells control.
  • the adjacent left side indicates day 0 of infection (d0)
  • the adjacent right side indicates 6 days after culture (d6).
  • MDCK/FCGRT was amplified 100-fold, while no significant difference was observed in MDCK/B2M, and approximately 1000-fold amplification was observed in MDCK/FCGRT/B2M.
  • FCGRT receptor protein and B2M protein form a heterodimer and are a type of membrane protein present on the cell surface.
  • the B2M protein improves the stability of itself and the FCGRT receptor protein by forming a heterodimer with the FCGRT receptor protein, and this heterodimer structure greatly contributes to the acquisition of HAstV infectivity.
  • HAstV particles were immobilized at 600 ng/well on a 96-well plate, and serially diluted heterodimers of recombinant FCGRT protein and B2M protein (ECD, His & AVI Tag) were seeded at 12 ⁇ g/ml at 50 ⁇ l/well, The proteins bound to the virus particles were bound with an HRP-labeled anti-histidine tag antibody to develop a color, and measured at 450 nm to measure the concentration-dependent binding amount of the heterodimer to HAstV. Since the recombinant receptor protein described above is produced by expressing it in HEK293 cells (derived from human embryonic kidney), the FCGRT protein and the B2M protein exist as heterodimers.
  • FIG. 11 shows the concentration dependence of HAstV (HAstV-1: FIG. 11-A (left), HAstV-4: FIG. 11-B (right)) on the heterodimer of FCGRT protein and B2M protein observed by ELISA.
  • Fig. 10 is a drawing showing an increase in the amount of binding between The horizontal axis is the concentration of the heterodimer. Both HAstV-1 and HAstV-4 showed an increase in absorbance with an increase in the concentration of the heterodimer, and the heterodimer of FCGRT protein and B2M protein was found to bind to HAstV particles in a concentration-dependent manner. rice field.
  • FCGRT protein is a HAstV receptor alone, but when combined with the B2M protein to form a heterodimer, both receptor proteins obtain structural stability, and the heterodimer Dimers were shown to confer de novo HAstV sensitivity to HAstV-insensitive cells in the most favorable manner.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Food Science & Technology (AREA)

Abstract

La présente invention a pour but de procurer un moyen d'identifier les molécules réceptrices de l'astrovirus humain (HAstV) et de développer un moyen de lutte contre l'infection par l'HAstV. Les inventeurs ont découvert qu'une protéine réceptrice FCGRT et une protéine B2M possèdent des molécules réceptrices du HAstV. Les inventeurs ont réussi à obtenir des cellules de mammifères recombinées et des mammifères recombinés dans lesquels des gènes codant pour les molécules réceptrices du HAstV sont introduits, ainsi qu'un procédé de production de virus, un procédé de criblage d'agents chimiques et d'autres procédés similaires les utilisant, et à obtenir en outre un procédé de détection du HAstV, un procédé de criblage d'agents chimiques et d'autres procédés similaires utilisant un support en phase solide sur lequel sont fixées les molécules réceptrices du HAstV.
PCT/JP2022/029580 2021-08-02 2022-08-02 Récepteur de l'infection par l'astrovirus humain et son application WO2023013618A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023540351A JPWO2023013618A1 (fr) 2021-08-02 2022-08-02

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021126962 2021-08-02
JP2021-126962 2021-08-02

Publications (1)

Publication Number Publication Date
WO2023013618A1 true WO2023013618A1 (fr) 2023-02-09

Family

ID=85155683

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/029580 WO2023013618A1 (fr) 2021-08-02 2022-08-02 Récepteur de l'infection par l'astrovirus humain et son application

Country Status (2)

Country Link
JP (1) JPWO2023013618A1 (fr)
WO (1) WO2023013618A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116970574A (zh) * 2023-06-26 2023-10-31 广东省农业科学院动物卫生研究所 一种鹅源波形蛋白的用途、促进增殖鹅星状病毒的方法及用途、疫苗

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012073992A1 (fr) * 2010-11-30 2012-06-07 中外製薬株式会社 Molécule de liaison à l'antigène, apte à se lier de façon répétée à une pluralité de molécules d'antigène
JP2015512034A (ja) * 2012-02-15 2015-04-23 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｆｃ受容体に基づくアフィニティークロマトグラフィー
JP2015525204A (ja) * 2012-05-14 2015-09-03 ユセベ ファルマ ソシエテ アノニム 抗ＦｃＲｎ抗体
JP2019536738A (ja) * 2016-09-16 2019-12-19 中外製薬株式会社 抗デングウイルス抗体、変異型Ｆｃ領域を含むポリペプチド、およびその使用方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012073992A1 (fr) * 2010-11-30 2012-06-07 中外製薬株式会社 Molécule de liaison à l'antigène, apte à se lier de façon répétée à une pluralité de molécules d'antigène
JP2015512034A (ja) * 2012-02-15 2015-04-23 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｆｃ受容体に基づくアフィニティークロマトグラフィー
JP2015525204A (ja) * 2012-05-14 2015-09-03 ユセベ ファルマ ソシエテ アノニム 抗ＦｃＲｎ抗体
JP2019536738A (ja) * 2016-09-16 2019-12-19 中外製薬株式会社 抗デングウイルス抗体、変異型Ｆｃ領域を含むポリペプチド、およびその使用方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ARIAS CARLOS, DUBOIS REBECCA: "The Astrovirus Capsid: A Review", VIRUSES, MDPI, CH, vol. 9, no. 1, 1 January 2017 (2017-01-01), CH , pages 15 - 13, XP093032484, ISSN: 1999-4915, DOI: 10.3390/v9010015 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116970574A (zh) * 2023-06-26 2023-10-31 广东省农业科学院动物卫生研究所 一种鹅源波形蛋白的用途、促进增殖鹅星状病毒的方法及用途、疫苗
CN116970574B (zh) * 2023-06-26 2024-02-09 广东省农业科学院动物卫生研究所 一种鹅源波形蛋白的用途、促进增殖鹅星状病毒的方法及用途、疫苗

Also Published As

Publication number Publication date
JPWO2023013618A1 (fr) 2023-02-09

Similar Documents

Publication Publication Date Title
Ma et al. LDLRAD3 is a receptor for Venezuelan equine encephalitis virus
Li et al. Porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response
Butel et al. Cell and molecular biology of simian virus 40: implications for human infections and disease
Schowalter et al. The Merkel cell polyomavirus minor capsid protein
Pudupakam et al. Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication
Krey et al. Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1
Gladue et al. Interaction of foot-and-mouth disease virus nonstructural protein 3A with host protein DCTN3 is important for viral virulence in cattle
To et al. Channel-inactivating mutations and their revertant mutants in the envelope protein of infectious bronchitis virus
Abente et al. The feline calicivirus leader of the capsid protein is associated with cytopathic effect
WO2023013618A1 (fr) Récepteur de l'infection par l'astrovirus humain et son application
Zhu et al. Interaction of E2 glycoprotein with heparan sulfate is crucial for cellular infection of Sindbis virus
Diller et al. Rotavirus species B encodes a functional fusion-associated small transmembrane protein
Boujon et al. Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine astrovirus
KR20170106473A (ko) 엔테로바이러스 71 동물 모델
Rauch et al. Mutations in herpes simplex virus glycoprotein D distinguish entry of free virus from cell-cell spread
García-Rodríguez et al. Parechovirus a infection of the intestinal epithelium: differences between genotypes A1 and A3
Arita et al. Quantitative analysis of poliomyelitis-like paralysis in mice induced by a poliovirus replicon
Helm et al. Environmentally-triggered contraction of the norovirus virion determines diarrheagenic potential
Yin et al. Isolation and characterization of a novel chicken astrovirus in China
Liao et al. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization
Kristen-Burmann et al. Reverse Genetic Assessment of the Roles Played by the Spike Protein and ORF3 in Porcine Epidemic Diarrhea Virus Pathogenicity
Sarmiento et al. Differential effects of three echovirus strains on cell lysis and insulin secretion in beta cell derived lines
Pu et al. Evaluate severe acute respiratory syndrome coronavirus 2 infectivity by pseudoviral particles
Méndez et al. Astrovirus
Haga et al. Neonatal Fc receptor is a functional receptor for human astrovirus

Legal Events

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

Ref document number: 22853037

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023540351

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE