WO2022231402A1 - Composition for preparing avian cell for production of antiviral vaccine and composition for preparing virus-resistant avian cell - Google Patents

Composition for preparing avian cell for production of antiviral vaccine and composition for preparing virus-resistant avian cell Download PDF

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WO2022231402A1
WO2022231402A1 PCT/KR2022/006261 KR2022006261W WO2022231402A1 WO 2022231402 A1 WO2022231402 A1 WO 2022231402A1 KR 2022006261 W KR2022006261 W KR 2022006261W WO 2022231402 A1 WO2022231402 A1 WO 2022231402A1
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virus
composition
avian
cells
b4galnt2
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French (fr)
Korean (ko)
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한재용
우승제
박진세
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서울대학교산학협력단
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Priority claimed from KR1020220054265A external-priority patent/KR20220149476A/en
Publication of WO2022231402A1 publication Critical patent/WO2022231402A1/en

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    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • 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
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Definitions

  • the present invention relates to a composition for preparing avian cells for the production of a virus vaccine and a composition for preparing virus-resistant avian cells.
  • Avian influenza virus belongs to influenza A virus, and the damage caused by AI is serious not only in Korea but also worldwide. In Korea, a total of six highly pathogenic avian influenza viruses occurred from December 2003 to April 2016, and in November of the same year, a new strain of H5N6 mutant highly pathogenic avian virus was reported. Overseas, since 2003, it mainly occurred in Southeast Asia, such as Thailand and Vietnam, but since then, it has spread all over the world, including Russia, Mongolia, Europe, America, Africa, and India, and the damage is increasing. In addition to the avian influenza virus, various viral diseases infecting birds can not only cause fatal damage to the livestock sector that raises chickens, ducks, geese, etc. , pneumonia, respiratory failure, and, in severe cases, death.
  • Influenza A viruses utilize a variety of host proteins for the transcription and translation of viral proteins (Aartian, 2016, Nature Reviews Microbiology ; Karlas, 2010, Nature Letters ). for chickens. There is no retinoic acid inducible gene 1 (RIG-I) receptor that recognizes avian influenza virus, and melanoma differentiation associated protein 5 (MDA5) is a major receptor that recognizes the virus. After virus recognition by MDA5, an immune response is induced to eliminate the virus.
  • RAG-I retinoic acid inducible gene 1
  • MDA5 melanoma differentiation associated protein 5
  • TMPRSS2 Type II transmembrane protease, serine 2 is a proteolytic enzyme similar to trypsin, and ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1) transfers sialic acid to glycoproteins or glycolipids to facilitate virus-cell membrane fusion.
  • human B4GALNT2 (beta-1,4 N-acetyl galactosaminyl transferase 2) is known to prevent virus from using sialic acid by attaching GalNAc to a sugar containing sialic acid, thereby disrupting the fusion of virus and cell membrane.
  • An object of the present invention is to provide a composition for preparing avian cells for virus vaccine production by controlling virus resistance.
  • Another object of the present invention is to provide a composition for producing a virus vaccine and a method for producing a virus vaccine, including the composition for producing avian cells for production of the virus vaccine.
  • An object of the present invention is to provide a composition for preparing avian cells having virus vaccine resistance.
  • Another object of the present invention is to provide a method for preparing virus-resistant avian cells, comprising the composition for preparing avian cells having virus vaccine resistance.
  • a composition for preparing avian cells for the production of a virus vaccine comprising a.
  • composition according to 1 above, wherein the substance for deleting MDA5 or TLR3 comprises gRNA that complementarily binds to MDA5 or TLR3.
  • composition for producing avian cells for virus vaccine production according to 1 above, wherein the substance overexpressing TMPRSS2 or ST3GAL1 is a vector containing a gene encoding TMPRSS2 or ST3GAL1.
  • the birds are chickens, ducks, geese, geese, pigeons, pheasants, bongguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwi birds, cuckoos, cranes, swallows, crows, storks,
  • a composition for preparing avian cells for the production of a virus vaccine selected from the group comprising herons, snipes, houndstooths, owls, owls, woodpeckers, eagles, hawks, parrots, sparrows, orioles or bunting.
  • composition for preparing avian cells for the production of a virus vaccine according to the above 1, wherein the virus is selected from the group comprising avian influenza virus, Newcastle virus and bronchitis virus.
  • composition for producing a virus vaccine comprising avian cells treated with the composition of any one of 1 to 6 above.
  • a composition for preparing virus-resistant algal cells comprising a substance overexpressing B4GALNT2 ( ⁇ -1,4 N-acetylgalactosaminyltransferase 2).
  • composition for preparing virus-resistant avian cells according to the above 14, wherein the B4GALNT2 is human B4GALNT2.
  • insects are chickens, ducks, geese, geese, pigeons, pheasants, bonguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwi birds, cuckoos, cranes, swallows, crows, storks, Herons, snipes, houndstooths, owls, owls, woodpeckers, eagles, hawks, parrots, sparrows, orioles or bunting birds.
  • a composition for preparing virus-resistant bird cells are chickens, ducks, geese, geese, pigeons, pheasants, bonguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwi birds, cuckoos, cranes, swallows, crows, stor
  • composition for preparing virus-resistant avian cells according to the above 14, wherein the virus is selected from the group comprising avian influenza virus, Newcastle virus and bronchitis virus.
  • a composition for preparing virus-resistant algae comprising primitive germ cells or blastoderm cells treated with the composition for preparing virus-resistant algal cells of any one of 14 to 19 above.
  • a method for producing a virus resistant avian cell comprising the step of overexpressing B4GALNT2 in the avian cell.
  • a composition for producing a virus vaccine is provided through the composition for producing avian cells for the production of a virus vaccine of the present invention. This can provide a virus vaccine production method having a higher production efficiency than the prior art, and can be preferably used in the virus vaccine production process.
  • a method for preparing virus-resistant algae through the composition for preparing virus-resistant algal cells of the present invention can be used in the livestock field because it can produce transgenic birds with resistance to avian influenza, Newcastle disease virus, chicken infectious bronchitis virus, and the like.
  • FIG. 1 is a schematic diagram of the present invention, specifically, when MDA5 is deleted or expression of TMPRSS2 and ST3GAL1 is induced, the production efficiency of a virus vaccine precursor increases, and when human-derived B4GALNT2 is expressed, virus production can be inhibited. have.
  • FIG. 2 is a diagram showing the pattern of gene editing compared to the wild-type genotype after inducing genetic modification by simultaneously targeting chicken MDA5, TLR3, or MDA5 and TLR3 in chicken fibroblasts (DF-1) using avian genome editing technology. .
  • Figure 3 shows the viral RNA analog poly I:C and low pathogenic influenza A virus when treated with wild-type chicken fibroblasts, chicken MDA5 or TLR3 depleted fibroblasts, and chicken MDA5 and TLR3 depleted fibroblasts at the same time, interferon and lower It is a graph showing the induction of activity/expression of genes.
  • Figure 4 is a graph comparing the effect of overexpressing chicken MDA5 in a chicken MDA5 cell line and the virus titer produced when the low pathogenic influenza A virus was infected with wild-type chicken fibroblasts and chicken MDA5 removed fibroblasts.
  • FIG. 5 is a graph showing that TMPRSS2 overexpression in chicken fibroblasts and an increase in TMPRSS2 gene expression level and TMPRSS2 overexpressing cell line increase the production of avian influenza virus.
  • FIG. 6 is a graph showing that ST3GAL1 overexpression in chicken fibroblasts and an increase in the expression level of ST3GAL1 gene, and that the ST3GAL1 overexpressing cell line increases the production of avian influenza virus.
  • FIG. 7 is a graph showing that a cell line that simultaneously overexpresses TMPRSS2 and ST3GAL1 in chicken fibroblasts increases the production of avian influenza virus.
  • FIG. 8 is a schematic diagram showing the function of human B4GALNT2, and is a diagram showing the expression when overexpressing human B4GALNT2 via PiggyBac.
  • FIG. 9 is a schematic diagram for knock-in of human B4GALNT2 followed by chicken GAPDH via CRISPR/Cas9 and a diagram showing the genotype of a gene-edited cell clone. It also shows the expression of human B4GALNT2 knocked-in after GAPDH.
  • FIG. 10 is a diagram showing that the human B4GALNT2 introduced chicken fibroblast line has significantly lower viral titer and higher cellular activity than wild-type chicken fibroblasts because binding to sialic acid used by the virus is inhibited.
  • the present invention relates to a substance that deletes melanoma differentiation associated protein 5 (MDA5) or toll like receptor 3 (TLR3); or a substance that overexpresses TMPRSS2 (type II transmembrane protease, serine 2) or ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1); It relates to a composition for preparing avian cells for the production of a virus vaccine comprising a.
  • MDA5 melanoma differentiation associated protein 5
  • TLR3 toll like receptor 3
  • the type of the substance for deleting MDA5 or TLR3 is not limited as long as it inhibits the synthesis of a protein product of the gene that performs a normal function.
  • gRNA guide RNA
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc-finger nuclease
  • the nucleic acid may correspond to a substance that deletes MDA5 or TLR3 of the present invention irrespective of the nucleotide sequence if it can bind complementarily to MDA5 or TLR3, or encode a protein that binds complementarily, and the corresponding sequence can be determined by methods known by those skilled in the art.
  • the gRNA is an RNA complementary to a nucleotide sequence of a target gene or a sequence corresponding thereto, and is complementary to all or part of a target DNA sequence to guide an endonuclease protein to the target DNA sequence.
  • the gRNA may be a dual RNA comprising two RNAs, namely, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA); or a single chain guide RNA (sgRNA) form comprising a first site comprising a sequence that is fully or partially complementary to a sequence in the target DNA and a second site comprising a sequence that interacts with an RNA-guided nuclease,
  • CRISPR RNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • sgRNA single chain guide RNA
  • sgRNA single chain guide RNA
  • the RNA-guided endonuclease may be included in the scope of the present invention without limitation as long as it is a form capable of having activity in the target DNA sequence.
  • the gRNA according to the present invention may preferably be in the form of a single-stranded guide RNA (sgRNA), but is not limited thereto, and may be appropriately selected according to the type of endonuclease used or a microorganism derived therefrom.
  • sgRNA single-stranded guide RNA
  • the MDA5 or TLR3 may play a role of activating an innate immune mechanism against a foreign antigen by recognizing a foreign RNA ligand in avian cells. Therefore, when the MDA5 or TLR3 is deleted, virus replication and Assembly can be further facilitated. On the other hand, when the expression of MDA5 is decreased by treatment with complementary siRNA, etc., the above effect may not be exhibited. Since MDA5 or TLR3 is conserved in algae, it may exert the above effect in all algae regardless of algae species, but may be more effective in algae in which Retinoic acid inducible gene 1 (RIG-1) is deleted. The bird in which the RIG-1 (Retinoic acid inducible gene 1) is deleted may be specifically chicken.
  • RIG-1 Retinoic acid inducible gene 1
  • avian cells when avian cells are treated with a substance that deletes either the MDA5 gene or the TLR3 gene, a higher viral titer may be exhibited compared to the case where the avian cells are not, preferably both MDA5 and TLR3 are deleted or When all of the expression is reduced, the virus titer may be about 30 times higher than that of the control group.
  • the present invention is not limited thereto.
  • the substance overexpressing TMPRSS2 or ST3GAL1 may be a vector including a gene encoding TMPRSS2 or ST3GAL1.
  • the vector comprising the gene encoding TMPRSS2 or ST3GAL1 may include the TMPRSS2 base sequence of SEQ ID NO: 1, the ST3GAL1 base sequence of SEQ ID NO: 2, or a homologue of each base sequence, respectively.
  • the homologue may include a nucleotide sequence encoding the same amino acid as the amino acid encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 even if there is a nucleotide sequence that is partially different from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, Even if some amino acid sequences are different, it may include a nucleotide sequence in which there is no difference in the function of the encoded protein or only a difference in the degree of increase or decrease of the effect exists.
  • the corresponding sequence may include a sequence that varies due to base variation between species, and even if there is some base variation, the gene can be identified through alignment and sequence comparison of the sequence.
  • nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and any nucleotide sequence are aligned, at least 60% homology, preferably 70%, more preferably 80%, even more preferably A sequence having 90%, most preferably 99% homology may correspond to the homologue of the present invention.
  • the TMPRSS2 or ST3GAL1 can promote the influx of a foreign virus into the cell in the avian cell, when the TMPRSS2 or ST3GAL1 is overexpressed, the virus replication and assembly in the avian cell can be further promoted. Since the TMPRSS2 or ST3GAL1 is conserved in algae, the above effect may be exerted regardless of the species of algae, but it may be more effective in algae in which RIG-1 is preferably deleted. The bird in which the RIG-1 is deleted may specifically be a chicken.
  • a higher viral titer when avian cells are treated with a substance overexpressing either TMPRSS2 or ST3GAL1, a higher viral titer may be exhibited than when not, preferably when both TMPRSS2 and ST3GAL12 are overexpressed, 10 to 100 fold viral titer.
  • the present invention is not limited thereto.
  • the composition for preparing avian cells for the production of a virus vaccine of the present invention includes a substance that deletes melanoma differentiation associated protein 5 (MDA5) or toll like receptor 3 (TLR3); or a substance that overexpresses TMPRSS2 (type II transmembrane protease, serine 2) or ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1); It may include a recombinant vector carrying a.
  • the recombinant vector can be used without limitation by those skilled in the art, regardless of the sequence, as long as it can deliver the substances to the target cell.
  • recombinant vectors examples include plasmid, bacteriophage, cosmid, BAC (bacteria artificial chromosome), YAC (yeast artificial chromosome), retrovirus, adenovirus, adeno-associated virus (adeno-associated) virus), or a non-viral vector.
  • BAC bacteria artificial chromosome
  • YAC yeast artificial chromosome
  • retrovirus retrovirus
  • adenovirus adeno-associated virus (adeno-associated) virus
  • non-viral vector examples include a non-viral vector.
  • the present invention is not limited thereto.
  • the algae may be included in the scope of the present invention without limitation as long as the algae belong to Avian .
  • Avian chickens, ducks, geese, geese, pigeons, pheasants, bonguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwis, cuckoos, cranes, swallows, crows, storks, herons, snipes, plovers, It may include an owl, an owl, a woodpecker, an eagle, a hawk, a parrot, a sparrow, an owl or a bunting, etc.
  • the bird may be a chicken, but is not limited thereto.
  • the virus may be included in the scope of the present invention regardless of the host species as long as the virus can be delivered to the avian cells. Specifically, it may be a virus using an alga as a host and RNA as a genome. Examples of viruses that host birds and have RNA as their genomes include avian influenza (AI), Newcastle disease virus (NDV) or chicken infectious bronchitis virus ( Avian infectious bronchitis virus ). . Preferably, the virus may be an avian influenza virus, but is not limited thereto.
  • the present invention relates to a substance that deletes the MDA5 or TLR3; or a substance that overexpresses TMPRSS2 or ST3GAL1; It relates to a composition for producing a virus vaccine comprising avian cells treated with the composition comprising a.
  • the algal cells treated with the composition containing the MDA5, TLR3, TMPRSS2 or ST3GAL1 in algae are not limited to cell types, and may be selected by those skilled in the art because the mechanism of action is conserved.
  • PPC primordial germ cells
  • endothelial cells egg cells etc.
  • it may be a fibroblast with a fast division rate and easy culture.
  • the present invention is not limited thereto.
  • the present invention relates to a first step of deleting MDA5 or TLR3 or overexpressing TMPRSS2 or ST3GAL1 in an avian cell;
  • It relates to a method for producing a virus vaccine comprising a second step of infecting the avian cells that have undergone the first step with the attenuated virus.
  • the first step is a material for deleting the MDA5 or TLR3 in the aforementioned avian cells;
  • it may be treating a composition containing a vector including a gene encoding TMPRSS2 or ST3GAL1.
  • the present invention is not limited thereto, and it may be performed through a gene expression control method used by those skilled in the art.
  • the infecting attenuated virus and the virus targeted by the produced vaccine may be the same virus or may share the same antigen.
  • the step of infecting the virus may include injecting the virus itself, which is the target of the vaccine, into the avian cells that have undergone the first step, or a vector carrying the genome of the virus that is the target of the vaccine is injected into the avian cells. It can also be delivered to the cell so that the virus is synthesized in the cell.
  • the vector carrying the viral genome that is the target of the vaccine is not limited to the type as mentioned above, but a plasmid, bacteriophage, cosmid, BAC, YAC, retrovirus, adenovirus, adeno-associated virus or non-viral
  • a person skilled in the art, such as a vector can select and use it without limitation.
  • the method for producing a virus vaccine may further include a third step of culturing the avian cells that have passed through the second step to obtain a virus therefrom.
  • a new viral protein can be synthesized in the cell by the previously injected virus or viral vector, and a new virus can be assembled therefrom.
  • the incubation time may be selected without limitation by those skilled in the art according to experimental conditions. An increase in the incubation time may increase the replication of the viral genome and assembly of the virus, but if it exceeds a certain value, it may not have an effect on the increase in the recovery rate of the virus vaccine due to cell death.
  • the incubation time may be culturing for at least 6 hours or more, and the upper limit is not limited, but may be, for example, 120 hours, 96 hours or 72 hours. However, the present invention is not limited thereto.
  • the assembled virus can be extracted and isolated by methods known by those skilled in the art. For example, it can be obtained by disrupting the algal cell culture solution and then concentrating the virus contained in the supernatant obtained by centrifugation. However, the present invention is not limited thereto.
  • a higher titer of a virus vaccine can be obtained compared to the conventional method.
  • a higher viral titer compared to the control group that is not The highest viral titer can be exhibited when both MDA5 and TLR3 are deleted or their expression is suppressed, and both TMPRSS2 and ST3GAL1 are overexpressed.
  • the present invention is not limited thereto.
  • the present invention relates to a composition for preparing virus-resistant algal cells comprising a substance overexpressing B4GALNT2 ( ⁇ -1,4 N-acetylgalactosaminyltransferase 2).
  • the virus resistance may be that the virus is blocked from being injected into the cell, the injected virus replicates the gene to inhibit the assembly of the next generation virus, or inhibits the release of the assembled virus it could be It is not limited to the above mechanism, and it means to have resistance to viral infection.
  • Viruses having resistance to the virus resistance may be included in the scope of the present invention without limitation as long as the virus is an avian host.
  • avian influenza virus, Newcastle virus or chicken infectious bronchitis virus, etc. may be included therein, and in addition, infectious laryngotracheitis virus (ILTV ) having DNA as a genome, herpes virus ( Herpes virus ), Marek may include Marek's disease virus (MDV), Avian leucosis virus (ALV) and infectious bursal of fabricius disease virus (IBDV).
  • MDV Marek's disease virus
  • ABV Avian leucosis virus
  • IBDV infectious bursal of fabricius disease virus
  • the present invention is not limited thereto.
  • the B4GALNT2 can suppress the influx of viral genetic material into the cell by controlling the binding affinity of the foreign virus to the cell in the avian cell. Therefore, when B4GALNT2 is overexpressed, Resistance may increase. Since algal cells generally do not express B4GALNT2, the effect of the present B4GALNT2 can be exerted regardless of the species of algae. For example, in one embodiment of the present invention, when human B4GALNT2 was overexpressed in chicken fibroblasts, it was confirmed that the virus titer was 3 to 4 times lower than that in the case of not. However, the present invention is not limited thereto.
  • the B4GALNT2 may be included without limitation, regardless of the species, as long as it is a gene encoding the B4GALNT2 protein or a homologue thereof.
  • the nucleotide sequence encoding the B4GALNT2 protein may be, for example, a B4GALNT2 gene sequence conserved in species such as humans, mice, goats, sheep, cats, pigs, frogs, monkeys, chimpanzees, hamsters, dogs, or lizards, and more Specifically, it may be the nucleotide sequence of SEQ ID NO: 3.
  • the present invention is not limited thereto.
  • the substance overexpressing B4GALNT2 may be a vector including a gene encoding B4GALNT2.
  • the vector containing the gene encoding B4GALNT2 is not limited to its type, and those skilled in the art such as plasmid, bacteriophage, cosmid, BAC, YAC, retrovirus, adenovirus, adeno-associated virus or non-viral vector can be used without limitation.
  • the alga is the aforementioned birds, for example, chickens, ducks, geese, geese, pigeons, pheasants, bongguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, It can be a kiwi, cuckoo, crane, swallow, crow, stork, heron, snipe, houndstooth, owl, owl, woodpecker, eagle, hawk, parrot, sparrow, oriole or bunting, and avian cells are invented regardless of cell type Although it may be included in the scope of, preferably, it may be an avian cell having totipotency, for example, a stem cell or a blastocyst. However, the present invention is not limited thereto.
  • the present invention also relates to a composition for preparing virus-resistant algae comprising primitive germ cells or blastoderm cells treated with a composition for preparing virus-resistant algal cells comprising a substance overexpressing B4GALNT2 ( ⁇ -1,4 N-acetylgalactosaminyltransferase 2). .
  • the primordial germ cells or blastoderm cells are totipotent cells capable of differentiating into avian individuals, and a composition for preparing virus-resistant avian cells containing a substance overexpressing the B4GALNT2 ( ⁇ -1,4 N-acetylgalactosaminyltransferase 2) is used as a raw material. It may be expressed by injection into germ cells or blastoderm cells.
  • the method for differentiating the primordial germ cells or blastoderm cells injected with the composition into an adult may be carried out without limitation according to a known method.
  • the present invention also relates to a method for producing a virus-resistant avian cell comprising the step of overexpressing B4GALNT2 in the avian cell.
  • the step of overexpressing B4GALNT2 in the avian cell may be performed by treating the avian cell with a gene overexpression method that can be used by a person skilled in the art, for example, a vector containing a gene encoding B4GALNT2. may be a step of treating chicken blastoderm cells with a vector containing a gene encoding human B4GALNT2.
  • a gene overexpression method that can be used by a person skilled in the art, for example, a vector containing a gene encoding B4GALNT2.
  • a vector containing a gene encoding B4GALNT2. may be a step of treating chicken blastoderm cells with a vector containing a gene encoding human B4GALNT2.
  • the present invention is not limited thereto.
  • the present invention also relates to a method for producing an alga from the virus-resistant algal cell.
  • the method of the present invention relates to a method for producing an alga from an avian cell overexpressing B4GALNT2, and a method for producing a transgenic organism known by those skilled in the art can be used without limitation.
  • blastocyst cells are treated with a vector containing the B4GALNT2 gene and a marker gene conferring resistance to a selection substance, and selection of cells containing the vector can be achieved by treating a selection substance such as an antibiotic or a metabolic inhibitor.
  • a selection substance such as an antibiotic or a metabolic inhibitor.
  • a chimeric individual having the transformed cells can be prepared.
  • an individual having the transgene homozygous can be produced.
  • the above process is a method known to those skilled in the art, and can be easily changed according to experimental conditions and the like.
  • the selection material include puromycin, neomycin, lactose or ganciclovir (GCV), and the marker gene includes puromycin acetyltransferase, neomycin phosphotransferase gene, LacZ or thymidine kinase. can be heard
  • the present invention is not limited thereto.
  • the synthetic cMDA5 gRNA Sense Oligo and antisense oligo (Bionics) of Table 1 were linked to the pX459 (Addgene) plasmid according to a known method (Lee 2017, Dev Comp Immunol).
  • the synthesized cMDA5 gRNA Sense Oligo and antisense oligo were attached to each single-stranded double-stranded under the conditions of 95°C for 30 seconds, 72°C for 2 minutes, 37°C for 2 minutes, and 25°C for 2 minutes, and 2.5 ⁇ l of double-stranded Oligo; 25 ng/ ⁇ l Mix 1.5 ⁇ l of Px459, 1 ⁇ l of 10X T4 DNA Ligase buffer (Takara), 0.5 ⁇ l of BbsI (NEB), 0.5 ⁇ l of T4 DNA Ligase (Takara), and 4 ⁇ l of Distilled water (DW) at 37 °C for 5 minutes, 16 Double-stranded Oligo was ligated to the Px459 vector under 12 cycle conditions for 10 minutes at °C.
  • cMDA5 CRISPR/Cas9 was completed by extracting the plasmid using the QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's method.
  • cMDA5 CRISPR/Cas9 was transfected into a chicken fibroblast line (DF-1) (ATCC) grown in 12-well cell culture plates using Lipofectamine® Transfection Reagent (ThermoFisher) according to the manufacturer's method.
  • DF-1 chicken fibroblast line
  • DF-1 ATCC
  • Lipofectamine® Transfection Reagent ThermoFisher
  • puromycin GIBCO
  • concentration of 1 ⁇ g/ml is treated and cultured for 3 to 4 days.
  • the cells were washed with Phosphate-Buffered Saline (PBS, ThermoFisher), and then the cell attachment was detached with 0.05% TE solution diluted 10 times with 0.5% Trypsin/EDTA (TE) Solution (ThermoFisher). After counting the cells by counting the cells, dilute the cells so that one cell per well can be put in a 96-well plate, and the cell clones are cultured to establish a chicken fibroblast cell line from which chicken MDA5 has been removed.
  • PBS Phosphate-Buffered Saline
  • TE Trypsin/EDTA
  • Lipofectamine® Transfection Reagent by ligating and transforming the synthetic cTLR3 gRNA Sense Oligo and antisense oligo (Bionics) in Table 1 to pX459 vector in the same way to create “cTLR3 CRISPR/Cas9” targeting chicken Toll like receptor 3 (TLR3) (ThermoFisher) was used to transfect according to the manufacturer's method.
  • puromycin GIBCO
  • a single clone was established in a 96-well plate, genomic DNA was extracted, and then PCR was performed.
  • the target DNA was extracted using the Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's method. After PCR was performed using the "cTLR3 T7 F/R" primers in Table 1, pGEM®-T Easy Vector Systems (Promega) was used to ligate the pGEM®-T Easy Vector according to the manufacturer's method. After transforming into Competent E. coli, using the QIAprep Spin Miniprep Kit (QIAGEN), the plasmid was extracted according to the manufacturer's method and sequence analysis was performed (Bionics, Korea) to confirm whether chicken TLR3 was edited (Fig. 2). ).
  • cTLR3 CRISPR/Cas9 was introduced into fibroblasts from which chicken MDA5 was removed to establish a monoclonal cell line in which both chicken MDA5 and TLR3 were removed, and then it was confirmed that both chicken MDA5 and TLR3 were removed through sequencing (Fig. 2).
  • BioFACT 2X Pfu PCR Master Mix1 BIOFACT
  • 1 ⁇ l each of the “IFNB promoter F/R” primer 1 ⁇ l of the previously extracted gDNA
  • 22 ⁇ l of UltraPureTM distilled Water Invitrogen
  • PCR was performed according to the manufacturer's method.
  • the part was cut with a blade, and the target DNA was extracted using the Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's method.
  • the IFNB promoter was ligated to the pGEM®-T Easy Vector using pGEM®-T Easy Vector Systems (Promega) according to the manufacturer's method. After transforming with a sample linked to Competent E. coli, the plasmid was extracted using the QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's method to complete "IFNB_T vector". The IFNB promoter sequence was confirmed by entrusting the extracted plasmid to Bionics (Korea) for sequencing. Thereafter, "IFNB_T vector" was used at the 5'-TCGA-3' sticky end to complement the XhoI recognition site in Table 1, and the 5'-AGCT-3' sticky end to complement the HindIII-HF recognition site.
  • the cell line from which chicken MDA5 was removed did not significantly induce the activity of interferon beta to poly I:C stimulation and influenza A virus infection when compared to the wild-type cell line (FIG. 3).
  • the plasmid was linearized by treating 5 ⁇ g of Piggy Bac (Addgene) plasmid with 1 ⁇ l of AgeI, BsrGI (NEB) and 5 ⁇ l of 1X CutSmart® Buffer (New England Biolabs).
  • TMPRSS2 and ST3GAL1 genes were synthesized in Bionics (Korea) and ligated to the linearized Piggy Bac using the In-Fusion® HD cloning Kit (Takara) according to the manufacturer's method, and "TMPRSS2_PB", “ST3GAL1_PB”, “ST3T2_PB” plasmids were obtained. Written.
  • Chicken fibroblast (DF-1) cell lines were transfected with "TMPRSS2_PB", “ST3GAL1_PB” or “ST3T2_PB” 1.2 ⁇ g and 0.8 ⁇ g PiggyBac transposon (pCyL50) using Lipofectamine® Transfection Reagent (ThermoFisher) according to the manufacturer's method. . After transfection, 1 ⁇ g/ml puromycin was treated to maintain passage 15 times. Then, RNA was extracted according to the manufacturer's method using Tri-reagent (Molecular Research Center Inc), and complementary DNA (cDNA) was synthesized according to the manufacturer's method using Superscript IV (Thermo Fisher).
  • Example 4 Measurement of virus titer in TMPRSS2, ST3GAL1 overexpressing cell lines
  • TMPRSS2 and ST3GAL1 overexpressing cell lines showed significantly higher viral titers than wild-type fibroblasts. (FIGS. 5 and 6).
  • a cell line that simultaneously overexpresses TMPRSS2 and ST3GAL1 had a significantly higher virus than the wild-type fibroblast line at 72 hours post-infection without trypsin treatment. titers were shown ( FIG. 7 ).
  • the plasmid was linearized by treating 5 ⁇ g of Piggy Bac (Addgene) plasmid with 1 ⁇ l of HindIII, NotI (NEB) and 5 ⁇ l of 1X CutSmart® Buffer (New England Biolabs).
  • the human B4GALNT2 gene was ligated to the linearized Piggy Bac synthesized by Bioneer (Korea) using the In-Fusion® HD cloning Kit (Takara) according to the manufacturer's method to create "PB_B4GALNT2".
  • the T2A and B4GALNT2 genes were synthesized on the pBHA plasmid by Bioneer (Korea).
  • CRISPR/Cas9 targeting the GAPDH gene intron 10 was based on the pX459 vector as described in Example 1.
  • GAPDH gRNA Sense/anti-sense in Table 1 It was written by linking oligo.
  • 3 ⁇ g of the PiggyBac transposon (pCyL50) of Example 3 and 3 ⁇ g of CRISPR/Cas9 were transfected into a vector chicken fibroblast cell line using Lipofectamine® Transfection Reagent (ThermoFisher) as described in Example 1 according to the manufacturer's method.
  • Example 6 Determination of virus titer in human B4GALNT2 overexpressing cell line
  • Green fluorescent protein Green fluorescent protein
  • the cell line into which B4GALNT2 was introduced showed significantly higher cellular activity than the wild-type fibroblast cell line (FIG. 10).
  • the cell line in which the human B4GALNT2 gene was inserted after GAPDH showed a 4.4 and 4.6 fold decrease in virus titer, respectively, compared to the wild-type fibroblast cell line (Fig. 10).

Abstract

The present invention relates to a composition for preparing avian cells for production of an antiviral vaccine and a composition for preparing virus-resistant avian cells. The composition for preparing avian cells for production of an antiviral vaccine according to the present invention can be preferably utilized as a composition for production of an antiviral vaccine and used to provide a method for production of an antiviral vaccine having a high titer. In addition, the composition for preparing virus-resistant avian cells can preferably provide a method for preparing virus-resistant avian cells or virus-resistant birds.

Description

바이러스 백신 생산용 조류 세포 제조용 조성물 및 바이러스 저항성 조류 세포 제조용 조성물Composition for preparing avian cells for virus vaccine production and composition for preparing virus-resistant avian cells
본 발명은 바이러스 백신 생산용 조류 세포 제조용 조성물 및 바이러스 저항성 조류 세포 제조용 조성물에 관한 것이다.The present invention relates to a composition for preparing avian cells for the production of a virus vaccine and a composition for preparing virus-resistant avian cells.
조류 인플루엔자 바이러스 (Avian influenza virus, AI)는 인플루엔자 A 바이러스 (Influenza A virus)에 속하며 AI로 인한 피해는 국내뿐만 아니라 전 세계적으로도 심각하다. 우리나라에서는 2003년 12월부터 2016년 4월까지 총 6차례 고병원성 조류 인플루엔자 바이러스가 발병했고, 같은 해 11월에는 H5N6형의 새로운 변종 돌연변이 고병원성 조류 바이러스가 발병했다. 해외에서는 2003년부터 태국, 베트남 등 동남아시아에서 주로 발병했으나 이후 러시아, 몽골, 유럽, 아메리카, 아프리카, 인도 등 전 세계적으로 발병하며 그 피해가 커지고 있다. 조류 인플루엔자 바이러스 이외에도, 조류에서 감염되는 여러 바이러스 질환들은 발병할 경우 닭, 오리, 거위 등을 사육하는 축산 분야에 치명적인 피해를 끼칠 수 있을 뿐 아니라, 사람에게 전파될 경우, 발열, 오한, 기침, 인후통, 폐렴, 호흡부전, 심하게는 사망에 이르는 경우가 있어 조절이 필요하다.Avian influenza virus (AI) belongs to influenza A virus, and the damage caused by AI is serious not only in Korea but also worldwide. In Korea, a total of six highly pathogenic avian influenza viruses occurred from December 2003 to April 2016, and in November of the same year, a new strain of H5N6 mutant highly pathogenic avian virus was reported. Overseas, since 2003, it mainly occurred in Southeast Asia, such as Thailand and Vietnam, but since then, it has spread all over the world, including Russia, Mongolia, Europe, America, Africa, and India, and the damage is increasing. In addition to the avian influenza virus, various viral diseases infecting birds can not only cause fatal damage to the livestock sector that raises chickens, ducks, geese, etc. , pneumonia, respiratory failure, and, in severe cases, death.
기존의 인플루엔자 A 바이러스 백신은 주로 달걀에 바이러스를 접종하여 배양하는 방식으로 생산되었다. 하지만 기존의 방법은 고병원성 인플루엔자를 생산하기 어려웠고, 빈번한 바이러스 유전자 변형이 일어났으며, 생산 비용이 높고 가금 질병이 도래했을 때는 달걀을 이용한 백신 생산이 어려운 문제점이 있었다. 이에 따라 달걀 기반의 백신을 대체할 목적으로 세포 배양을 매개로 한 백신이 개발되었다. 하지만 인플루엔자 바이러스 Hemaglutinin (HA) 단백질의 가공을 위해 외부에서 트립신(Trypsin)을 처리해야 하고 높은 트립신 농도는 바이러스 생산량에 영향을 끼친다는 문제점이 있었다.Existing influenza A virus vaccines were mainly produced by inoculating eggs with the virus and culturing them. However, the existing method was difficult to produce highly pathogenic influenza, frequent viral genetic modification, high production cost, and difficulty in producing a vaccine using eggs when poultry diseases arrived. Accordingly, cell culture-based vaccines have been developed to replace egg-based vaccines. However, there was a problem that trypsin had to be processed from the outside for processing the influenza virus hemaglutinin (HA) protein, and a high trypsin concentration had an effect on virus production.
인플루엔자 A 바이러스는 바이러스 단백질의 전사 및 번역을 위해 다양한 숙주 단백질을 이용한다 (Aartian, 2016, Nature Reviews Microbiology; Karlas, 2010, Nature Letters). 닭의 경우. 조류 인플루엔자 바이러스를 인지하는 RIG-I(Retinoic acid inducible gene 1) 수용체가 없고 MDA5(Melanoma differentiation associated protein 5)가 바이러스를 인지하는 주요 수용체이다. MDA5에 의한 바이러스 인식 후 면역반응이 유도되어 바이러스를 제거한다. TMPRSS2(Type II transmembrane protease, serine 2)는 트립신과 비슷한 단백질 분해 효소이고, ST3GAL1(ST3 beta-galactoside alpha-2,3-sialyltransferase 1)은 당단백질 혹은 당지질로 Sialic acid를 옮겨 바이러스와 세포막의 융합을 돕는다.Influenza A viruses utilize a variety of host proteins for the transcription and translation of viral proteins (Aartian, 2016, Nature Reviews Microbiology ; Karlas, 2010, Nature Letters ). for chickens. There is no retinoic acid inducible gene 1 (RIG-I) receptor that recognizes avian influenza virus, and melanoma differentiation associated protein 5 (MDA5) is a major receptor that recognizes the virus. After virus recognition by MDA5, an immune response is induced to eliminate the virus. TMPRSS2 (Type II transmembrane protease, serine 2) is a proteolytic enzyme similar to trypsin, and ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1) transfers sialic acid to glycoproteins or glycolipids to facilitate virus-cell membrane fusion. help
반면, 사람의 B4GALNT2(beta-1,4 N-acetyl galactosaminyl transferase 2)는 Sialic acid를 포함한 당에 GalNAc를 붙여 바이러스가 Sialic acid를 이용하지 못하도록 하여 바이러스와 세포막의 융합을 방해한다고 알려져 있다.On the other hand, human B4GALNT2 (beta-1,4 N-acetyl galactosaminyl transferase 2) is known to prevent virus from using sialic acid by attaching GalNAc to a sugar containing sialic acid, thereby disrupting the fusion of virus and cell membrane.
따라서, 상기 세포의 바이러스 저항성을 조절하는 유전자를 촉진 또는 억제함으로써, 바이러스 백신을 생산하거나, 바이러스 자체에 대한 저항성을 가지는 형질전환 개체를 개발할 수 있다면, 바이러스 감염증에 대한 해결책을 제시할 수 있을 것이다.Therefore, if it is possible to produce a virus vaccine or to develop a transgenic organism having resistance to the virus itself by promoting or suppressing the gene that regulates the virus resistance of the cell, it will be possible to present a solution to the viral infection.
본 발명은 바이러스 저항성을 조절함으로써, 바이러스 백신 생산용 조류 세포 제조용 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a composition for preparing avian cells for virus vaccine production by controlling virus resistance.
또한 상기 바이러스 백신 생산용 조류 세포 제조용 조성물을 포함하는 바이러스 백신 생산용 조성물 및 바이러스 백신 생산 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a composition for producing a virus vaccine and a method for producing a virus vaccine, including the composition for producing avian cells for production of the virus vaccine.
본 발명은 바이러스 백신 저항성을 가진 조류 세포 제조용 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a composition for preparing avian cells having virus vaccine resistance.
또한, 상기 바이러스 백신 저항성을 가진 조류 세포 제조용 조성물을 포함하는 바이러스 저항성 조류 세포 제조 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a method for preparing virus-resistant avian cells, comprising the composition for preparing avian cells having virus vaccine resistance.
1. MDA5(melanoma differentiation associated protein 5) 또는 TLR3(toll like receptor 3)를 결실시키는 물질; 또는 TMPRSS2(type 켺rotease, serine 2) 또는 ST3GAL1(ST3 beta-galactoside alpha-2,3-sialyltransferase 1)를 과발현시키는 물질; 을 포함하는 바이러스 백신 생산용 조류 세포 제조용 조성물.1. A substance that deletes melanoma differentiation associated protein 5 (MDA5) or toll like receptor 3 (TLR3); or a substance that overexpresses TMPRSS2 (type krotease, serine 2) or ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1); A composition for preparing avian cells for the production of a virus vaccine comprising a.
2. 위 1에 있어서, 상기 MDA5 또는 TLR3를 결실시키는 물질은 MDA5 또는 TLR3에 상보적으로 결합하는 gRNA를 포함하는 바이러스 백신 생산용 조류 세포 제조용 조성물.2. The composition according to 1 above, wherein the substance for deleting MDA5 or TLR3 comprises gRNA that complementarily binds to MDA5 or TLR3.
3. 위 1에 있어서, 상기 TMPRSS2 또는 ST3GAL1을 과발현시키는 물질은 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터인 바이러스 백신 생산용 조류 세포 제조용 조성물.3. The composition for producing avian cells for virus vaccine production according to 1 above, wherein the substance overexpressing TMPRSS2 or ST3GAL1 is a vector containing a gene encoding TMPRSS2 or ST3GAL1.
4. 위 1에 있어서, 상기 조류는 닭, 오리, 거위, 기러기, 비둘기, 꿩, 봉관조, 칠면조, 메추라기, 고니, 공작, 원앙, 플라밍고, 키위새, 뻐꾸기, 두루미, 제비, 까마귀, 황새, 왜가리, 도요새, 물떼새, 올빼미, 부엉이, 딱따구리, 독수리, 매, 앵무새, 참새, 꾀꼬리 또는 멧새를 포함하는 군에서 선택되는 바이러스 백신 생산용 조류 세포 제조용 조성물.4. The above 1, wherein the birds are chickens, ducks, geese, geese, pigeons, pheasants, bongguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwi birds, cuckoos, cranes, swallows, crows, storks, A composition for preparing avian cells for the production of a virus vaccine selected from the group comprising herons, snipes, houndstooths, owls, owls, woodpeckers, eagles, hawks, parrots, sparrows, orioles or bunting.
5. 위 1에 있어서, 상기 바이러스는 조류 인플루엔자 바이러스, 뉴캐슬 바이러스 및 기관지염 바이러스를 포함하는 군에서 선택되는 바이러스 백신 생산용 조류 세포 제조용 조성물.5. The composition for preparing avian cells for the production of a virus vaccine according to the above 1, wherein the virus is selected from the group comprising avian influenza virus, Newcastle virus and bronchitis virus.
6. 위 1에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 백신 생산용 조류 세포 제조용 조성물.6. The composition for preparing avian cells for the production of a virus vaccine according to the above 1, wherein the bird is a chicken, and the virus is an avian influenza virus.
7. 위 1 내지 6 중 어느 하나의 조성물이 처리된 조류 세포를 포함하는 바이러스 백신 생산용 조성물.7. A composition for producing a virus vaccine comprising avian cells treated with the composition of any one of 1 to 6 above.
8. 조류 세포에서 MDA5 또는 TLR3를 결실시키거나, 또는 TMPRSS2 또는 ST3GAL1을 과발현시키는 제1 단계; 및 상기 제1 단계를 거친 조류 세포를 약독화한 바이러스에 감염시키는 제2 단계를 포함하는 바이러스 백신 생산 방법.8. A first step of deleting MDA5 or TLR3 or overexpressing TMPRSS2 or ST3GAL1 in avian cells; and a second step of infecting the avian cells that have undergone the first step with the attenuated virus.
9. 위 8에 있어서, 상기 MDA5 또는 TLR3의 결실은 상기 조류 세포에 MDA5 또는 TLR3에 상보적으로 결합하는 gRNA를 포함하는 물질을 처리하여 수행되는 바이러스 백신 생산 방법.9. The method for producing a virus vaccine according to the above 8, wherein the deletion of MDA5 or TLR3 is performed by treating the avian cells with a substance containing a gRNA that complementarily binds to MDA5 or TLR3.
10. 위 8에 있어서, 상기 TMPRSS2 또는 ST3GAL1의 과발현은 상기 조류 세포에 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터를 처리하여 수행되는 바이러스 백신 생산 방법.10. The method for producing a virus vaccine according to 8 above, wherein the overexpression of TMPRSS2 or ST3GAL1 is performed by treating the avian cells with a vector containing a gene encoding TMPRSS2 or ST3GAL1.
11. 위 8에 있어서, 상기 바이러스의 감염은 상기 조류 세포에 바이러스를 주입하거나 또는 바이러스 유전체 염기서열을 포함하는 벡터를 주입하여 수행되는 바이러스 백신 생산 방법.11. The method for producing a virus vaccine according to 8 above, wherein the virus infection is performed by injecting the virus into the avian cells or injecting a vector containing a viral genome sequence.
12. 위 8에 있어서, 상기 2단계를 거친 조류 세포를 배양하여 그로부터 바이러스를 얻는 제3 단계를 더 포함하는 바이러스 백신 생산 방법.12. The method for producing a virus vaccine according to 8 above, further comprising a third step of culturing the avian cells that have undergone the second step to obtain a virus therefrom.
13. 위 8에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 백신 생산 방법.13. The method for producing a virus vaccine according to 8 above, wherein the bird is a chicken, and the virus is an avian influenza virus.
14. B4GALNT2(β-1,4 N-acetylgalactosaminyltransferase 2)를 과발현시키는 물질을 포함하는 바이러스 저항성 조류 세포 제조용 조성물.14. A composition for preparing virus-resistant algal cells, comprising a substance overexpressing B4GALNT2 (β-1,4 N-acetylgalactosaminyltransferase 2).
15. 위 14에 있어서, 상기 B4GALNT2는 인간 B4GALNT2인 바이러스 저항성 조류 세포 제조용 조성물.15. The composition for preparing virus-resistant avian cells according to the above 14, wherein the B4GALNT2 is human B4GALNT2.
16. 위 14에 있어서, 상기 B4GALNT2를 과발현시키는 물질은 B4GALNT2를 코딩하는 유전자를 포함하는 벡터인 바이러스 저항성 조류 세포 제조용 조성물.16. The composition for producing virus-resistant avian cells according to the above 14, wherein the substance overexpressing B4GALNT2 is a vector containing a gene encoding B4GALNT2.
17. 위 14에 있어서, 상기 조류는 닭, 오리, 거위, 기러기, 비둘기, 꿩, 봉관조, 칠면조, 메추라기, 고니, 공작, 원앙, 플라밍고, 키위새, 뻐꾸기, 두루미, 제비, 까마귀, 황새, 왜가리, 도요새, 물떼새, 올빼미, 부엉이, 딱따구리, 독수리, 매, 앵무새, 참새, 꾀꼬리 또는 멧새를 포함하는 군에서 선택되는 바이러스 저항성 조류 세포 제조용 조성물.17. The above 14 above, wherein the birds are chickens, ducks, geese, geese, pigeons, pheasants, bonguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwi birds, cuckoos, cranes, swallows, crows, storks, Herons, snipes, houndstooths, owls, owls, woodpeckers, eagles, hawks, parrots, sparrows, orioles or bunting birds. A composition for preparing virus-resistant bird cells.
18. 위 14에 있어서, 상기 바이러스는 조류 인플루엔자 바이러스, 뉴캐슬 바이러스 및 기관지염 바이러스를 포함하는 군에서 선택되는 바이러스 저항성 조류 세포 제조용 조성물.18. The composition for preparing virus-resistant avian cells according to the above 14, wherein the virus is selected from the group comprising avian influenza virus, Newcastle virus and bronchitis virus.
19. 위 14에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 저항성 조류 세포 제조용 조성물.19. The composition for preparing virus-resistant avian cells according to 14 above, wherein the bird is a chicken, and the virus is an avian influenza virus.
20. 위 14 내지 19 중 어느 하나의 바이러스 저항성 조류 세포 제조용 조성물이 처리된 원시생식세포 또는 배반엽세포를 포함하는 바이러스 저항성 조류 제조용 조성물.20. A composition for preparing virus-resistant algae comprising primitive germ cells or blastoderm cells treated with the composition for preparing virus-resistant algal cells of any one of 14 to 19 above.
21. 조류 세포에서 B4GALNT2를 과발현시키는 단계를 포함하는 바이러스 저항성 조류 세포 제조 방법.21. A method for producing a virus resistant avian cell comprising the step of overexpressing B4GALNT2 in the avian cell.
22. 위 21에 있어서, 상기 B4GALNT2는 인간 B4GALNT2인 바이러스 저항성 조류 세포 제조 방법.22. The method according to 21 above, wherein the B4GALNT2 is human B4GALNT2.
23. 위 21에 있어서, 상기 B4GALNT2의 과발현은 B4GALNT2를 코딩하는 유전자를 포함하는 벡터를 상기 조류 세포에 처리하여 수행되는 바이러스 저항성 조류 세포 제조 방법.23. The method according to 21 above, wherein the overexpression of B4GALNT2 is performed by treating the avian cells with a vector comprising a gene encoding B4GALNT2.
24. 위 21에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 저항성 조류 세포 제조 방법.24. The method for producing virus-resistant avian cells according to 21 above, wherein the bird is a chicken, and the virus is an avian influenza virus.
본 발명의 바이러스 백신 생산용 조류 세포 제조용 조성물을 통해 바이러스 백신 생산용 조성물을 제공한다. 이는 종래보다 더 높은 생산 효율을 가지는 바이러스 백신 생산 방법을 제공할 수 있어, 바람직하게는 바이러스 백신 생산 공정에 활용할 수 있다.A composition for producing a virus vaccine is provided through the composition for producing avian cells for the production of a virus vaccine of the present invention. This can provide a virus vaccine production method having a higher production efficiency than the prior art, and can be preferably used in the virus vaccine production process.
또한, 본 발명의 바이러스 저항성 조류 세포 제조용 조성물을 통해 바이러스 저항성 조류 제조 방법을 제공한다. 이는 바람직하게는 조류 인플루엔자, 뉴캐슬병 바이러스, 닭 전염성 기관지염 바이러스 등에 대한 저항성을 가진 형질전환 조류를 제조할 수 있어 축산 분야에서 활용할 수 있다.In addition, there is provided a method for preparing virus-resistant algae through the composition for preparing virus-resistant algal cells of the present invention. Preferably, it can be used in the livestock field because it can produce transgenic birds with resistance to avian influenza, Newcastle disease virus, chicken infectious bronchitis virus, and the like.
도 1은 본 발명에 대한 모식도로서, 구체적으로는 MDA5를 결실시키거나, TMPRSS2, ST3GAL1의 발현을 유도하면 바이러스 백신의 전구체의 생산 효율이 증가하고, 인간 유래 B4GALNT2를 발현시키면 바이러스 생산을 억제할 수 있다.1 is a schematic diagram of the present invention, specifically, when MDA5 is deleted or expression of TMPRSS2 and ST3GAL1 is induced, the production efficiency of a virus vaccine precursor increases, and when human-derived B4GALNT2 is expressed, virus production can be inhibited. have.
도 2는 조류 유전체 편집 기술을 활용하여 닭 섬유아세포(DF-1)에서 닭 MDA5, TLR3 또는 MDA5와 TLR3를 동시에 타깃하여 유전자 변형을 유도한 후 야생형 유전자형과 대비해서 유전자 편집된 양상을 보여주는 그림이다.FIG. 2 is a diagram showing the pattern of gene editing compared to the wild-type genotype after inducing genetic modification by simultaneously targeting chicken MDA5, TLR3, or MDA5 and TLR3 in chicken fibroblasts (DF-1) using avian genome editing technology. .
도 3은 바이러스 RNA 유사체인 poly I:C 및 저병원성 인플루엔자 A 바이러스를 야생형 닭 섬유아세포, 닭 MDA5 혹은 TLR3가 제거된 섬유아세포, 그리고 닭 MDA5, TLR3가 동시에 제거된 섬유아세포에 처리했을 때 인터페론 및 하위 유전자들의 활성/발현 유도를 나타낸 그래프이다.Figure 3 shows the viral RNA analog poly I:C and low pathogenic influenza A virus when treated with wild-type chicken fibroblasts, chicken MDA5 or TLR3 depleted fibroblasts, and chicken MDA5 and TLR3 depleted fibroblasts at the same time, interferon and lower It is a graph showing the induction of activity/expression of genes.
도 4는 닭 MDA5가 제거된 세포주에 닭 MDA5를 과발현하면 나타나는 효과와 저병원성 인플루엔자 A 바이러스를 야생형 닭 섬유아세포, 닭 MDA5가 제거된 섬유아세포에 감염시켰을 때 생산된 바이러스 역가를 비교한 그래프이다.Figure 4 is a graph comparing the effect of overexpressing chicken MDA5 in a chicken MDA5 cell line and the virus titer produced when the low pathogenic influenza A virus was infected with wild-type chicken fibroblasts and chicken MDA5 removed fibroblasts.
도 5는 닭 섬유아세포에 TMPRSS2를 과발현하고 TMPRSS2 유전자 발현량 증가를 보여주고 TMPRSS2 과발현 세포주가 조류 인플루엔자 바이러스 생산을 증가시킴을 보여주는 그래프이다.5 is a graph showing that TMPRSS2 overexpression in chicken fibroblasts and an increase in TMPRSS2 gene expression level and TMPRSS2 overexpressing cell line increase the production of avian influenza virus.
도 6은 닭 섬유아세포에 ST3GAL1을 과발현하고 ST3GAL1 유전자 발현량 증가를 보여주고 ST3GAL1 과발현 세포주가 조류 인플루엔자 바이러스 생산을 증가시킴을 보여주는 그래프이다.6 is a graph showing that ST3GAL1 overexpression in chicken fibroblasts and an increase in the expression level of ST3GAL1 gene, and that the ST3GAL1 overexpressing cell line increases the production of avian influenza virus.
도 7은 닭 섬유아세포에 TMPRSS2, ST3GAL1을 동시에 과발현 하는 세포주가 조류 인플루엔자 바이러스 생산을 증가시킴을 보여주는 그래프이다.7 is a graph showing that a cell line that simultaneously overexpresses TMPRSS2 and ST3GAL1 in chicken fibroblasts increases the production of avian influenza virus.
도 8은 사람 B4GALNT2의 기능을 모식도로 보여주고 PiggyBac을 매개로 사람 B4GALNT2를 과발현 했을 때 그 발현을 보여주는 그림이다.8 is a schematic diagram showing the function of human B4GALNT2, and is a diagram showing the expression when overexpressing human B4GALNT2 via PiggyBac.
도 9는 CRISPR/Cas9을 매개로 닭 GAPDH 뒤에 사람 B4GALNT2를 knock-in하기 위한 모식도 및 유전자 편집된 세포 클론의 유전자형을 보여주는 그림이다. 또한 GAPDH 뒤에 knock-in된 사람 B4GALNT2의 발현을 보여준다.9 is a schematic diagram for knock-in of human B4GALNT2 followed by chicken GAPDH via CRISPR/Cas9 and a diagram showing the genotype of a gene-edited cell clone. It also shows the expression of human B4GALNT2 knocked-in after GAPDH.
도 10은 사람 B4GALNT2가 도입된 닭 섬유아세포주가 야생형 닭 섬유아세포보다 바이러스가 이용하는 Sialic acid와의 결합이 저해되어 바이러스 역가가 유의적으로 낮고 세포 활성이 높음을 보여주는 그림이다.10 is a diagram showing that the human B4GALNT2 introduced chicken fibroblast line has significantly lower viral titer and higher cellular activity than wild-type chicken fibroblasts because binding to sialic acid used by the virus is inhibited.
이하 본 발명을 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적인 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 의미를 따른다.Hereinafter, the present invention will be described in detail. Unless otherwise defined, all terms in this specification have the same general meaning as understood by those of ordinary skill in the art to which the present invention belongs, and in case of conflict with the meaning of the terms used in this specification, the The meaning used in the specification is followed.
본 발명은 MDA5(melanoma differentiation associated protein 5) 또는 TLR3(toll like receptor 3)를 결실시키는 물질; 또는 TMPRSS2(type Ⅱ transmembrane protease, serine 2) 또는 ST3GAL1(ST3 beta-galactoside alpha-2,3-sialyltransferase 1)를 과발현시키는 물질; 을 포함하는 바이러스 백신 생산용 조류 세포 제조용 조성물에 관한 것이다.The present invention relates to a substance that deletes melanoma differentiation associated protein 5 (MDA5) or toll like receptor 3 (TLR3); or a substance that overexpresses TMPRSS2 (type II transmembrane protease, serine 2) or ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1); It relates to a composition for preparing avian cells for the production of a virus vaccine comprising a.
본 발명에서 상기 MDA5 또는 TLR3를 결실시키는 물질은 정상적인 기능을 수행하는 상기 유전자의 산물 단백질의 합성을 저해하는 것이라면, 그 종류는 제한되지 않는다. 예를 들면, MDA5 또는 TLR3에 상보적으로 결합하는 gRNA(guide RNA)를 들 수 있고, 상기 MDA5 또는 TLR3에 상보적으로 결합하는 TALEN(transcription activator-like effector nuclease) 또는 ZFN(zinc-finger nuclease)을 코딩하는 핵산일 수 있다. 상기 핵산은 상기 MDA5 또는 TLR3에 상보적으로 결합할 수 있거나, 상보적으로 결합하는 단백질을 코딩할 수 있다면 염기 서열에 관계 없이 본 발명의 MDA5 또는 TLR3를 결실시키는 물질에 해당할 수 있고, 해당 서열은 당업자에 의해 공지된 방법에 의해 결정될 수 있다.In the present invention, the type of the substance for deleting MDA5 or TLR3 is not limited as long as it inhibits the synthesis of a protein product of the gene that performs a normal function. For example, gRNA (guide RNA) that complementarily binds to MDA5 or TLR3 is mentioned, and TALEN (transcription activator-like effector nuclease) or ZFN (zinc-finger nuclease) that complementarily binds to MDA5 or TLR3 is mentioned. It may be a nucleic acid encoding The nucleic acid may correspond to a substance that deletes MDA5 or TLR3 of the present invention irrespective of the nucleotide sequence if it can bind complementarily to MDA5 or TLR3, or encode a protein that binds complementarily, and the corresponding sequence can be determined by methods known by those skilled in the art.
상기 gRNA는 타깃 유전자의 염기서열 또는 이에 대응되는 서열에 상보적인 RNA로서, 표적 DNA 서열과 전부 또는 일부 상보적으로 결합하여 해당 표적 DNA 서열로 엔도뉴클레아제(endonuclease) 단백질을 이끄는 역할을 하는 리보핵산을 의미한다. 상기 gRNA는 두 개의 RNA, 즉 crRNA(CRISPR RNA) 및 tracrRNA(trans-activating crRNA)를 구성 요소로 포함하는 이중 RNA(dual RNA); 또는 표적 DNA 내 서열과 전부 또는 일부 상보적인 서열을 포함하는 제1 부위 및 RNA-가이드 뉴클레아제와 상호작용하는 서열을 포함하는 제2 부위를 포함하는 단일 사슬 가이드 RNA(sgRNA) 형태를 말하나, RNA-가이드 엔도뉴클레아제가 표적 DNA 서열에서 활성을 가질 수 있는 형태라면 제한 없이 본 발명의 범위에 포함될 수 있다. 본 발명에 따른 gRNA는 바람직하게는, 단일 사슬 가이드 RNA(sgRNA) 형태일 수 있으나, 이에 제한되지 않으며, 사용된 엔도뉴클레아제의 종류 또는 그 유래 미생물 등에 따라서 적절히 선택할 수 있다.The gRNA is an RNA complementary to a nucleotide sequence of a target gene or a sequence corresponding thereto, and is complementary to all or part of a target DNA sequence to guide an endonuclease protein to the target DNA sequence. means nucleic acids. The gRNA may be a dual RNA comprising two RNAs, namely, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA); or a single chain guide RNA (sgRNA) form comprising a first site comprising a sequence that is fully or partially complementary to a sequence in the target DNA and a second site comprising a sequence that interacts with an RNA-guided nuclease, The RNA-guided endonuclease may be included in the scope of the present invention without limitation as long as it is a form capable of having activity in the target DNA sequence. The gRNA according to the present invention may preferably be in the form of a single-stranded guide RNA (sgRNA), but is not limited thereto, and may be appropriately selected according to the type of endonuclease used or a microorganism derived therefrom.
본 발명에서 상기 MDA5 또는 TLR3는 조류 세포에서 외부 RNA 리간드를 인식하여 외부 항원에 대한 선천성 면역기전을 활성화하는 역할을 수행할 수 있으므로, 상기 MDA5 또는 TLR3가 결실되는 경우, 조류 세포에서 바이러스의 복제 및 조립이 더욱 촉진될 수 있다. 반면, MDA5의 발현이 상보적인 siRNA를 처리하는 등으로 감소하는 경우에는, 상기 효과가 발휘되지 않을 수 있다. 상기 MDA5 또는 TLR3는 조류에서 보존되어 있어, 조류의 종에 관계 없이 모든 조류에서 상기 효과를 발휘할 수 있으나, RIG-1(Retinoic acid inducible gene 1)이 결실된 조류에서 더욱 효과적일 수 있다. 상기 RIG-1(Retinoic acid inducible gene 1)이 결실된 조류는 구체적으로 닭일 수 있다. 본 발명의 일 실시예에서, MDA5 유전자 또는 TLR3 유전자 중 하나를 결실시키는 물질을 조류 세포에 처리하는 경우 그렇지 않은 경우에 비해 더 높은 바이러스 역가를 나타낼 수 있고, 바람직하게는 MDA5 및 TLR3가 모두 결실되거나 그 발현이 모두 감소된 경우, 대조군 대비 30 배 가량의 바이러스 역가를 나타낼 수 있다. 다만 이에 제한되지 않는다.In the present invention, the MDA5 or TLR3 may play a role of activating an innate immune mechanism against a foreign antigen by recognizing a foreign RNA ligand in avian cells. Therefore, when the MDA5 or TLR3 is deleted, virus replication and Assembly can be further facilitated. On the other hand, when the expression of MDA5 is decreased by treatment with complementary siRNA, etc., the above effect may not be exhibited. Since MDA5 or TLR3 is conserved in algae, it may exert the above effect in all algae regardless of algae species, but may be more effective in algae in which Retinoic acid inducible gene 1 (RIG-1) is deleted. The bird in which the RIG-1 (Retinoic acid inducible gene 1) is deleted may be specifically chicken. In one embodiment of the present invention, when avian cells are treated with a substance that deletes either the MDA5 gene or the TLR3 gene, a higher viral titer may be exhibited compared to the case where the avian cells are not, preferably both MDA5 and TLR3 are deleted or When all of the expression is reduced, the virus titer may be about 30 times higher than that of the control group. However, the present invention is not limited thereto.
본 발명에서 상기 TMPRSS2 또는 ST3GAL1을 과발현시키는 물질은 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터일 수 있다. 상기 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터는 각각 서열번호 1의 TMPRSS2 염기서열 또는 서열번호 2의 ST3GAL1 염기서열 또는 각각의 염기서열의 상동체를 포함할 수 있다. 상기 상동체는 서열번호 1 또는 서열번호 2의 염기서열과 일부 상이한 염기서열이 있더라도 서열번호 1 또는 서열번호 2의 염기서열에 의해 코딩되는 아미노산과 동일한 아미노산을 코딩하는 염기서열을 포함할 수 있고, 일부 아미노산 서열이 상이하더라도 코딩된 단백질의 기능에 차이가 없거나 효과의 일부 증감 정도의 차이만이 존재하는 염기 서열을 포함할 수도 있다. 상기 대응되는 서열은 종간 염기 변이에 의해 달라지는 서열을 포함할 수 있고, 일부 염기 변이가 있더라도, 그 서열의 얼라인(align) 및 서열 대비를 통해 그 유전자의 확인이 가능하다. 예를 들면, 서열번호 1 또는 서열번호 2의 염기서열과 임의의 염기서열을 얼라인했을 때, 최소 60 %의 상동성, 바람직하게는 70 %, 보다 바람직하게는 80 %, 보다 더 바람직하게는 90 %, 가장 바람직하게는 99 %의 상동성을 가지는 서열이 본 발명의 상기 상동체에 해당할 수 있다.In the present invention, the substance overexpressing TMPRSS2 or ST3GAL1 may be a vector including a gene encoding TMPRSS2 or ST3GAL1. The vector comprising the gene encoding TMPRSS2 or ST3GAL1 may include the TMPRSS2 base sequence of SEQ ID NO: 1, the ST3GAL1 base sequence of SEQ ID NO: 2, or a homologue of each base sequence, respectively. The homologue may include a nucleotide sequence encoding the same amino acid as the amino acid encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 even if there is a nucleotide sequence that is partially different from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, Even if some amino acid sequences are different, it may include a nucleotide sequence in which there is no difference in the function of the encoded protein or only a difference in the degree of increase or decrease of the effect exists. The corresponding sequence may include a sequence that varies due to base variation between species, and even if there is some base variation, the gene can be identified through alignment and sequence comparison of the sequence. For example, when the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and any nucleotide sequence are aligned, at least 60% homology, preferably 70%, more preferably 80%, even more preferably A sequence having 90%, most preferably 99% homology may correspond to the homologue of the present invention.
본 발명에서 상기 TMPRSS2 또는 ST3GAL1는 조류 세포에서 외부 바이러스가 세포 내부로 유입되는 것을 촉진할 수 있으므로, 상기 TMPRSS2 또는 ST3GAL1이 과발현되는 경우, 조류 세포에서 바이러스 복제 및 조립이 더욱 촉진될 수 있다. 상기 TMPRSS2 또는 ST3GAL1이 조류에서 보존되어 있으므로, 조류의 종에 관계 없이 상기 효과를 발휘할 수 있으나, 바람직하게는 RIG-1이 결실된 조류에서 더욱 효과적일 수 있다. 상기 RIG-1이 결실된 조류는 구체적으로 닭일 수 있다. 본 발명의 일 실시예에서, TMPRSS2 또는 ST3GAL1 중 하나가 과발현되는 물질을 조류 세포에 처리하는 경우 그렇지 않은 경우에 비해 더 높은 바이러스 역가를 나타낼 수 있고, 바람직하게는 TMPRSS2 및 ST3GAL12 모두를 과발현 하는 경우, 10 내지 100 배의 바이러스 역가를 나타낼 수 있다. 다만 이에 제한되지 않는다.In the present invention, since the TMPRSS2 or ST3GAL1 can promote the influx of a foreign virus into the cell in the avian cell, when the TMPRSS2 or ST3GAL1 is overexpressed, the virus replication and assembly in the avian cell can be further promoted. Since the TMPRSS2 or ST3GAL1 is conserved in algae, the above effect may be exerted regardless of the species of algae, but it may be more effective in algae in which RIG-1 is preferably deleted. The bird in which the RIG-1 is deleted may specifically be a chicken. In an embodiment of the present invention, when avian cells are treated with a substance overexpressing either TMPRSS2 or ST3GAL1, a higher viral titer may be exhibited than when not, preferably when both TMPRSS2 and ST3GAL12 are overexpressed, 10 to 100 fold viral titer. However, the present invention is not limited thereto.
본 발명의 상기 바이러스 백신 생산용 조류 세포 제조용 조성물은 MDA5(melanoma differentiation associated protein 5) 또는 TLR3(toll like receptor 3)를 결실시키는 물질; 또는 TMPRSS2(type Ⅱ transmembrane protease, serine 2) 또는 ST3GAL1(ST3 beta-galactoside alpha-2,3-sialyltransferase 1)를 과발현시키는 물질; 을 담지한 재조합 벡터를 포함할 수 있다. 상기 재조합 벡터는 상기 물질들을 타겟 세포로 전달할 수 있는 것이라면, 그 서열에 관계 없이 당업자에 의해 제한 없이 사용될 수 있다. 재조합 벡터의 예를 들면, 플라스미드(plasmid), 박테리오파지(bacteriophage), 코스미드(cosmid), BAC(bacteria artificial chromosome), YAC(yeast artificial chromosome), 레트로바이러스, 아데노바이러스, 아데노연관바이러스(adeno-associated virus), 비바이러스성 벡터일 수 있다. 다만 이에 제한되지 않는다.The composition for preparing avian cells for the production of a virus vaccine of the present invention includes a substance that deletes melanoma differentiation associated protein 5 (MDA5) or toll like receptor 3 (TLR3); or a substance that overexpresses TMPRSS2 (type II transmembrane protease, serine 2) or ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1); It may include a recombinant vector carrying a. The recombinant vector can be used without limitation by those skilled in the art, regardless of the sequence, as long as it can deliver the substances to the target cell. Examples of recombinant vectors, plasmid, bacteriophage, cosmid, BAC (bacteria artificial chromosome), YAC (yeast artificial chromosome), retrovirus, adenovirus, adeno-associated virus (adeno-associated) virus), or a non-viral vector. However, the present invention is not limited thereto.
본 발명에서 상기 조류는 조강(Avian)에 속하는 조류라면 제한 없이 본 발명의 범위에 포함될 수 있다. 예를 들면, 닭, 오리, 거위, 기러기, 비둘기, 꿩, 봉관조, 칠면조, 메추라기, 고니, 공작, 원앙, 플라밍고, 키위새, 뻐꾸기, 두루미, 제비, 까마귀, 황새, 왜가리, 도요새, 물떼새, 올빼미, 부엉이, 딱따구리, 독수리, 매, 앵무새, 참새, 꾀꼬리 또는 멧새 등을 포함할 수 있다, 바람직하게 상기 조류는 닭일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the algae may be included in the scope of the present invention without limitation as long as the algae belong to Avian . For example, chickens, ducks, geese, geese, pigeons, pheasants, bonguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwis, cuckoos, cranes, swallows, crows, storks, herons, snipes, plovers, It may include an owl, an owl, a woodpecker, an eagle, a hawk, a parrot, a sparrow, an owl or a bunting, etc. Preferably, the bird may be a chicken, but is not limited thereto.
상기 바이러스는 상기 조류 세포에 전달될 수 있는 바이러스라면 숙주의 종에 관계 없이 본 발명의 범위에 포함될 수 있다. 구체적으로는 조류를 숙주로 하고, RNA를 유전체로 하는 바이러스일 수 있다. 조류를 숙주로 하고, RNA를 유전체로 하는 바이러스의 예를 들면, 조류 인플루엔자(Avian influenza, AI), 뉴캐슬병 바이러스(Newcastle disease virus, NDV) 또는 닭 전염성 기관지염 바이러스(Avian infectious bronchitis virus) 가 포함될 수 있다. 바람직하게는, 상기 바이러스는 조류 인플루엔자 바이러스 일 수 있으나, 이에 제한되는 것은 아니다.The virus may be included in the scope of the present invention regardless of the host species as long as the virus can be delivered to the avian cells. Specifically, it may be a virus using an alga as a host and RNA as a genome. Examples of viruses that host birds and have RNA as their genomes include avian influenza (AI), Newcastle disease virus (NDV) or chicken infectious bronchitis virus ( Avian infectious bronchitis virus ). . Preferably, the virus may be an avian influenza virus, but is not limited thereto.
본 발명은 상기 MDA5또는 TLR3를 결실시키는 물질; 또는 TMPRSS2또는 ST3GAL1을 과발현시키는 물질; 을 포함하는 조성물이 처리된 조류 세포를 포함하는 바이러스 백신 생산용 조성물에 관한 것이다.The present invention relates to a substance that deletes the MDA5 or TLR3; or a substance that overexpresses TMPRSS2 or ST3GAL1; It relates to a composition for producing a virus vaccine comprising avian cells treated with the composition comprising a.
상기 MDA5 또는 TLR3를 결실시키는 물질; 또는 TMPRSS2또는 ST3GAL1을 과발현시키는 물질; 을 포함하는 조성물이 처리된 조류 세포는 앞서 언급하였듯, 조류에서 상기 MDA5, TLR3, TMPRSS2 또는 ST3GAL1의 작용기전이 보존되어 있으므로 세포 종류에 제한되지 않고, 당업자에 의하여 선택될 수 있다. 예를 들면, 표피세포(epithelial cell), 간세포(hepatocyte), 림프구(lymphocyte), 섬유아세포(fibroblast), 원시생식세포(primordial germ cell, PGC), 내피세포(endothelial cell), 알세포(egg cell) 등일 수 있다. 다만 바람직하게는, 분열 속도가 빠르고, 배양이 용이한 섬유아세포(fibroblast) 일 수 있다. 다만 이에 제한되는 것은 아니다.a substance that deletes the MDA5 or TLR3; or a substance that overexpresses TMPRSS2 or ST3GAL1; As mentioned above, the algal cells treated with the composition containing the MDA5, TLR3, TMPRSS2 or ST3GAL1 in algae are not limited to cell types, and may be selected by those skilled in the art because the mechanism of action is conserved. For example, epithelial cells, hepatocytes, lymphocytes, fibroblasts, primordial germ cells (PGC), endothelial cells, egg cells etc. However, preferably, it may be a fibroblast with a fast division rate and easy culture. However, the present invention is not limited thereto.
본 발명은 조류 세포에서 MDA5 또는 TLR3를 결실시키거나, 또는 TMPRSS2 또는 ST3GAL1을 과발현시키는 제1 단계; 및The present invention relates to a first step of deleting MDA5 or TLR3 or overexpressing TMPRSS2 or ST3GAL1 in an avian cell; and
상기 제1 단계를 거친 조류 세포를 약독화한 바이러스에 감염시키는 제2 단계를 포함하는 바이러스 백신 생산 방법에 관한 것이다.It relates to a method for producing a virus vaccine comprising a second step of infecting the avian cells that have undergone the first step with the attenuated virus.
상기 바이러스 백신 생산 방법 과정에 있어서, 상기 제1 단계는 앞서 언급한 조류 세포에 상기 MDA5 또는 TLR3를 결실시키는 물질; 예를 들면 MDA5 또는 TLR3에 상보적으로 결합하는 gRNA가 포함된 조성물을 처리하거나, TMPRSS2 또는 ST3GAL1을 과발현시키는 물질; 예를 들면 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터를 포함하는 조성물을 처리하는 것일 수 있다. 다만 이에 제한되지 않고, 당업자가 사용하는 유전자 발현 조절 방법을 통하여 수행될 수도 있다.In the process of the virus vaccine production method, the first step is a material for deleting the MDA5 or TLR3 in the aforementioned avian cells; For example, a substance that treats a composition containing a gRNA that complementarily binds to MDA5 or TLR3, or overexpresses TMPRSS2 or ST3GAL1; For example, it may be treating a composition containing a vector including a gene encoding TMPRSS2 or ST3GAL1. However, the present invention is not limited thereto, and it may be performed through a gene expression control method used by those skilled in the art.
상기 제 2단계의 약독화한 바이러스에 감염시키는 단계에서, 감염시키는 약독화 바이러스와 생산되는 백신이 타깃으로 하는 바이러스는 동일한 바이러스이거나, 동일한 항원을 공유하는 것일 수 있다. 또한, 상기 바이러스에 감염시키는 단계는 앞서 언급한 백신의 타깃이 되는 바이러스 자체를 상기 제 1 단계를 거친 조류 세포에 주입하는 것일 수 있고, 또는 백신의 타깃이 되는 바이러스 유전체를 담지한 벡터를 조류 세포에 전달하여, 바이러스가 세포 내에서 합성되도록 하는 것일 수도 있다. 상기 백신의 타깃이 되는 바이러스 유전체를 담지한 벡터는 앞서 언급하였던 것처럼, 그 종류에 제한되는 것은 아니고, 플라스미드, 박테리오파지, 코스미드, BAC, YAC, 레트로바이러스, 아데노바이러스, 아데노연관바이러스 또는 비바이러스성 벡터 등 당업자가 제한 없이 선택해서 사용할 수 있다.In the second step of infecting the attenuated virus, the infecting attenuated virus and the virus targeted by the produced vaccine may be the same virus or may share the same antigen. In addition, the step of infecting the virus may include injecting the virus itself, which is the target of the vaccine, into the avian cells that have undergone the first step, or a vector carrying the genome of the virus that is the target of the vaccine is injected into the avian cells. It can also be delivered to the cell so that the virus is synthesized in the cell. The vector carrying the viral genome that is the target of the vaccine is not limited to the type as mentioned above, but a plasmid, bacteriophage, cosmid, BAC, YAC, retrovirus, adenovirus, adeno-associated virus or non-viral A person skilled in the art, such as a vector, can select and use it without limitation.
본 발명에서 상기 바이러스 백신 생산 방법은 상기 2단계를 거친 조류 세포를 배양하여, 그로부터 바이러스를 얻는 제 3단계를 더 포함할 수도 있다. 상기 2단계를 거친 조류 세포를 배양하면, 앞서 주입한 바이러스 또는 바이러스 벡터에 의해 세포에서 새로운 바이러스 단백질이 합성될 수 있고, 그로부터 새로운 바이러스가 조립될 수 있다. 상기 배양하는 시간은 실험 조건에 따라 당업자에 의하여 제한 없이 선택될 수 있다. 배양 시간의 증가는 바이러스 유전체의 복제 및 바이러스의 조립을 증가시킬 수 있으나, 일정 수치를 초과하는 경우, 세포의 사멸로 인하여 바이러스 백신의 회수율의 증가에 영향이 없을 수 있다. 배양 시간은 적어도 6 시간 이상 배양하는 것 일수 있고, 상한은 제한되지 않으나 예를 들면, 120 시간, 96 시간 또는 72 시간 일 수 있다. 다만 이에 제한되지 않는다. 이후 상기 조립된 바이러스는 당업자에 의해 공지된 방법에 의하여, 추출 및 분리될 수 있다. 예를 들면, 조류 세포 배양액을 파쇄한 후, 원심분리하여 얻은 상층액에 포함된 바이러스를 농축하여 얻을 수 있다. 다만 이에 제한되는 것은 아니다.In the present invention, the method for producing a virus vaccine may further include a third step of culturing the avian cells that have passed through the second step to obtain a virus therefrom. When the algal cells that have undergone the above two steps are cultured, a new viral protein can be synthesized in the cell by the previously injected virus or viral vector, and a new virus can be assembled therefrom. The incubation time may be selected without limitation by those skilled in the art according to experimental conditions. An increase in the incubation time may increase the replication of the viral genome and assembly of the virus, but if it exceeds a certain value, it may not have an effect on the increase in the recovery rate of the virus vaccine due to cell death. The incubation time may be culturing for at least 6 hours or more, and the upper limit is not limited, but may be, for example, 120 hours, 96 hours or 72 hours. However, the present invention is not limited thereto. Then, the assembled virus can be extracted and isolated by methods known by those skilled in the art. For example, it can be obtained by disrupting the algal cell culture solution and then concentrating the virus contained in the supernatant obtained by centrifugation. However, the present invention is not limited thereto.
본 발명의 상기 바이러스 백산 생산 방법에 의하는 경우, 종래의 방법에 비하여 더 높은 역가의 바이러스 백신을 수득할 수 있다. 앞서 언급하였던 것처럼 일 실시예에서 본 발명의 상기 조류 세포에서 MDA5 또는 TLR3를 결실시키거나, 또는 TMPRSS2 또는 ST3GAL1을 과발현시키는 제1 단계를 거친 조류 세포의 경우, 그렇지 않은 대조군에 비하여 더 높은 바이러스 역가를 보이는 것을 확인하였고, 바람직하게는 상기 MDA5 및 TLR3 가 모두 결실되거나 또는 그 발현이 억제되고, TMPRSS2 및 ST3GAL1이 모두 과발현되는 경우에, 가장 높은 바이러스 역가를 보일 수 있다. 다만 이에 제한되는 것은 아니다.In the case of the method for producing vaccinia virus of the present invention, a higher titer of a virus vaccine can be obtained compared to the conventional method. As mentioned above, in the case of avian cells that have undergone the first step of deleting MDA5 or TLR3 or overexpressing TMPRSS2 or ST3GAL1 in the avian cells of the present invention in one embodiment, a higher viral titer compared to the control group that is not The highest viral titer can be exhibited when both MDA5 and TLR3 are deleted or their expression is suppressed, and both TMPRSS2 and ST3GAL1 are overexpressed. However, the present invention is not limited thereto.
본 발명은 B4GALNT2(β-1,4 N-acetylgalactosaminyltransferase 2)를 과발현시키는 물질을 포함하는 바이러스 저항성 조류 세포 제조용 조성물에 관한 것이다.The present invention relates to a composition for preparing virus-resistant algal cells comprising a substance overexpressing B4GALNT2 (β-1,4 N-acetylgalactosaminyltransferase 2).
본 발명에서 상기 바이러스 저항성은 바이러스가 세포 내로 주입되는 것이 차단되는 것일 수 있고, 주입된 바이러스가 유전자를 복제하여 다음 세대의 바이러스의 조립을 억제하는 것일 수 있고, 또는 조립된 바이러스의 방출을 억제하는 것일 수 있다. 상기 기전에 제한되지 않고, 바이러스 감염에 대해 저항성을 가지는 것을 의미한다.In the present invention, the virus resistance may be that the virus is blocked from being injected into the cell, the injected virus replicates the gene to inhibit the assembly of the next generation virus, or inhibits the release of the assembled virus it could be It is not limited to the above mechanism, and it means to have resistance to viral infection.
상기 바이러스 저항성에서 저항성을 가지는 바이러스는 조류를 숙주로 하는 바이러스라면 제한 없이 본 발명의 범위에 포함될 수 있다. 앞서 언급한 것과 같이, 조류 인플루엔자 바이러스, 뉴캐슬 바이러스 또는 닭 전염성 기관지염 바이러스 등이 이에 포함될 수 있고, 이외에도 DNA를 유전체로 가지는 전염성 후두 기관염 바이러스(infectious laryngotracheitis virus, ILTV), 허피스바이러스(Herpes virus), 마렉병바이러스(Marek's disease virus, MDV), 조류 백혈병 바이러스(Avian leucosis virus, ALV) 및 전염성 훼브리셔스낭병 바이러스(infectious bursal of fabricius disease virus, IBDV)가 포함될 수 있다. 다만 이에 제한되는 것은 아니다.Viruses having resistance to the virus resistance may be included in the scope of the present invention without limitation as long as the virus is an avian host. As mentioned above, avian influenza virus, Newcastle virus or chicken infectious bronchitis virus, etc. may be included therein, and in addition, infectious laryngotracheitis virus ( ILTV ) having DNA as a genome, herpes virus ( Herpes virus ), Marek may include Marek's disease virus (MDV), Avian leucosis virus (ALV) and infectious bursal of fabricius disease virus (IBDV). However, the present invention is not limited thereto.
본 발명에서 상기 B4GALNT2는 조류 세포에서 외부 바이러스의 세포와의 결합 친화도(binding affinity)를 조절하여 바이러스 유전물질의 세포 내 유입을 억제할 수 있으므로, B4GALNT2가 과발현되는 경우, 조류 세포에서 바이러스에 대한 저항성이 증가할 수 있다. 조류 세포는 일반적으로 B4GALNT2가 발현되지 않으므로, 조류의 종에 관계 없이, 본 B4GALNT2에 의한 효과가 발휘될 수 있다. 예를 들면 본 발명의 일 실시예에서, 닭 섬유아세포에 인간 B4GALNT2를 과발현 시키는 경우 그렇지 않은 경우에 비해 3배 내지 4배 낮은 바이러스 역가를 나타내는 것을 확인하였다. 다만 이에 제한되는 것은 아니다.In the present invention, the B4GALNT2 can suppress the influx of viral genetic material into the cell by controlling the binding affinity of the foreign virus to the cell in the avian cell. Therefore, when B4GALNT2 is overexpressed, Resistance may increase. Since algal cells generally do not express B4GALNT2, the effect of the present B4GALNT2 can be exerted regardless of the species of algae. For example, in one embodiment of the present invention, when human B4GALNT2 was overexpressed in chicken fibroblasts, it was confirmed that the virus titer was 3 to 4 times lower than that in the case of not. However, the present invention is not limited thereto.
본 발명에서 상기 B4GALNT2는 종에 관계없이, B4GALNT2 단백질을 코딩하는 유전자 또는 이의 상동체라면 제한 없이 포함될 수 있다. B4GALNT2 단백질을 코딩하는 염기서열은 예를 들면 인간, 쥐, 염소, 양, 고양이, 돼지, 개구리, 원숭이, 침팬지, 햄스터, 개, 또는 도마뱀 등의 종에서 보존되는 B4GALNT2 유전자 염기서열일 수 있고, 더욱 구체적으로는 서열번호 3의 염기서열일 수 있다. 다만, 이에 제한되는 것은 아니다.In the present invention, the B4GALNT2 may be included without limitation, regardless of the species, as long as it is a gene encoding the B4GALNT2 protein or a homologue thereof. The nucleotide sequence encoding the B4GALNT2 protein may be, for example, a B4GALNT2 gene sequence conserved in species such as humans, mice, goats, sheep, cats, pigs, frogs, monkeys, chimpanzees, hamsters, dogs, or lizards, and more Specifically, it may be the nucleotide sequence of SEQ ID NO: 3. However, the present invention is not limited thereto.
본 발명에서 상기 B4GALNT2를 과발현시키는 물질은 B4GALNT2를 코딩하는 유전자를 포함하는 벡터일 수 있다. 앞서 언급하였듯, B4GALNT2를 코딩하는 유전자를 포함하는 벡터는 그 종류에 제한되는 것은 아니고, 플라스미드, 박테리오파지, 코스미드, BAC, YAC, 레트로바이러스, 아데노바이러스, 아데노연관바이러스 또는 비바이러스성 벡터 등 당업자가 제한 없이 선택해서 사용할 수 있다.In the present invention, the substance overexpressing B4GALNT2 may be a vector including a gene encoding B4GALNT2. As mentioned above, the vector containing the gene encoding B4GALNT2 is not limited to its type, and those skilled in the art such as plasmid, bacteriophage, cosmid, BAC, YAC, retrovirus, adenovirus, adeno-associated virus or non-viral vector can be used without limitation.
본 발명의 상기 바이러스 저항성 조류 세포 제조용 조성물에서 상기 조류는 앞서 언급한 조류, 예를 들면, 닭, 오리, 거위, 기러기, 비둘기, 꿩, 봉관조, 칠면조, 메추라기, 고니, 공작, 원앙, 플라밍고, 키위새, 뻐꾸기, 두루미, 제비, 까마귀, 황새, 왜가리, 도요새, 물떼새, 올빼미, 부엉이, 딱따구리, 독수리, 매, 앵무새, 참새, 꾀꼬리 또는 멧새일 수 있고, 조류 세포는 세포의 종류와 무관하게 발명의 범위에 포함될 수 있으나, 바람직하게는 만능성(totipotency)을 가진 조류 세포, 예를 들면 줄기세포 또는 배반엽 세포(blastocyst)일 수 있다. 다만 이에 제한되는 것은 아니다.In the composition for preparing virus-resistant algal cells of the present invention, the alga is the aforementioned birds, for example, chickens, ducks, geese, geese, pigeons, pheasants, bongguan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, It can be a kiwi, cuckoo, crane, swallow, crow, stork, heron, snipe, houndstooth, owl, owl, woodpecker, eagle, hawk, parrot, sparrow, oriole or bunting, and avian cells are invented regardless of cell type Although it may be included in the scope of, preferably, it may be an avian cell having totipotency, for example, a stem cell or a blastocyst. However, the present invention is not limited thereto.
본 발명은 또한 B4GALNT2(β-1,4 N-acetylgalactosaminyltransferase 2)를 과발현시키는 물질을 포함하는 바이러스 저항성 조류 세포 제조용 조성물이 처리된 원시생식세포 또는 배반엽 세포를 포함하는 바이러스 저항성 조류 제조용 조성물에 관한 것이다.The present invention also relates to a composition for preparing virus-resistant algae comprising primitive germ cells or blastoderm cells treated with a composition for preparing virus-resistant algal cells comprising a substance overexpressing B4GALNT2 (β-1,4 N-acetylgalactosaminyltransferase 2). .
상기 원시생식세포 또는 배반엽 세포는 조류 개체로서 분화할 수 있는 전능성을 가진 세포로서, 상기 B4GALNT2(β-1,4 N-acetylgalactosaminyltransferase 2)를 과발현시키는 물질을 포함하는 바이러스 저항성 조류 세포 제조용 조성물을 원시생식세포 또는 배반엽 세포에 주입하여 발현시킨 것 일 수 있다. 상기 조성물이 주입된 원시생식세포 또는 배반엽 세포를 성체로 분화시키는 방법은 공지된 방법에 따라 제한 없이 실시될 수 있다. The primordial germ cells or blastoderm cells are totipotent cells capable of differentiating into avian individuals, and a composition for preparing virus-resistant avian cells containing a substance overexpressing the B4GALNT2 (β-1,4 N-acetylgalactosaminyltransferase 2) is used as a raw material. It may be expressed by injection into germ cells or blastoderm cells. The method for differentiating the primordial germ cells or blastoderm cells injected with the composition into an adult may be carried out without limitation according to a known method.
본 발명은 또한 조류 세포에서 B4GALNT2를 과발현시키는 단계를 포함하는 바이러스 저항성 조류 세포 제조 방법에 관한 것이다.The present invention also relates to a method for producing a virus-resistant avian cell comprising the step of overexpressing B4GALNT2 in the avian cell.
상기 조류 세포에서 B4GALNT2를 과발현시키는 단계는 앞서 언급하였듯, 당업자가 사용할 수 있는 유전자 과발현 방법, 예를 들면 B4GALNT2를 코딩하는 유전자를 포함하는 벡터를 상기 조류 세포에 처리하여 수행될 수도 있고, 바람직하게는 닭의 배반엽 세포에 인간 B4GALNT2를 코딩하는 유전자를 포함하는 벡터를 처리하는 단계일 수 있다. 다만 이에 제한되는 것은 아니다.As mentioned above, the step of overexpressing B4GALNT2 in the avian cell may be performed by treating the avian cell with a gene overexpression method that can be used by a person skilled in the art, for example, a vector containing a gene encoding B4GALNT2. may be a step of treating chicken blastoderm cells with a vector containing a gene encoding human B4GALNT2. However, the present invention is not limited thereto.
또한, 본 발명은 상기 바이러스 저항성 조류 세포로부터 조류를 제조하는 방법에 관한 것이다.The present invention also relates to a method for producing an alga from the virus-resistant algal cell.
본 발명의 방법은 B4GALNT2 과발현된 조류 세포로부터 조류를 제조하는 방법에 관한 것으로서, 당업자에 의해 공지된 형질전환 개체 제조 방법을 제한 없이 이용할 수 있다. 예를 들면, 상기 B4GALNT2 유전자와 선별 물질에 내성을 부여하는 표지 유전자를 포함하는 벡터를 배반포 세포에 처리하고, 상기 벡터를 포함하는 세포의 선별을 항생제 또는 대사 억제제 등의 선별 물질을 처리하여 이루어질 수 있고, 상기 선별된 배반포 세포를 암컷 성체 또는 핵을 제거한 미수정란 등에 주입하여, 상기 형질전환된 세포를 가지는 키메라(chimeric) 개체를 제조할 수 있다. 이후 교배과정을 거쳐, 상기 형질전환 유전자를 동형 접합으로 가지는 개체를 제조할 수 있다. 상기 과정은 해당 분야의 동업자에게 공지된 방법으로서, 실험 조건 등에 따라 용이하게 변경이 가능하다. 상기 선별 물질의 예를 들면, 퓨로마이신, 네오마이신, lactose 또는 ganciclovir(GCV)를 들 수 있고, 상기 표지 유전자로는 퓨로마이신 아세틸트렌스퍼라아제, 네오마이신 포스포트랜스퍼라아제 유전자, LacZ 또는 thymidine kinase를 들 수 있다. 다만 이에 제한되는 것은 아니다.The method of the present invention relates to a method for producing an alga from an avian cell overexpressing B4GALNT2, and a method for producing a transgenic organism known by those skilled in the art can be used without limitation. For example, blastocyst cells are treated with a vector containing the B4GALNT2 gene and a marker gene conferring resistance to a selection substance, and selection of cells containing the vector can be achieved by treating a selection substance such as an antibiotic or a metabolic inhibitor. In addition, by injecting the selected blastocyst cells into a female adult or an unfertilized egg from which the nucleus has been removed, a chimeric individual having the transformed cells can be prepared. Thereafter, through a mating process, an individual having the transgene homozygous can be produced. The above process is a method known to those skilled in the art, and can be easily changed according to experimental conditions and the like. Examples of the selection material include puromycin, neomycin, lactose or ganciclovir (GCV), and the marker gene includes puromycin acetyltransferase, neomycin phosphotransferase gene, LacZ or thymidine kinase. can be heard However, the present invention is not limited thereto.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.Hereinafter, examples will be given to describe the present invention in detail.
실시예 1. 닭 면역 수용체 유전자 편집 벡터 작성 및 면역 수용체 유전자 편집 세포주의 개발Example 1. Creation of chicken immune receptor gene editing vector and development of immune receptor gene editing cell line
본 발명에서는 pX459(Addgene) 플라스미드에 표 1의 합성 cMDA5 gRNA Sense Oligo, antisense oligo (Bionics)를 공지된 방법에 따라 연결했다(Lee 2017, Dev Comp Immunol). 구체적으로, 합성된 cMDA5 gRNA Sense Oligo, antisense oligo를 95℃ 30초, 72℃ 2분, 37℃ 2분, 25℃2분 조건으로 각각의 단일가닥을 이중가닥으로 붙이고, 이중가닥 Oligo 2.5 μl, 25 ng/μl Px459 1.5 μl, 10X T4 DNA Ligase buffer (Takara) 1 μl, BbsI (NEB) 0.5 μl, T4 DNA Ligase (Takara) 0.5 μl, Distilled water (DW) 4 μl를 섞어 37 ℃ 5분, 16℃ 10 분간 12 사이클 조건으로 Px459 벡터에 이중가닥 Oligo를 연결시켰다. Competent E. coli에 연결된 벡터를 넣어 형질전환 한 뒤 QIAprep Spin Miniprep Kit (QIAGEN)을 이용하여 제조사의 방법에 따라 플라스미드를 추출하여"cMDA5 CRISPR/Cas9" 벡터를 완성하였다. 12-well 세포 배양 플레이트에서 자라는 닭 섬유아세포주 (DF-1) (ATCC)에 cMDA5 CRISPR/Cas9을 Lipofectamine® Transfection Reagent(ThermoFisher)을 이용하여 제조사의 방법에 따라 트랜스팩션 했다. 트랜스펙션 후 하루 뒤 1 μg/ml 농도의 퓨로마이신(GIBCO)을 처리하여 3~4일간 배양한다. 배양 후 세포를 Phosphate-Buffered Saline(PBS, ThermoFisher)로 washing해준 뒤 0.5% Trypsin/EDTA (TE) Solution (ThermoFisher)를 10배 희석한 0.05% TE solution으로 세포 부착을 떼어냈다. 세포를 걷어 세포수를 측정한 뒤 96-well 플레이트에 웰 한 개당 세포 한 개가 들어갈 수 있도록 희석하여 세포 클론을 배양하여 닭 MDA5를 제거한 닭 섬유아세포주를 확립한다. In the present invention, the synthetic cMDA5 gRNA Sense Oligo and antisense oligo (Bionics) of Table 1 were linked to the pX459 (Addgene) plasmid according to a known method (Lee 2017, Dev Comp Immunol). Specifically, the synthesized cMDA5 gRNA Sense Oligo and antisense oligo were attached to each single-stranded double-stranded under the conditions of 95°C for 30 seconds, 72°C for 2 minutes, 37°C for 2 minutes, and 25°C for 2 minutes, and 2.5 μl of double-stranded Oligo; 25 ng/μl Mix 1.5 μl of Px459, 1 μl of 10X T4 DNA Ligase buffer (Takara), 0.5 μl of BbsI (NEB), 0.5 μl of T4 DNA Ligase (Takara), and 4 μl of Distilled water (DW) at 37 °C for 5 minutes, 16 Double-stranded Oligo was ligated to the Px459 vector under 12 cycle conditions for 10 minutes at °C. After transforming with the vector linked to Competent E. coli, the "cMDA5 CRISPR/Cas9" vector was completed by extracting the plasmid using the QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's method. cMDA5 CRISPR/Cas9 was transfected into a chicken fibroblast line (DF-1) (ATCC) grown in 12-well cell culture plates using Lipofectamine® Transfection Reagent (ThermoFisher) according to the manufacturer's method. One day after transfection, puromycin (GIBCO) at a concentration of 1 μg/ml is treated and cultured for 3 to 4 days. After incubation, the cells were washed with Phosphate-Buffered Saline (PBS, ThermoFisher), and then the cell attachment was detached with 0.05% TE solution diluted 10 times with 0.5% Trypsin/EDTA (TE) Solution (ThermoFisher). After counting the cells by counting the cells, dilute the cells so that one cell per well can be put in a 96-well plate, and the cell clones are cultured to establish a chicken fibroblast cell line from which chicken MDA5 has been removed.
확립된 세포주를 걷어 New England Biolabs에서 제공하는 프로토콜 (https://international.neb.com/protocols/2019/04/30/quick-protocol-for-extraction-and-purification-of-genomic-dna-using-the-monarch-genomic-dna-purification-kit-neb-t3010, 2021. 4. 16., 17:51)대로 Genomic DNA를 추출한 뒤 표 1의 "cMDA5 T7 F/R" 프라이머와 Taq polymerase 0.1 μl, dNTP 0.4 μl, 10X buffer 1 μl (BIOFACT)로 제조사의 방법대로 중합효소 연쇄 반응 (Polymerase Chain Reaction, PCR)을 수행한 뒤 1% agarose A (Biotech) gel에 전기영동하여 밴드를 블레이드로 잘라 Wizard® SV Gel and PCR Clean-Up System (Promega)을 이용하여 제조사의 방법에 따라 타깃 DNA를 추출했다. pGEM®-T Easy Vector Systems (Promega)를 이용하여 제조사의 방법에 따라 pGEM®-T Easy Vector에 라이게이션 (ligation)한 뒤 Competent E.coli에 트랜스포메이션하고 QIAprep Spin Miniprep Kit (QIAGEN)을 이용하여 제조사의 방법에 따라 플라스미드를 추출하여 Bionics (한국)에 시퀀싱 (Sequencing)을 의뢰하여 닭 MDA5 편집 여부를 확인했다(도 2). The protocol provided by New England Biolabs (https://international.neb.com/protocols/2019/04/30/quick-protocol-for-extraction-and-purification-of-genomic-dna-using by kicking established cell lines) -the-monarch-genomic-dna-purification-kit-neb-t3010, 2021. 4. 16., 17:51) After extracting genomic DNA, "cMDA5 T7 F/R" primer and Taq polymerase 0.1 μl of Table 1 , dNTP 0.4 μl, 10X buffer 1 μl (BIOFACT) according to the manufacturer's method, polymerase chain reaction (PCR) is performed, and then electrophoresed on 1% agarose A (Biotech) gel to cut the band with a blade Wizard ® SV Gel and PCR Clean-Up System (Promega) was used to extract target DNA according to the manufacturer's method. After ligation to pGEM®-T Easy Vector according to the manufacturer's method using pGEM®-T Easy Vector Systems (Promega), transform into Competent E.coli, and use QIAprep Spin Miniprep Kit (QIAGEN). According to the manufacturer's method, the plasmid was extracted and sequencing was requested to Bionics (Korea) to confirm whether chicken MDA5 was edited (FIG. 2).
같은 방식으로 pX459 벡터에 표 1의 합성 cTLR3 gRNA Sense Oligo, antisense oligo (Bionics)를 연결 및 형질전환하여 닭 Toll like receptor 3 (TLR3)를 타깃하는 "cTLR3 CRISPR/Cas9"를 작성하여 Lipofectamine® Transfection Reagent (ThermoFisher)을 이용하여 제조사의 방법에 따라 트랜스팩션했다. 트랜스펙션 후 하루 뒤 1 μg/ml 농도의 퓨로마이신(GIBCO)을 처리하여 3~4일간 배양했다. 96-well 플레이트에 단일 클론을 확립하여 genomic DNA를 추출한 뒤 PCR을 수행했다. Wizard® SV Gel and PCR Clean-Up System (Promega)을 이용하여 제조사의 방법에 따라 타깃 DNA를 추출했다. 표 1의 "cTLR3 T7 F/R" 프라이머를 이용하여 PCR을 수행한 뒤, pGEM®-T Easy Vector Systems (Promega)를 이용하여 제조사의 방법에 따라 pGEM®-T Easy Vector에 라이게이션 (ligation)한 뒤 Competent E.coli에 트랜스포메이션하고 QIAprep Spin Miniprep Kit (QIAGEN)을 이용하여 제조사의 방법에 따라 플라스미드를 추출하여 서열 분석을 진행하여 (Bionics, 한국), 닭 TLR3 편집 여부를 확인했다(도 2).Lipofectamine® Transfection Reagent by ligating and transforming the synthetic cTLR3 gRNA Sense Oligo and antisense oligo (Bionics) in Table 1 to pX459 vector in the same way to create “cTLR3 CRISPR/Cas9” targeting chicken Toll like receptor 3 (TLR3) (ThermoFisher) was used to transfect according to the manufacturer's method. One day after transfection, puromycin (GIBCO) at a concentration of 1 μg/ml was treated and cultured for 3 to 4 days. A single clone was established in a 96-well plate, genomic DNA was extracted, and then PCR was performed. The target DNA was extracted using the Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's method. After PCR was performed using the "cTLR3 T7 F/R" primers in Table 1, pGEM®-T Easy Vector Systems (Promega) was used to ligate the pGEM®-T Easy Vector according to the manufacturer's method. After transforming into Competent E. coli, using the QIAprep Spin Miniprep Kit (QIAGEN), the plasmid was extracted according to the manufacturer's method and sequence analysis was performed (Bionics, Korea) to confirm whether chicken TLR3 was edited (Fig. 2). ).
같은 방식으로 닭 MDA5가 제거된 섬유아세포에 "cTLR3 CRISPR/Cas9"을 도입하여 닭 MDA5, TLR3가 모두 제거된 단일클론 세포주를 확립한 뒤 시퀀싱을 통해 닭 MDA5, TLR3가 모두 제거됨을 확인했다(도 2).In the same way, "cTLR3 CRISPR/Cas9" was introduced into fibroblasts from which chicken MDA5 was removed to establish a monoclonal cell line in which both chicken MDA5 and TLR3 were removed, and then it was confirmed that both chicken MDA5 and TLR3 were removed through sequencing (Fig. 2).
명칭designation 서열(5'-3’)Sequence (5'-3') 번호number
cMDA5 gRNA Sense Oligo forwardcMDA5 gRNA Sense Oligo forward CACCGAACCGAGGTCGAAACGTACGCACCGAACCGAGGTCGAAACGTACG 서열번호 4SEQ ID NO: 4
cMDA5 gRNA anti-sense Oligo reversecMDA5 gRNA anti-sense Oligo reverse AAACCGTACGTTTCGACCTCGGTTCAAACCGTACGTTTCGACCTCGGTTC 서열번호 5SEQ ID NO: 5
cMDA5 T7 forwardcMDA5 T7 forward GAGACGAGCGCTTCCTCTACGAGACGAGCGCTTCCTCTAC 서열번호 6SEQ ID NO: 6
cMDA5 T7 reversecMDA5 T7 reverse CCTTATTGCTGGCCCACTGACCTTATTGCTGGCCCACTGA 서열번호 7SEQ ID NO: 7
cTLR3 gRNA Sense Oligo forwardcTLR3 gRNA Sense Oligo forward CACCGAAAGGATCCACTTAGAGCTGCACCGAAAGGATCCACTTAGAGCTG 서열번호 8SEQ ID NO: 8
cTLR3 gRNA anti-sense Oligo reversecTLR3 gRNA anti-sense Oligo reverse AAACCAGCTCTAAGTGGATCCTTTCAAACCAGCTCTAAGTGGATCCTTTC 서열번호 9SEQ ID NO: 9
cTLR3 T7 forwardcTLR3 T7 forward TGACCGAGTACAGCAATCTGTGACCGAGTACAGCAATCTG 서열번호 10SEQ ID NO: 10
cTLR3 T7 reversecTLR3 T7 reverse ATCCCCAAAGCCCTGGGAGAATCCCCAAAGCCCTGGGAGA 서열번호 11SEQ ID NO: 11
IFNB promoter forwardIFNB promoter forward ACAGCCACCACATGGTCTCACCTTGCCAGACAGCCACCACATGGTCTCACCTTGCCAG 서열번호 12SEQ ID NO: 12
IFNB promoter reverseIFNB promoter reverse GATGGTGTTTGGGGTGTTGCGATGGTGTTTGGGGTGTTGC 서열번호 13SEQ ID NO: 13
IFNB promoter infusion forwardIFNB promoter infusion forward TCGAACAGCCACCACATGGTCTCACCTTGCCAGTCGAACAGCCACCACATGGTCTCACCTTGCCAG 서열번호 14SEQ ID NO: 14
IFNB promoter infusion reverseIFNB promoter infusion reverse AGCTGATGGTGTTTGGGGTGTTGCAGCTGATGGTGTTTGGGGTGTTGC 서열번호 15SEQ ID NO: 15
ACTB forwardACTB forward AGGAGATCACAGCCCTGGCAAGGAGATCACAGCCCTGGCA 서열번호 16SEQ ID NO: 16
ACTB reverseACTB reverse CAATGGAGGGTCCGGATTCACAATGGAGGGTCCGGATTCA 서열번호 17SEQ ID NO: 17
TMPRSS2 forwardTMPRSS2 forward TTCTGCCAGGCCACAAGTAGTTCTGCCAGGCCACAAGTAG 서열번호 18SEQ ID NO: 18
TMPRSS2 reverseTMPRSS2 reverse GGAGAAATGCACACTCCCGAGGAGAAATGCACACTCCCGA 서열번호 19SEQ ID NO: 19
ST3GAL1 forwardST3GAL1 forward CACCCACCATTGGCTACGAACACCCACCATTGGCTACGAA 서열번호 20SEQ ID NO: 20
ST3GAL2 reverseST3GAL2 reverse AGGCCTGTGGAAGGGTATCTAGGCCTGTGGAAGGGTATCT 서열번호 21SEQ ID NO: 21
Human B4GALNT2 RT-PCR forwardHuman B4GALNT2 RT-PCR forward GGTGGTGCTAAGCGTGTTATGGTGGTGCTAAGCGTGTTAT 서열번호 22SEQ ID NO: 22
Human B4GALNT2 RT-PCR reverseHuman B4GALNT2 RT-PCR reverse ACCTCTGCCATCTCTCCACAACCTCTGCCATCTCTCCACA 서열번호 23SEQ ID NO: 23
GAPDH gRNA Sense Oligo forwardGAPDH gRNA Sense Oligo forward CACCGCTATTCCTTATAAAGAAAGTCACCGCTATTCCTTATAAAGAAAGT 서열번호 24SEQ ID NO: 24
GAPDH gRNA anti-sense Oligo reverseGAPDH gRNA anti-sense Oligo reverse AAACACTTTCTTTATAAGGAATAGCAAACACTTTCTTTATAAGGAATAGC 서열번호 25SEQ ID NO: 25
실시예 2. 닭 MDA5 편집 세포주에서의 생합성 리간드, 바이러스 처리 및 항 바이러스 반응 측정Example 2. Measurement of biosynthetic ligand, viral treatment and antiviral response in chicken MDA5 editing cell line
본 발명에서는 "pNL 1.2 [NlucP]" (Promega) 플라스미드 5 μg을 HindIII-HF, XhoI (New England Biolabs) 1μl, 1X CutSmart® Buffer (New England Biolabs) 5 μl처리하여 플라스미드를 선형화시켰다. 표 1의 "IFNB promoter F/R" 프라이머를 이용하여 닭 섬유아세포에서 추출한 genomic DNA (gDNA)로부터 인터페론 베타 (Interferon Beta) 프로모터 (Promoter)를 클로닝했다. 보다 더욱 상세히 설명하면 BioFACT 2X Pfu PCR Master Mix1 (BIOFACT) 25 μl, "IFNB promoter F/R" 프라이머 각각 1 μl, 앞서 추출한 gDNA 1μl, UltraPureTM distilled Water (Invitrogen) 22 μl를 섞어 제조사의 방법대로 PCR을 수행한 뒤 1% agarose A (Biotech) gel에 전기영동하여 성공적으로 인터페론 베타 (Interferon Beta) 프로모터 (Promoter) 밴드를 얻을 수 있었다. 해당 부분을 블레이드로 자르고, Wizard® SV Gel and PCR Clean-Up System (Promega)을 이용하여 제조사의 방법에 따라 타깃 DNA를 추출했다. pGEM®-T Easy Vector Systems (Promega)를 이용하여 제조사의 방법에 따라 IFNB promoter를 pGEM®-T Easy Vector에 연결시켰다. Competent E. coli에 연결된 샘플을 넣어 형질전환 한 뒤 QIAprep Spin Miniprep Kit(QIAGEN)를 이용하여 제조사의 방법에 따라 플라스미드를 추출하여 "IFNB_T vector"를 완성했다. 추출한 플라스미드를 Bionics (한국)에 시퀀싱 의뢰를 맡겨 IFNB 프로모터 시퀀스를 확인했다. 이후 "IFNB_T vector"를 표 1의 XhoI 인지부위와 상보결합을 할 수 있도록 5'-TCGA-3' 점착성 말단, HindIII-HF 인지부위와 상보결합을 할 수 있도록 5'-AGCT-3' 점착성 말단이 삽입된 "IFNB promoter infusion F/R"을 이용하여 선형화된 플라스미드 HindIII-HF, XhoI의 인지 부위에 상보적으로 결합 할 수 있는 인서트 (insert)를 클로닝 했다. XhoI, HindIII-HF로 선형화된 "pNL 1.2 [NlucP]" (Promega) 플라스미드, 앞서 클로닝한 IFNB 프로모터 인서트를 In-Fusion® HD cloning Kit (Takara)를 이용하여 제조사의 방법대로 연결시켜 "IFNB_pNL1.2"를 완성했다.In the present invention, 5 μg of “pNL 1.2 [NlucP]” (Promega) plasmid was treated with HindIII-HF, 1 μl of XhoI (New England Biolabs), and 5 μl of 1X CutSmart® Buffer (New England Biolabs) to linearize the plasmid. The interferon beta promoter (Promoter) was cloned from genomic DNA (gDNA) extracted from chicken fibroblasts using the "IFNB promoter F/R" primer in Table 1. In more detail, 25 μl of BioFACT 2X Pfu PCR Master Mix1 (BIOFACT), 1 μl each of the “IFNB promoter F/R” primer, 1 μl of the previously extracted gDNA, and 22 μl of UltraPure™ distilled Water (Invitrogen) were mixed and PCR was performed according to the manufacturer's method. After performing electrophoresis on 1% agarose A (Biotech) gel, interferon beta (Interferon Beta) promoter band was successfully obtained. The part was cut with a blade, and the target DNA was extracted using the Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's method. The IFNB promoter was ligated to the pGEM®-T Easy Vector using pGEM®-T Easy Vector Systems (Promega) according to the manufacturer's method. After transforming with a sample linked to Competent E. coli, the plasmid was extracted using the QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's method to complete "IFNB_T vector". The IFNB promoter sequence was confirmed by entrusting the extracted plasmid to Bionics (Korea) for sequencing. Thereafter, "IFNB_T vector" was used at the 5'-TCGA-3' sticky end to complement the XhoI recognition site in Table 1, and the 5'-AGCT-3' sticky end to complement the HindIII-HF recognition site. An insert capable of complementary binding to the recognition site of the linearized plasmid HindIII-HF and XhoI was cloned using the inserted "IFNB promoter infusion F/R". The "pNL 1.2 [NlucP]" (Promega) plasmid linearized with XhoI, HindIII-HF, the previously cloned IFNB promoter insert was ligated using the In-Fusion® HD cloning Kit (Takara) according to the manufacturer's method, and "IFNB_pNL1.2" " was completed.
닭 MDA5가 제거된 섬유아세포, 닭 TLR3가 제거된 섬유아세포, 닭 MDA5, TLR3 모두 제거된 섬유아세포 그리고 야생형 (wild type) 섬유아세포에 "IFN-Beta_pNL1.2" 플라스미드 200ng, "pGL 4.53" (Promega) 25ng을 닭 MDA5 적중 태아 섬유아세포에 Lipofectamine® Transfection Reagent (ThermoFisher)를 이용하여 제조사의 방법에 따라 트랜스팩션했다.200ng of "IFN-Beta_pNL1.2" plasmid, "pGL 4.53" (Promega ) 25ng of chicken MDA5-targeted fetal fibroblasts were transfected using Lipofectamine® Transfection Reagent (ThermoFisher) according to the manufacturer's method.
트랜스팩션 수행 후 24시간동안 세포를 배양하고 double strand RNA (dsRNA)의 일종으로 바이러스 RNA 유사체인 포유류 MDA5의 리간드인 Poly (I:C) (HMW)/LyoVecTM (InvivoGen, 이하 long poly I:C라고 명명) 혹은 Park (2019, JID)의 보고에서 만들어진 저병원성 인플루엔자 A 바이러스 (PR8-H5N8)를 Multiplicity of infection (MOI) = 0.1로 처리 후 각각 24시간, 8시간 후 세포를 걷어 Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega)를 이용해서 인터페론 베타(IFNB) 프로모터 활성을 제조사의 방법에 따라 측정했다. Promoter assay 결과 닭 MDA5를 제거한 세포주는 야생형 세포주와 비교할 때 poly I:C 자극과 인플루엔자 A 바이러스 감염에 인터페론 베타의 활성을 유의적으로 유도하지 못했다(도 3). 또한, 닭 MDA5를 제거한 세포주에 PR8-H5N8을 MOI=0.1로 Park (2019, JID)에 나타난 방식으로 바이러스를 감염시키고 TICD50을 수행했을 때 30배 정도의 바이러스 역가가 증가했음을 확인했다(도 4).Cells were cultured for 24 hours after transfection, and Poly (I:C) (HMW)/LyoVecTM (InvivoGen, hereinafter referred to as long poly I:C Name) or Park (2019, JID) of low pathogenicity influenza A virus (PR8-H5N8) made in the report of Multiplicity of infection (MOI) = 0.1, after 24 hours and 8 hours, respectively, the cells were collected and Nano-Glo® Dual- Interferon beta (IFNB) promoter activity was measured using the Luciferase® Reporter Assay System (Promega) according to the manufacturer's method. As a result of the promoter assay, the cell line from which chicken MDA5 was removed did not significantly induce the activity of interferon beta to poly I:C stimulation and influenza A virus infection when compared to the wild-type cell line (FIG. 3). In addition, it was confirmed that the virus titer increased by about 30 times when the chicken MDA5-removed cell line was infected with PR8-H5N8 in the manner shown in Park (2019, JID) with MOI = 0.1 and TICD50 was performed (Fig. 4). .
실시예 3. 닭 TMPRSS2, ST3GAL1 과발현 벡터의 작성Example 3. Construction of chicken TMPRSS2, ST3GAL1 overexpression vectors
Piggy Bac (Addgene) 플라스미드 5μg를 AgeI, BsrGI (NEB) 1μl, 1X CutSmart® Buffer (New England Biolabs) 5μl처리하여 플라스미드를 선형화시켰다. TMPRSS2, ST3GAL1 유전자는 Bionics (한국)에서 합성되어 선형화된 Piggy Bac에 In-Fusion® HD cloning Kit (Takara)를 이용하여 제조사의 방법대로 라이게이션 하여 "TMPRSS2_PB", "ST3GAL1_PB", "ST3T2_PB" 플라스미드를 작성했다. 닭 섬유아세포 (DF-1) 세포주에 "TMPRSS2_PB", "ST3GAL1_PB" 혹은 "ST3T2_PB" 1.2 μg와 0.8 μg PiggyBac transposon (pCyL50)을 Lipofectamine® Transfection Reagent (ThermoFisher)를 이용하여 제조사의 방법에 따라 트랜스팩션했다. 트랜스펙션 후 1 μg/ml 퓨로마이신을 처리하여 15번의 계대배양을 유지했다. 이후 Tri-reagent (Molecular Research Center Inc)을 이용하여 제조사의 방법대로 RNA를 추출하고 Superscript IV (Thermo Fisher)룰 이용하여 제조사의 방법대로 complementary DNA (cDNA)를 합성했다. 표 1에 나온 "ACTB-F/R", "ST3GAL1-F/R", "TMPRSS2-F/R" 프라이머와 Taq polymerase 0.1 μl, dNTP 0.4 μl, 10X buffer 1 μl (BIOFACT), 20X EvaGreen (Biotium) 1 μl로 StepOnePlus real time PCR system (Applied Biosystems)에 제시된 방법대로 유전자 발현을 조사했다. TMPRSS2는 야생형 섬유아세포주 대조군에 비해 350배 발현이 증가했고(도 5), ST3GAL1은 1,500배 발현이 증가했다(도 6). 또한, TMPRSS2, ST3GAL1을 동시에 발현시킨 결과, TMPRSS2, ST3GAL1 모두 120배 유전자 발현이 증가되었다(도 7). The plasmid was linearized by treating 5 μg of Piggy Bac (Addgene) plasmid with 1 μl of AgeI, BsrGI (NEB) and 5 μl of 1X CutSmart® Buffer (New England Biolabs). TMPRSS2 and ST3GAL1 genes were synthesized in Bionics (Korea) and ligated to the linearized Piggy Bac using the In-Fusion® HD cloning Kit (Takara) according to the manufacturer's method, and "TMPRSS2_PB", "ST3GAL1_PB", "ST3T2_PB" plasmids were obtained. Written. Chicken fibroblast (DF-1) cell lines were transfected with "TMPRSS2_PB", "ST3GAL1_PB" or "ST3T2_PB" 1.2 μg and 0.8 μg PiggyBac transposon (pCyL50) using Lipofectamine® Transfection Reagent (ThermoFisher) according to the manufacturer's method. . After transfection, 1 μg/ml puromycin was treated to maintain passage 15 times. Then, RNA was extracted according to the manufacturer's method using Tri-reagent (Molecular Research Center Inc), and complementary DNA (cDNA) was synthesized according to the manufacturer's method using Superscript IV (Thermo Fisher). "ACTB-F/R", "ST3GAL1-F/R", "TMPRSS2-F/R" primers from Table 1, 0.1 μl of Taq polymerase, 0.4 μl of dNTP, 1 μl of 10X buffer (BIOFACT), 20X EvaGreen (Biotium) ) in 1 μl, gene expression was investigated according to the method presented in the StepOnePlus real time PCR system (Applied Biosystems). The expression of TMPRSS2 was increased 350-fold compared to the control of the wild-type fibroblast line ( FIG. 5 ), and the expression of ST3GAL1 was increased 1,500-fold ( FIG. 6 ). In addition, as a result of simultaneous expression of TMPRSS2 and ST3GAL1, gene expression of both TMPRSS2 and ST3GAL1 was increased 120-fold ( FIG. 7 ).
실시예 4. TMPRSS2, ST3GAL1 과발현 세포주에서의 바이러스 역가 측정Example 4. Measurement of virus titer in TMPRSS2, ST3GAL1 overexpressing cell lines
앞서 Park (2019, JID)에 제시된 PR8-H5N8, PR8-H9N2 저병원성 조류 인플루엔자 바이러스를 감염시킨 뒤 TICD50를 수행한 결과, TMPRSS2, ST3GAL1 과발현된 세포주에서 야생형 섬유아세포주보다 유의적으로 높은 바이러스 역가를 보였다(도 5 및 도 6). 또한, PR8-H5N8, PR8-H9N2 저병원성 조류 인플루엔자 바이러스를 감염시킨 뒤 TICD50를 수행한 결과, TMPRSS2, ST3GAL1을 동시에 과발현하는 세포주에서 트립신 처리 없이 감염 후 72시간때 야생형 섬유아세포주보다 유의적으로 높은 바이러스 역가를 보였다(도 7).As a result of performing TICD50 after infection with PR8-H5N8 and PR8-H9N2 low pathogenic avian influenza viruses presented in Park (2019, JID), TMPRSS2 and ST3GAL1 overexpressing cell lines showed significantly higher viral titers than wild-type fibroblasts. (FIGS. 5 and 6). In addition, as a result of performing TICD50 after infection with PR8-H5N8 and PR8-H9N2 low pathogenic avian influenza viruses, a cell line that simultaneously overexpresses TMPRSS2 and ST3GAL1 had a significantly higher virus than the wild-type fibroblast line at 72 hours post-infection without trypsin treatment. titers were shown ( FIG. 7 ).
실시예 5. 사람 B4GALNT2 과발현 벡터의 작성 및 닭 게놈으로의 도입Example 5. Construction of human B4GALNT2 overexpression vector and introduction into chicken genome
Piggy Bac (Addgene) 플라스미드 5μg를 HindIII, NotI (NEB) 1μl, 1X CutSmart® Buffer (New England Biolabs) 5μl처리하여 플라스미드를 선형화시켰다. 사람 B4GALNT2 유전자는 Bioneer (한국)에서 합성되어 선형화된 Piggy Bac에 In-Fusion® HD cloning Kit (Takara)를 이용하여 제조사의 방법대로 라이게이션 하여 "PB_B4GALNT2"를 작성했다. GAPDH유전자 뒤에 T2A, B4GALNT2 유전자를 Bioneer (한국)에서 pBHA 플라스미드에 합성했다 GAPDH 유전자 인트론 10을 타깃하는 CRISPR/Cas9은 실시예 1에서 기술하듯 pX459 벡터를 기반으로 표 1의 GAPDH gRNA Sense/anti-sense oligo을 연결시켜 작성했다. 실시예 3의 PiggyBac transposon (pCyL50) 3 μg, CRISPR/Cas9 3 μg를 벡터 닭 섬유아세포주에 실시예 1에 기술하듯 Lipofectamine® Transfection Reagent (ThermoFisher)을 이용하여 제조사의 방법에 따라 트랜스팩션했다. 트랜스펙션 후 300 μg/ml G418을 처리하여 7일간 배양 후 표 1의 "Human B4GALNT2 RT-PCR F/R"로 실시예 3에서 기술하듯 사람 B4GALNT2 발현량을 분석했다. "PB_B4GALNT2"를 도입한 후 발현량을 조사한 결과 사람 B4GALNT2의 전사체가 관찰되었다(도 8). 또한, 닭 GAPDH 유전자 뒤에 사람 B4GALNT2 유전자를 CRISPR/Cas9을 매개로 한 Knock-in을 수행한 결과, 사람 B4GALNT2의 전사체가 관찰되었다(도 9).The plasmid was linearized by treating 5 μg of Piggy Bac (Addgene) plasmid with 1 μl of HindIII, NotI (NEB) and 5 μl of 1X CutSmart® Buffer (New England Biolabs). The human B4GALNT2 gene was ligated to the linearized Piggy Bac synthesized by Bioneer (Korea) using the In-Fusion® HD cloning Kit (Takara) according to the manufacturer's method to create "PB_B4GALNT2". After the GAPDH gene, the T2A and B4GALNT2 genes were synthesized on the pBHA plasmid by Bioneer (Korea). CRISPR/Cas9 targeting the GAPDH gene intron 10 was based on the pX459 vector as described in Example 1. GAPDH gRNA Sense/anti-sense in Table 1 It was written by linking oligo. 3 μg of the PiggyBac transposon (pCyL50) of Example 3 and 3 μg of CRISPR/Cas9 were transfected into a vector chicken fibroblast cell line using Lipofectamine® Transfection Reagent (ThermoFisher) as described in Example 1 according to the manufacturer's method. After transfection, 300 μg/ml G418 was treated and cultured for 7 days, and then human B4GALNT2 expression level was analyzed as described in Example 3 with “Human B4GALNT2 RT-PCR F/R” in Table 1. After introducing "PB_B4GALNT2", as a result of examining the expression level, a transcript of human B4GALNT2 was observed (FIG. 8). In addition, as a result of performing CRISPR/Cas9-mediated knock-in of the human B4GALNT2 gene after the chicken GAPDH gene, a transcript of human B4GALNT2 was observed ( FIG. 9 ).
실시예 6. 사람 B4GALNT2 과발현 세포주에서의 바이러스 역가 측정Example 6. Determination of virus titer in human B4GALNT2 overexpressing cell line
PB_B4GALNT2를 도입하여 무작위적으로 사람 B4GALNT를 닭 게놈에 도입하거나 CRISPR/Cas9을 통해 GAPDH 유전자 뒤에 사람 B4GALNT2를 knock-in한 후, 형광 유전자 (Green fluorescent protein)이 라벨링된 alpha-2,3 linked sialic acids에 붙는 Maackia Amurensis (MMA) Lectin I (Vector Laboratories)를 제조사의 방법대로 처리하여 형광을 측정한 결과, MMA-lectin binding이 사람 B4GALNT2가 도입되었을 때 형광이 유의적으로 감소함을 보였다(도 10).alpha-2,3 linked sialic acids labeled with a fluorescent gene (Green fluorescent protein) after random introduction of human B4GALNT into the chicken genome by introducing PB_B4GALNT2 or knock-in of human B4GALNT2 after the GAPDH gene via CRISPR/Cas9 Maackia Amurensis (MMA) Lectin I (Vector Laboratories) attached to the fluorescence was measured according to the manufacturer's method. As a result, MMA-lectin binding showed a significant decrease in fluorescence when human B4GALNT2 was introduced (FIG. 10) .
실시예 4와 같이 저병원성 인플루엔자 바이러스 (PR8-H5N8, PR8-H9N2)를 MOI=0.1로 감염시킨 뒤 Premix WST-1 Cell Proliferation Assay System (Takara)를 제조사의 방법에 따라 세포 활성을 측정한 결과, 사람 B4GALNT2가 도입된 세포주가 야생형 섬유아세포주보다 유의적으로 높은 세포활성을 보였다(도 10). 또한 PR8-H5N8, PR8-H9N2를 감염시켜 TCID50을 통해 바이러스 역가를 측정한 결과, GAPDH 뒤에 사람 B4GALNT2 유전자를 삽입한 세포주에서 야생형 섬유아세포주에 비해 각각 4.4, 4.6배 감소된 바이러스 역가를 보였다(도 10).As in Example 4, after infecting the low pathogenic influenza virus (PR8-H5N8, PR8-H9N2) at MOI = 0.1, the Premix WST-1 Cell Proliferation Assay System (Takara) was used to measure cell activity according to the manufacturer's method. The cell line into which B4GALNT2 was introduced showed significantly higher cellular activity than the wild-type fibroblast cell line (FIG. 10). In addition, as a result of infecting PR8-H5N8 and PR8-H9N2 and measuring the virus titer through TCID50, the cell line in which the human B4GALNT2 gene was inserted after GAPDH showed a 4.4 and 4.6 fold decrease in virus titer, respectively, compared to the wild-type fibroblast cell line (Fig. 10).

Claims (24)

  1. MDA5(melanoma differentiation associated protein 5) 또는 TLR3(toll like receptor 3)를 결실시키는 물질; 또는 TMPRSS2(type Ⅱ transmembrane protease, serine 2) 또는 ST3GAL1(ST3 beta-galactoside alpha-2,3-sialyltransferase 1)를 과발현시키는 물질; 을 포함하는 바이러스 백신 생산용 조류 세포 제조용 조성물.a substance that deletes melanoma differentiation associated protein 5 (MDA5) or toll like receptor 3 (TLR3); or a substance that overexpresses TMPRSS2 (type II transmembrane protease, serine 2) or ST3GAL1 (ST3 beta-galactoside alpha-2,3-sialyltransferase 1); A composition for preparing avian cells for the production of a virus vaccine comprising a.
  2. 청구항 1에 있어서, 상기 MDA5 또는 TLR3를 결실시키는 물질은 MDA5 또는 TLR3에 상보적으로 결합하는 gRNA를 포함하는 바이러스 백신 생산용 조류 세포 제조용 조성물.The composition according to claim 1, wherein the substance that deletes MDA5 or TLR3 comprises gRNA that complementarily binds to MDA5 or TLR3.
  3. 청구항 1에 있어서, 상기 TMPRSS2 또는 ST3GAL1을 과발현시키는 물질은 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터인 바이러스 백신 생산용 조류 세포 제조용 조성물.The composition of claim 1, wherein the substance overexpressing TMPRSS2 or ST3GAL1 is a vector containing a gene encoding TMPRSS2 or ST3GAL1.
  4. 청구항 1에 있어서, 상기 조류는 닭, 오리, 거위, 기러기, 비둘기, 꿩, 봉관조, 칠면조, 메추라기, 고니, 공작, 원앙, 플라밍고, 키위새, 뻐꾸기, 두루미, 제비, 까마귀, 황새, 왜가리, 도요새, 물떼새, 올빼미, 부엉이, 딱따구리, 독수리, 매, 앵무새, 참새, 꾀꼬리 또는 멧새를 포함하는 군에서 선택되는 바이러스 백신 생산용 조류 세포 제조용 조성물.The method according to claim 1, wherein the birds are chickens, ducks, geese, geese, pigeons, pheasants, feng guan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwis, cuckoos, cranes, swallows, crows, storks, herons, Snipe, houndstooth, owl, owl, woodpecker, eagle, hawk, parrot, sparrow, oriole or bunting, a composition for producing avian cells for the production of a virus vaccine selected from the group comprising.
  5. 청구항 1에 있어서, 상기 바이러스는 조류 인플루엔자 바이러스, 뉴캐슬 바이러스 및 기관지염 바이러스를 포함하는 군에서 선택되는 바이러스 백신 생산용 조류 세포 제조용 조성물.The composition according to claim 1, wherein the virus is selected from the group comprising avian influenza virus, Newcastle virus and bronchitis virus.
  6. 청구항 1에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 백신 생산용 조류 세포 제조용 조성물.The composition of claim 1, wherein the bird is a chicken, and the virus is an avian influenza virus.
  7. 청구항 1 내지 6 중 어느 한 항의 조성물이 처리된 조류 세포를 포함하는 바이러스 백신 생산용 조성물.A composition for producing a virus vaccine comprising avian cells treated with the composition of any one of claims 1 to 6.
  8. 조류 세포에서 MDA5 또는 TLR3를 결실시키거나, 또는 TMPRSS2 또는 ST3GAL1을 과발현시키는 제1 단계; 및a first step of deleting MDA5 or TLR3 or overexpressing TMPRSS2 or ST3GAL1 in an avian cell; and
    상기 제1 단계를 거친 조류 세포를 약독화한 바이러스에 감염시키는 제2 단계를 포함하는 바이러스 백신 생산 방법.and a second step of infecting the avian cells that have undergone the first step with the attenuated virus.
  9. 청구항 8에 있어서, 상기 MDA5 또는 TLR3의 결실은 상기 조류 세포에 MDA5 또는 TLR3에 상보적으로 결합하는 gRNA를 포함하는 물질을 처리하여 수행되는 바이러스 백신 생산 방법.The method according to claim 8, wherein the deletion of MDA5 or TLR3 is performed by treating the avian cells with a substance containing gRNA that complementarily binds to MDA5 or TLR3.
  10. 청구항 8에 있어서, 상기 TMPRSS2 또는 ST3GAL1의 과발현은 상기 조류 세포에 TMPRSS2 또는 ST3GAL1을 코딩하는 유전자를 포함하는 벡터를 처리하여 수행되는 바이러스 백신 생산 방법.The method according to claim 8, wherein the overexpression of TMPRSS2 or ST3GAL1 is performed by treating the avian cells with a vector containing a gene encoding TMPRSS2 or ST3GAL1.
  11. 청구항 8에 있어서, 상기 바이러스의 감염은 상기 조류 세포에 바이러스를 주입하거나 또는 바이러스 유전체 염기서열을 포함하는 벡터를 주입하여 수행되는 바이러스 백신 생산 방법.The method for producing a virus vaccine according to claim 8, wherein the virus infection is performed by injecting the virus into the avian cells or injecting a vector containing a viral genome sequence.
  12. 청구항 8에 있어서, 상기 2단계를 거친 조류 세포를 배양하여 그로부터 바이러스를 얻는 제3 단계를 더 포함하는 바이러스 백신 생산 방법.The method according to claim 8, further comprising a third step of culturing the avian cells that have undergone the second step to obtain a virus therefrom.
  13. 청구항 8에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 백신 생산 방법.The method of claim 8 , wherein the avian is a chicken and the virus is an avian influenza virus.
  14. B4GALNT2(β-1,4 N-acetylgalactosaminyltransferase 2)를 과발현시키는 물질을 포함하는 바이러스 저항성 조류 세포 제조용 조성물.A composition for preparing virus-resistant algal cells, comprising a substance overexpressing B4GALNT2 (β-1,4 N-acetylgalactosaminyltransferase 2).
  15. 청구항 14에 있어서, 상기 B4GALNT2는 인간 B4GALNT2인 바이러스 저항성 조류 세포 제조용 조성물.The composition according to claim 14, wherein the B4GALNT2 is human B4GALNT2.
  16. 청구항 14에 있어서, 상기 B4GALNT2를 과발현시키는 물질은 B4GALNT2를 코딩하는 유전자를 포함하는 벡터인 바이러스 저항성 조류 세포 제조용 조성물.The composition according to claim 14, wherein the substance overexpressing B4GALNT2 is a vector containing a gene encoding B4GALNT2.
  17. 청구항 14에 있어서, 상기 조류는 닭, 오리, 거위, 기러기, 비둘기, 꿩, 봉관조, 칠면조, 메추라기, 고니, 공작, 원앙, 플라밍고, 키위새, 뻐꾸기, 두루미, 제비, 까마귀, 황새, 왜가리, 도요새, 물떼새, 올빼미, 부엉이, 딱따구리, 독수리, 매, 앵무새, 참새, 꾀꼬리 또는 멧새를 포함하는 군에서 선택되는 바이러스 저항성 조류 세포 제조용 조성물.15. The method of claim 14, wherein the birds are chickens, ducks, geese, geese, pigeons, pheasants, feng guan birds, turkeys, quails, cygnus, peacocks, mandarin ducks, flamingos, kiwis, cuckoos, cranes, swallows, crows, storks, herons, Snipe, houndstooth, owl, owl, woodpecker, eagle, hawk, parrot, sparrow, oriole or bunting, a composition for preparing virus-resistant avian cells selected from the group consisting of.
  18. 청구항 14에 있어서, 상기 바이러스는 조류 인플루엔자 바이러스, 뉴캐슬 바이러스 및 기관지염 바이러스를 포함하는 군에서 선택되는 바이러스 저항성 조류 세포 제조용 조성물.The composition for preparing virus-resistant avian cells according to claim 14, wherein the virus is selected from the group comprising avian influenza virus, Newcastle virus and bronchitis virus.
  19. 청구항 14에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 저항성 조류 세포 제조용 조성물.The composition for preparing virus-resistant avian cells according to claim 14, wherein the bird is a chicken, and the virus is an avian influenza virus.
  20. 위 14 내지 19 중 어느 하나의 바이러스 저항성 조류 세포 제조용 조성물이 처리된 원시생식세포 또는 배반엽세포를 포함하는 바이러스 저항성 조류 제조용 조성물.A composition for preparing virus-resistant algae comprising primitive germ cells or blastoderm cells treated with the composition for preparing virus-resistant algal cells of any one of the above 14 to 19.
  21. 조류 세포에서 B4GALNT2를 과발현시키는 단계를 포함하는 바이러스 저항성 조류 세포 제조 방법.A method for producing a virus-resistant avian cell comprising the step of overexpressing B4GALNT2 in the avian cell.
  22. 청구항 21에 있어서, 상기 B4GALNT2는 인간 B4GALNT2인 바이러스 저항성 조류 세포 제조 방법.The method of claim 21 , wherein the B4GALNT2 is human B4GALNT2.
  23. 청구항 21에 있어서, 상기 B4GALNT2의 과발현은 B4GALNT2를 코딩하는 유전자를 포함하는 벡터를 상기 조류 세포에 처리하여 수행되는 바이러스 저항성 조류 세포 제조 방법.The method of claim 21, wherein the overexpression of B4GALNT2 is performed by treating the avian cells with a vector including a gene encoding B4GALNT2.
  24. 청구항 21에 있어서, 상기 조류는 닭이고, 상기 바이러스는 조류 인플루엔자 바이러스인 바이러스 저항성 조류 세포 제조 방법.The method of claim 21 , wherein the avian is a chicken, and the virus is an avian influenza virus.
PCT/KR2022/006261 2021-04-30 2022-05-02 Composition for preparing avian cell for production of antiviral vaccine and composition for preparing virus-resistant avian cell WO2022231402A1 (en)

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