WO1998044093A1 - Novel dna vector and vaccines containing novel recombinant dna vectors as the active ingredient - Google Patents
Novel dna vector and vaccines containing novel recombinant dna vectors as the active ingredient Download PDFInfo
- Publication number
- WO1998044093A1 WO1998044093A1 PCT/JP1998/001358 JP9801358W WO9844093A1 WO 1998044093 A1 WO1998044093 A1 WO 1998044093A1 JP 9801358 W JP9801358 W JP 9801358W WO 9844093 A1 WO9844093 A1 WO 9844093A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna vector
- virus
- dna
- gene
- vector
- Prior art date
Links
- 108020004414 DNA Proteins 0.000 title claims abstract description 192
- 239000013598 vector Substances 0.000 title claims abstract description 189
- 108020004511 Recombinant DNA Proteins 0.000 title claims abstract description 62
- 229960005486 vaccine Drugs 0.000 title claims abstract description 54
- 239000004480 active ingredient Substances 0.000 title claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 106
- 241000700662 Fowlpox virus Species 0.000 claims abstract description 39
- 108700005075 Regulator Genes Proteins 0.000 claims abstract description 10
- 241000700605 Viruses Species 0.000 claims description 64
- 239000013612 plasmid Substances 0.000 claims description 63
- 238000000034 method Methods 0.000 claims description 37
- 241000588724 Escherichia coli Species 0.000 claims description 32
- 239000002773 nucleotide Substances 0.000 claims description 25
- 125000003729 nucleotide group Chemical group 0.000 claims description 25
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 22
- 208000015181 infectious disease Diseases 0.000 claims description 11
- 108700026244 Open Reading Frames Proteins 0.000 claims description 9
- 241000711404 Avian avulavirus 1 Species 0.000 claims description 8
- 230000002238 attenuated effect Effects 0.000 claims description 7
- 244000052769 pathogen Species 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 230000000890 antigenic effect Effects 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 101800003158 5 kDa peptide Proteins 0.000 claims description 4
- 108091023045 Untranslated Region Proteins 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000007918 intramuscular administration Methods 0.000 claims description 4
- 230000001717 pathogenic effect Effects 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 claims description 3
- 208000035473 Communicable disease Diseases 0.000 claims description 3
- 101710204837 Envelope small membrane protein Proteins 0.000 claims description 3
- 241000701047 Gallid alphaherpesvirus 2 Species 0.000 claims description 3
- 102000003886 Glycoproteins Human genes 0.000 claims description 3
- 108090000288 Glycoproteins Proteins 0.000 claims description 3
- 101710133291 Hemagglutinin-neuraminidase Proteins 0.000 claims description 3
- 241000710842 Japanese encephalitis virus Species 0.000 claims description 3
- 101710145006 Lysis protein Proteins 0.000 claims description 3
- 101710172711 Structural protein Proteins 0.000 claims description 3
- 230000010354 integration Effects 0.000 claims description 3
- 238000007912 intraperitoneal administration Methods 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 101500001532 Avian infectious bursal disease virus Capsid protein VP2 Proteins 0.000 claims 1
- 108091026890 Coding region Proteins 0.000 claims 1
- 238000007920 subcutaneous administration Methods 0.000 claims 1
- 230000003449 preventive effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 112
- 239000012634 fragment Substances 0.000 description 98
- 239000000243 solution Substances 0.000 description 45
- 239000002609 medium Substances 0.000 description 43
- 238000010276 construction Methods 0.000 description 35
- 108020004707 nucleic acids Proteins 0.000 description 23
- 102000039446 nucleic acids Human genes 0.000 description 23
- 150000007523 nucleic acids Chemical class 0.000 description 23
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 21
- 238000012869 ethanol precipitation Methods 0.000 description 21
- 230000014509 gene expression Effects 0.000 description 20
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 17
- 229960000723 ampicillin Drugs 0.000 description 17
- 238000005520 cutting process Methods 0.000 description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 102000003960 Ligases Human genes 0.000 description 15
- 108090000364 Ligases Proteins 0.000 description 15
- 238000000246 agarose gel electrophoresis Methods 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 14
- 108091008146 restriction endonucleases Proteins 0.000 description 14
- 229920001817 Agar Polymers 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 208000000666 Fowlpox Diseases 0.000 description 12
- 241000700618 Vaccinia virus Species 0.000 description 12
- 239000008272 agar Substances 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 239000011543 agarose gel Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 10
- 101150066555 lacZ gene Proteins 0.000 description 10
- 241001643084 Cyrtanthus elatus virus A Species 0.000 description 9
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 9
- 101150008820 HN gene Proteins 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 238000007796 conventional method Methods 0.000 description 9
- 241000287828 Gallus gallus Species 0.000 description 8
- 239000004744 fabric Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 7
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 7
- 101710163270 Nuclease Proteins 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- -1 5-bromo-4-chloro-3-indolyl Chemical group 0.000 description 6
- 210000004102 animal cell Anatomy 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 102000012410 DNA Ligases Human genes 0.000 description 5
- 108010061982 DNA Ligases Proteins 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 239000006142 Luria-Bertani Agar Substances 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 108010093031 Galactosidases Proteins 0.000 description 4
- 210000004748 cultured cell Anatomy 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 241000700663 Avipoxvirus Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 102000004594 DNA Polymerase I Human genes 0.000 description 3
- 108010017826 DNA Polymerase I Proteins 0.000 description 3
- 102000002464 Galactosidases Human genes 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108020003215 DNA Probes Proteins 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 241000702626 Infectious bursal disease virus Species 0.000 description 2
- 102100021244 Integral membrane protein GPR180 Human genes 0.000 description 2
- 101710081079 Minor spike protein H Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- LINMATFDVHBYOS-MBJXGIAVSA-N (2s,3r,4s,5r,6r)-2-[(5-bromo-1h-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C=C12 LINMATFDVHBYOS-MBJXGIAVSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 102100039819 Actin, alpha cardiac muscle 1 Human genes 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 240000000662 Anethum graveolens Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 201000006082 Chickenpox Diseases 0.000 description 1
- UNPLRYRWJLTVAE-UHFFFAOYSA-N Cloperastine hydrochloride Chemical compound Cl.C1=CC(Cl)=CC=C1C(C=1C=CC=CC=1)OCCN1CCCCC1 UNPLRYRWJLTVAE-UHFFFAOYSA-N 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 101000959247 Homo sapiens Actin, alpha cardiac muscle 1 Proteins 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 241000209027 Ilex aquifolium Species 0.000 description 1
- DIWRORZWFLOCLC-UHFFFAOYSA-N Lorazepam Chemical compound C12=CC(Cl)=CC=C2NC(=O)C(O)N=C1C1=CC=CC=C1Cl DIWRORZWFLOCLC-UHFFFAOYSA-N 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 208000010359 Newcastle Disease Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000700629 Orthopoxvirus Species 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 241000700646 Vaccinia virus WR Species 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 206010046980 Varicella Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000003711 chorioallantoic membrane Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 230000002431 foraging effect Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003067 hemagglutinative effect Effects 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- FRGKKTITADJNOE-UHFFFAOYSA-N sulfanyloxyethane Chemical compound CCOS FRGKKTITADJNOE-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- 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
- C12N15/86—Viral vectors
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24041—Use of virus, viral particle or viral elements as a vector
- C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a novel DNA vector, and more particularly, to a novel DNA vector useful as a vector having high expression in animal cells or a vaccine.
- Conventional technology a novel DNA vector useful as a vector having high expression in animal cells or a vaccine.
- Gene engineering is often aimed at introducing foreign genes into bacteria and cells for expression. In this case, it is not possible to separate and purify the DNA fragment containing the specific gene of interest and put it directly into bacteria or cells to increase it. DNA fragments cannot normally be replicated by themselves and may be degraded by various DNA degrading enzymes contained in cells.
- a target foreign gene is covalently linked to a carrier that can be transferred to a host cell and that can multiply autonomously in the host cell (this carrier is generally called a vector). Let them act together.
- Various vectors are known depending on the combination with the host cell. For example, when a microorganism such as Escherichia coli or yeast is used as a host, a DNA vector called a plasmid is generally used. There are many specific examples of this, such as PBR322 and pUC18, which are also described in detail in “Gene Engineering Experiments”, pp. 75-128 (Yasutaka Takagi, Kodansha Scientifiq).
- viruses are used as vectors depending on the purpose and the host cell.
- SV40 and poliovirus which have been used less recently because they are oncoviruses, but have been used since the late 1970s, or vaccinia virus, fowlpox virus, and baculo, which have been commonly used since the 1980s.
- viral vectors such as viruses and retroviruses.
- the disadvantages common to viral vectors that use animal cells as a host are: The point is that it takes time to produce the recombinant virus, and the expression level of the foreign gene is not always sufficient. Regarding the expression level, the viral vector has a much lower copy number (the ratio of the replicated vector per host cell) than the plasmid vector. The use of a strong promoter can improve the expression a little, but the expression level is large. The increase cannot be expected.
- the present inventors have made intensive studies to develop a new vector using an animal cell as a host which has overcome the above-mentioned drawbacks, and as a result, a novel DNA vector which gives an animal cell an extremely higher copy number than a conventional virus vector. They found one and completed the present invention. Furthermore, they have found a method of using the recombinant novel DNA vector as a vaccine, and have completed the present invention.
- the first embodiment of the present invention is a fowlpox virus-derived DNA vector of about 5 kb, which is replicated only in box virus-infected cells.
- the DNA vector is characterized in that it contains an inverted terminal repeat sequence containing a sequence obtained by repeating the nucleotide sequence of SEQ ID NO: 1 twice or more. Further, the DNA vector is characterized by containing a regulatory gene and at least three or more protein coding regions.
- a second aspect of the present invention is a recombinant DNA vector obtained by incorporating at least one or more foreign genes into the above DNA vector.
- the at least one foreign gene is under the control of a foreign regulatory gene.
- the integration site of the foreign gene is located in the untranslated region or the fourth codeic region.
- the foreign gene is preferably a gene encoding an antigenic protein of a pathogen.
- the gene encoding the antigenic protein of the pathogen may be a gene encoding the E protein of Japanese encephalitis virus, or an HN protein of the Newcastle disease virus.
- Gene encoding protein, gene encoding F protein, gene encoding glycoprotein gB of Marek's disease virus, and infectious bursal disease virus It is preferably a gene selected from the group consisting of the genes encoding the structural protein VP2 of luz.
- the regulatory gene may be a promoter of a vaccinia virus gene encoding a 7.5 kDa peptide or an 11K polypeptide or a variant thereof, a synthetic promoter having sequences of both an early promoter and a late promoter, and a base sequence described below. (SEQ ID NO: 5)
- the promoter is selected from the group consisting of a promoter having a DNA sequence represented by
- a third aspect of the present invention is a vaccine containing the above-mentioned recombinant DNA vector as an active ingredient.
- the vaccine of the present invention preferably contains the above-mentioned recombinant DNA vector and an attenuated box virus.
- a fourth aspect of the present invention is a method for transforming Escherichia coli with a plasmid containing a recombinant DNA vector containing a foreign gene, culturing the transformant to amplify the recombinant vector, And a step of introducing the vaccine into a box virus-infected cell to further amplify the vaccine.
- the recombinant DNA vector contains a foreign gene and DNA represented by the nucleotide sequence of SEQ ID NO: 2.
- a fifth aspect of the present invention is the use of the vaccine described above.
- a sixth aspect of the present invention is a method for preventing and / or treating infectious diseases, comprising infecting a recombinant DNA vector of lOng lig with a poxvirus selected from the group consisting of orthopoxvirus and fowlpox virus.
- a poxvirus selected from the group consisting of orthopoxvirus and fowlpox virus.
- the above vaccine containing 1 ⁇ 10 3 to 1 ⁇ 10 5 pfu of attenuated poxvirus and 10 ng to 1 ⁇ g of the recombinant DNA vector is orally administered, intradermally administered, subcutaneously administered, intravenously administered, It is preferable to administer by an administration route selected from the group consisting of intramuscular administration and intraperitoneal administration.
- a novel DNA vector that can be replicated only in a box virus-infected cell is provided, and (2) a recombinant DNA vector in which a foreign gene is inserted into the DNA vector is provided. And (3) the recombination A vaccine comprising a DNA vector as an active ingredient is provided.
- FIG. 1 shows the construction of the vectors pNZ66, pNZ76, and pNZ76 used in the examples.
- the 3.3 kb fragment of pMAOO1 cut with ⁇ anii I is ligated with pUC19 cut with BSIIM I to construct PNZ66. This is cut with T / '/ JcII, cut out from p-volume P-1 with pal I and £ cd l, and ligated with a DNA polymerase treated 7.5K promoter gene to construct PNZ76.
- pNZ76 is further digested with dll, and PNZ87 is constructed by ligating the ⁇ a; rfil- / II fragment obtained from mplO-HN18RF.
- Figure 2 shows the restriction map of the novel DNA vector and the positional relationship between the subcloned clones.
- the restriction enzyme cleavage site of about 5 kb of DNA obtained from the fowlpox virus Sashimi strain and the position of each fragment subcloned into commercially available pUC18 plasmid are shown.
- the subclones of the 0.6 kb, l. lkb, 711 of the coRI digestion fragment and the 2.4 kb fragment of the ⁇ al double digestion fragment are No. 17, No. 16, and No. 72-2, respectively.
- No. 20 and No. 22 are the subclones of the 1.3 kb and 2.0 kb fragments that appeared after digestion with coRI after DNA polymerase I treatment.
- No. 73-9 is a subclone containing a 1.53 ⁇ 4 fragment that appeared after double-cutting the .22 clone with 1 and 51.
- the 0.8kb and 0.9kb subclones that appeared after double-cutting the clone of No. 20 with Si and ⁇ ; ⁇ 1 ⁇ and Stu ⁇ hi Bgl ⁇ I were No. 73-10 and No. 73-12, respectively. It is.
- Figure 3 shows the construction method of pPADOl.
- FIG. 4 shows a method for constructing a plasmid pPADin (4541-4759) P7.5: 1 acZ containing a recombinant DNA vector.
- pPADOl was cut with coSlI / Spel to construct pPADdI (454-4922).
- pPADOl was cut with o81I / 5 "peI, and pPA DrepL (454-4922) was constructed using a synthetic DNA linker.
- PPADrepL (454-4922) was cut with // « II, and 4.
- a 5 kb fragment was obtained.
- P7.5: lacZ was constructed with a 3.3 kb fragment obtained by cutting 1 with //// 7dIII / 7; l.
- FIG. 5 shows a method for constructing a plasmid pPADin (377) P7.5: HN containing a recombinant DNA vector.
- PNZ87 Using a 1.9 kb fragment obtained by digesting with S1 nuclease and a fragment obtained by digesting pPADOl with ssHII and treating with S1 nuclease and BAP, pPADin (377) P7.5: HN Was built.
- FIG. 6 shows the construction of pNZ1029 used to obtain fNZ1029 used in the examples.
- the 7.3 kb fragment obtained by digesting the abipox virus with oRI is ligated with pUC18 digested with fcd and treated with alkaline phosphatase to construct pNZ133.
- This is partially digested with oRV, and the; -3 ⁇ 4 ⁇ fragment cut out from pNZ76 is treated with DNA polymerase and ligated to construct PNZ1029.
- FIG. 7 shows the construction of PNZ2237 used in the examples.
- a 7.3 kb fragment obtained by treating Avibox virus with £ coRI and pUC18 treated with £ coRI and alkaline phosphatase were ligated to construct pNZ136, which was partially digested with Hind111 and £ coRV. The fragment is ligated with PVC18 to obtain PNZ136S. This is further processed to construct pNZl36SL, treated with HindIII and BadlI, and ligated with a fragment containing HN obtained from pNZ87 or the like to construct PNZ2237.
- the DNA vector of the present invention (hereinafter, simply referred to as a DNA vector) is an approximately 5 kb DNA vector derived from fowlpox virus, but cannot replicate in box virus-uninfected cells, and does not replicate in box virus-infected cells. It is a concomitant DNA vector because it can
- This DNA vector is a linear DNA, and its sequence contains the nucleotide sequence of SEQ ID NO: 1 twice or more.
- the terminal structure of the vector of the present invention is such that the base sequence described in SEQ ID NO: 1 is defined as one unit, and the sequence includes this sequence two or more times, preferably three times. Then salt It is preferable that the base sequence is an inverted terminal repeat sequence (Inverted Terminal Repeat; hereinafter, it may be referred to as ITR).
- ITR Inverted Terminal Repeat
- the DNA vector of the present invention more preferably has the nucleotide sequence of SEQ ID NO: 2. That is, the adenine and thymidine content (AT content) of the present invention is high, and in addition to the above-mentioned inverted terminal repeat sequence, at least three protein coding regions (Open Reading Frame: hereinafter referred to as RF), promoters and other Those containing sequences are preferred.
- RF Open Reading Frame
- the AT content is preferably 60 to 65%.
- the DNA vector of the present invention preferably has at least three or more ORFs, and preferably has four ORFs.If there are four ORFs, it may have the following nucleotide sequence. preferable.
- 0RF1 is the 1,207-671st nucleotide sequence of SEQ ID NO: 2
- 0RF2 is also the 1,257-7,776th nucleotide sequence
- 0RF3 is the 1,806-6,397th nucleotide sequence
- 0RF4 is the nucleotide sequence at positions 4,991 to 4,422.
- the translation directions of 0RF1 and 0RF4 are opposite.
- a promoter is preferably present before the translation initiation codon of the ORF in order to translate and express the ORF.
- the promoter and other sequences include the vaccinia virus 7.5 kDa promoter, the vaccinia virus llkDa promoter, the synthetic promoter from Moss et al. (Moss et al., J. Mol. Biol., 215: 749-769 (1989)) and These variants are exemplified.
- the vector of the present invention having the above structure has a high replication efficiency in a box virus-infected cell, and the replication efficiency is about 30 times or more the copy number of a natural box virus. preferable.
- one or more bases in the base sequence are caused by a spontaneous mutation or the like. Mutations such as substitution / deletion / addition / insertion may be caused artificially by a known technique using a heterogenous substance or a restriction enzyme.
- 0RF4 the nucleotide sequence at positions 4,991 to 4,422 of SEQ ID NO: 2
- Various mutations such as deletion or integration of a foreign gene described below can be generated.
- the function of the vector of the present invention is not impaired, so that similar mutations can be caused in these regions.
- Examples of the mutation site in the untranslated region include a mutation at the 377th restriction enzyme II site of SEQ ID NO: 2.
- Such a DNA vector is a livestock hygiene strain of fowlpox virus Sashimi (strain number: VA0101 / Japan Association of Biologicals for Animals) and a fowlpox virus Nishig ahara strain (strain number: VA0104, same) It can be obtained from fowlpox virus strains that are readily available, such as the Japan Association for Aging.
- these preserved strains are infected with chicken embryo fibroblasts (CEF cells) according to a standard method, cultured at about 37 ° C for one week, and then the DNA present in the cytoplasm of the infected cells is recovered.
- the fraction may be obtained by electrophoresis or the like, and a DNA fragment of about 5 kb other than the fowlpox virus genomic DNA confirmed thereby may be obtained.
- the DNA vector of the present invention does not replicate when introduced into a box virus-uninfected cell, but replicates when introduced into a box virus-infected cell.
- the DNA vector of the present invention is preferably a vector containing the DNA sequence represented by the nucleotide sequence of SEQ ID NO: 2.
- box virus mentioned here examples include an orthobox virus represented by vaccinia virus and an avibox virus represented by fowlpox virus.
- avibox virus used in the present invention include fowlpox virus, ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nishigahara strain, Sashimi strain, and Examples include viruses such as the CEVA strain which is commercially available as a live fowlpox vaccine strain. These include the depository institutions such as the American Type Culture Collection. It is available as a commercial vaccine from Seki or from manufacturers such as Intervet In.
- box virus infected cells Cells artificially infected with these box viruses or cells naturally infected with these viruses are called box virus infected cells.
- aviboxvirus-susceptible infections include primary culture cells prepared from embryonated chicken eggs such as CEF cells and chorioallantoic membrane of embryonated chicken eggs.
- Vaccinia virus-infected cells include cultured cell lines derived from animals such as Vero cells and RK13 cells, and epidermal cells and muscle cells of animal individuals instead of cultured cells.
- the method of infecting these cells with the box virus may be a method of infecting cells with a normal virus. Specifically, for example, CEF cells are grown to subconfluence in a medium such as Eagle's MEM, D-MEM, and the like, and the medium is discarded and the box virus described above is m. Infect to be 1-10.
- the DNA vector of the present invention has an extremely high replication efficiency in box virus-infected cells, and has the advantage that, when a foreign gene is inserted into the DNA vector, the foreign gene is also replicated very efficiently. More specifically, the replication efficiency is about 30 times or more the number of copies of the genome of the Avibox virus strain, and more than 10,000 copies per cell.
- the DNA vector of the present invention is inserted into an appropriate vector in a conventional manner (Molecular Cloning; A Laboratory Manual (1982) to obtain a Cold Spring Harbor Laboratory recombinant vector. If one is used, it is possible to replicate the DNA vector (gene) even in a box virus-uninfected cell.
- the vector into which the DNA vector of the present invention is incorporated is arbitrarily selected depending on the purpose. be able to.
- examples of such vectors include plasmids such as pBR322, pBR325, pUC7, pUC8, pUC18, pUCBM-20, phages such as M13 phage, and cosmids such as pHC79.
- a modified vector in which a gene such as a gene encoding _____________________________________________________________________________________________ either of which is incorporated in these vectors may be used.
- These vectors may be treated with an appropriate restriction enzyme, and the DNA vector of the present invention may be incorporated according to a conventional method.
- the recombinant vector into which the DNA vector of the present invention has been incorporated can replicate various Escherichia coli and other bacteria such as TG1 and JM103 as hosts.
- the recombinant DNA vector of the present invention contains the DNA vector described in (1) above, a foreign gene, a promoter and other regulatory genes, and the like.
- PNZ76 is constructed as described in Izukahei No. 1-157381 (US Pat. No. 5,286,639) and JP-A No. 1-168279 (US Pat. No. 5,387,519). (refer graph1) .
- pMAOOKShirakawa et al., Gene, 28: 127- (1984)) is digested with a! I, extracted with phenol: chloroform, and recovered pMAOOl that has been cleaved by ethanol precipitation.
- the 5'-terminal phosphate is removed by alkaline phosphatase treatment, and the DNA is again extracted with phenol: chloroform. Then, about 3.3 kb of the ⁇ -galactosidase gene (/ acZ) is recovered by ethanol precipitation.
- PUC19 is digested with Sa! I, extracted with phenol: chloroform as described above, and recovered by ethanol precipitation.
- the recovered fragment from PUC19 and 7acZ are ligated with ligase to obtain plasmid pNZ66.
- M13-mplORF DNA manufactured by Amersham
- ⁇ ; zill and ⁇ al extraction with phenol: cloth form is performed, and the cleaved M13-mpl0 RF DNA is recovered by ethanol precipitation.
- an adapter consisting of the following nucleotide sequence (SEQ ID NO: 8) and consisting of a 40 bp oligodoxynucleotide partially having a single strand is chemically synthesized using Genet AIII (manufactured by Zeon Corporation).
- This adapter includes site, site,: 1 site, A3 ⁇ 4oI site, and Badll site cleavage sites.
- This adapter and the cleaved M13-mpl0 RF DNA are mixed, ligated with a ligase, and transduced into Escherichia coli, which is convenient according to a conventional method (Methods in Enzymology, vol. 101).
- This transformant is cultured on 2 XYT agar medium containing 5-bromo-4-chloro-3-indolyl / 3 / 3-D-galactopyranoside and isopropyl-3-indolyl-) 3-D-galactoviranoside.
- the phage RF DNA is recovered, digested with Bg! Ll, and phage mp10-mpa into which the adapter DNA is inserted is obtained by agarose electrophoresis.
- Plasmid XLIII-10H containing the NDV HN gene (Virus Research, 7: 241-255 (1977)) was distributed by Assistant Professor Masao Kawakita of the University of Tokyo. Digest XLin-lOH with Irall, extract by agarose gel electrophoresis, and recover the cleaved mplO-mpa by ethanol precipitation.
- the cleaved raplO-mpaRF DNA is mixed with ⁇ l all of the above HN gene DNA, the cohesive ends are blunt-ended with DNA polymerase, and the mixture is extracted with phenol: black form and then recovered by ethanol precipitation. .
- the recovered DNA is ligated with ligase, transduced into Escherichia coli as described above, and grown on 2 XYT agar medium.
- the phage RF DNA is recovered from the formed plaque, cut with ⁇ al and ftol, and mp10-HN180 containing about 1.8 kb of HN gene fragment is obtained by agarose gel electrophoresis. (2-3) Construction of pNZ87
- PNZ76 is digested with Badi I, and an approximately 2.9 kb fragment containing no ⁇ -galactosidase gene is recovered by agarose gel electrophoresis.
- the hybrid phage mpl 0-HN180 After that, a DNA fragment of about 1.8 kb of the HN gene is recovered by agarose gel electrophoresis. Both were ligated with ligase, transformed into E. coli, and plasmids were extracted according to the method of Birnboim and Dori (Nucleic Acid Research, 7: 1513- (1979)), and HN was extracted by agarose gel electrophoresis. The plasmid containing the gene, PNZ87, is obtained.
- CEF Chicken embryo fibroblasts
- No.16, No.17 and No.72-2 are obtained by obtaining fragments digested with Eco I and fragments digested with I and J ⁇ al and ligating them to pUC18XG (Fig. 1). ).
- the DNA vector of the present invention was treated with DNA polymerase, and the fragment cut with coRI was ligated to PUC18XG treated with 3 ⁇ 4 / aI and £ coRI to obtain subclones No. 20 and No. 22 is obtained.
- Subclones No.73-1, No.73-2 and No.73-3 are obtained by cutting No.22 with / «II II and ligating it to pUC18 treated with / ⁇ .
- the recombinant vector of the present invention is constructed as follows (see FIGS. 3 to 5).
- Subclone No. 20 was cut with BgJlI.
- the subclones No.22 and No.72-2 are cut with and £ a U, and these fragments are ligated to obtain a subclone No.72-22.
- pPADOl was obtained by ligating the fragment obtained by digesting No.72-22 with I and treating with BAP, and the ⁇ II fragment of subclone No.20 (Fig. 2).
- the pPADOl obtained in the above (4-1) was cut with co81I and Spel, treated with DNA polymerase to make the ends and ends, self-ligated to transform Escherichia coli, and selected to obtain pPADdl (454 to 4922). ) Can be obtained.
- PPADOl obtained in (4-1) above was cut with ⁇ : o81I and ⁇ 9el to synthesize SEQ ID NO: 4.
- Escherichia coli is transformed by ligating with an NA linker and, when selected, pPADrepl (454-4922) can be obtained.
- the pNZ87 constructed in (2) above was cleaved with the moieties' ⁇ and 1 and treated with S1 nuclease to recover the fragment. Also, the pPADOl constructed in (3) above is cut with 5 ⁇ , treated with S1 nuclease and BAP, ligated with the fragment from pNZ87 recovered earlier, and transformed into E. coli. If selected, pPADin (377) p7.5: HN can be obtained (Fig. 5).
- a foreign gene and a promoter which will be described later, can also be incorporated into the vector obtained as described above.
- fNZ1029 is constructed as described in JP-A-168279 (FIG. 6).
- PUC18 (Pharmacia) was digested with EcoRI and Hindlll, and extracted with phenol: chloroform, and PUC18 cleaved by ethanol precipitation was recovered. The 5'-terminal phosphoric acid is removed by alkaline phosphatase treatment, and the DNA is recovered again by extraction with phenol: chloroform and ethanol precipitation. The cleaved ⁇ 8 and the EcoRI fragment of the purified Aviboxvirus (strain) DNA were ligated with ligase, transformed into a competent Escherichia coli, and then treated with 5-bromo-4-chloro-3-. Incubate on LB agar medium containing indolyl 1 / 3-D-galactovyranoside, isopropyl 1 / 9-D-galactopyranoside and ampicillin.
- the white colonies grown on the agar medium were cultured in LB liquid medium containing ampicillin,
- the plasmid was extracted by the method of Birnboim and Doli (Nucleic Acid Research, 7: 1513- (1979)), digested with ⁇ 1 and // 3 ⁇ 41111, and then subjected to agarose gel electrophoresis to obtain the original Avibox virus DN.
- a hybrid plasmid having a fragment of the same length as the CORI_7 /// K1III fragment of A is detected, and this is designated as pNZ133.
- pNZ133 constructed in (5-1) was partially digested with oRV, extracted with phenol: cloth form, and recovered by ethanol precipitation.
- the cleaved DNA of pNZ133 was mixed with the above fragment (ligated fragment of the 7.5K promoter gene and 3-galactosidase gene), the cohesive end was made blunt with DNA polymerase, and phenol: cloth form was used. After extraction, recover by ethanol precipitation.
- the recovered DNA was ligated with ligase, transformed into E. coli, and grown on LB agar medium containing ampicillin.
- Plasmid was collected from the grown E. coli by the method of Birnboim and Doley (Nucleic Acid Research, 7: 1513- (1979)). Then, a hybrid plasmid containing a mono-galactosidase gene fragment was selected by agarose gel electrophoresis and named PNZ1029.
- PNZ1029 obtained as described above, a DNA fragment containing a 7.5K promoter gene and a / 3-galactosidase gene in avibox virus (Hatopox Nakano strain (also referred to as NP strain)) was prepared by a conventional method. To obtain fNZ1029 by homologous recombination.
- PNZ2237 The construction of PNZ2237 is performed as follows according to the description in JP-A No. 157381 (FIG. 7).
- PNZ87 is cut with ⁇ al and ////] dill, and 1 — /// ⁇ 11 fragment ( ⁇ ) is recovered. Similarly, ⁇ 87 is cleaved with 0al and rall, and the al— ⁇ rall fragment (B) is recovered. further, Cut PNZ87 with //// 7dIII and // rflll to obtain / ⁇ ⁇ ! ⁇ I — /// — rfl 11 fragment (C).
- plasmid was extracted by the method of Birnboim and Dawley (described above), digested with coRI and // 7dIII, and then agarose gel electrophoresed. By electrophoresis, a hybrid plasmid having a fragment of the same length as the AcoRI-Hindi II fragment of the original avipox virus DNA was detected and designated as pNZ136.
- the plasmid pNZ136S obtained in the above (6-2-2) is cut with a! I at / ⁇ , extracted with phenol: chloroform, and recovered by ethanol precipitation.
- pNZ87, pNZ87-22 and pNZ87-37 were respectively cleaved with ⁇ ⁇ ; and a; rfll, subjected to agarose gel electrophoresis, and containing a vaccinia virus 7.5K promoter gene and HN gene DNA //// ⁇ — Recover a / rfll fragments.
- PNZ2237 By ligating both with ligase and selecting as described above, PNZ2237 can be obtained.
- the foreign gene to be incorporated into the recombinant DNA vector of the present invention is not particularly limited.
- Preferred examples of such foreign genes include various enzyme proteins and antigenic proteins, structural genes encoding peptides, and regulatory genes that regulate gene expression.
- examples of the enzyme protein include a gene encoding / 3-galactosidase and a gene encoding an enzyme protein such as human tissue plasminogen (tPA).
- tPA human tissue plasminogen
- antigen gene examples include those derived from mammalian cells, mammalian infectious pathogens, bird cells, bird infectious pathogens, fish cells, fish pathogens, and the like.
- a gene encoding the E protein of Japanese encephalitis virus US Pat. No. 5,021,347 and a gene encoding the HN protein of Newcastle disease virus (Miller et al., J. Gen. Virol., ⁇ 2, 1917-). 1927 (1986)
- F protein McGinnes et al., Virus Res., 5343-5356 (1986)
- Marek's disease virus glycoprotein gB Rosss et al., J. Biol.
- antigen genes Genes encoding antigens involved in the protection of infection, such as the gene encoding the structural protein VP2 of infectious bursal disease virus (Bay liss et al., J. Gen. Virol., 1303-1321 (1990)) Hereinafter, these genes are simply referred to as antigen genes).
- the regulatory gene examples include a promoter of a vaccinia virus gene that encodes a 7.5 kDa peptide, a 11K polypeptide, and the like.
- these promoters It may be a modified product in which a part is deleted.
- synthetic promoters for example, synthetic promoters having both the early and late promoter sequences (J. Mol. Biol., 215, 749-769 (1989), ibid, 215, 771 781 (1989)) ) Or some of them are modified to the extent that promoter activity is not lost, deletions, modification or substitution of bases, for example, the base sequence is as follows (SEQ ID NO: 5)
- Regulated genes that regulate gene expression used in gene recombination techniques such as
- exogenous genes can be incorporated alone or, if necessary, in combination of two or more.
- the insertion site of these genes is not particularly limited, as long as the DNA vector of the present invention does not lose its ability to replicate in a box virus-infected cell.
- a region is preferably a non-translated region or a region of 0RF4, more preferably a 395th restriction enzyme site of SEQ ID NO: 2 or a 4,541 to 4,922th region.
- the method for producing a recombinant DNA vector containing the novel DNA vector of the present invention is not particularly limited.
- a recombinant plasmid is prepared by incorporating the DNA vector of the present invention into a plasmid, and incorporating a foreign gene into an insertable site in the DNA vector sequence according to a conventional method.
- This recombinant plasmid is introduced into an appropriate cell such as E. coli, and the recombinant plasmid is propagated and purified.
- a recombinant DNA vector containing a foreign gene from the recombinant plasmid with an appropriate restriction enzyme, for example, 1 is used.
- an appropriate restriction enzyme for example, 1
- the method for introducing the recombinant DNA vector and the box virus into cells is not particularly limited.
- Escherichia coli is transformed and grown in a recombinant vector in which the recombinant DNA vector obtained as described above is incorporated into an appropriate vector, and the recombinant vector is prepared in a large amount.
- the recombinant vector may be directly or cut out from the recombinant vector by a conventional method into a cell previously infected with a box virus by an electroporation method, a lipofection method, etc., and introduced.
- the method of infecting the cell with the box virus is not particularly limited, and may be performed by a known method such as contacting the virus with the cell at a desired m.o.i.
- a recombinant DNA vector containing an antigen gene as a foreign gene is introduced into cells infected with a live box virus strain of a box virus, and transferred from the cell. Produced by repeating purification.
- the antigen gene to be inserted is not particularly limited as described in (2) above.
- the box virus to be used is not particularly limited as long as it is a strain recognized as a live vaccine strain.
- a fowlpox live vaccine strain of avibox virus which is still widely used, is a preferred example.
- the selection method at the time of subculture / purification is not particularly limited.However, whether or not the foreign gene shows a posi signal by the hybridization method using the foreign gene inserted into the recombinant DNA vector as a probe, or whether the foreign gene It is preferable to use an antibody against the encoded antigen to confirm whether the antigen is expressed. Then, let the box virus plaques form and recombine in all the plaques It is more preferable to repeat the purification work of subculturing and selecting the selected plaques until the presence of the foreign gene inserted into the DNA vector or the expression of the antigen can be confirmed.
- the vaccine solution of the present invention can be obtained by using the preparation solution containing the recombinant DNA vector and the box virus obtained as described above in the same manner as in a method for preparing a normal box virus live vaccine.
- This preparation method is a general method and is not particularly limited.
- cells capable of proliferating the recombinant DNA vector of the present invention are simultaneously infected with a box virus, and the cells are cultured.
- the cells are collected, disrupted, and then centrifuged to obtain a high-titer recombinant DNA vector.
- a centrifugal supernatant containing box virus and a precipitate containing box virus and a precipitate.
- This centrifuged supernatant which is essentially free of host cells and no or debris thereof, but contains the cell culture medium, the recombinant DNA vector, and the box virus, can be used as the vaccine of the present invention.
- an attenuated box virus may be appropriately added to a preparation containing the recombinant DNA vector and box virus prepared as described above, and used as a vaccine.
- the attenuated box virus is not particularly limited, but an avibox virus, an orthobox virus and the like can be preferably used.
- Avipoxvirus include fowlpox virus: ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nisshi gahara strain, Sashimi strain And viruses such as the CEVA strain which is commercially available as a fowlpox vaccine strain.
- vaccinia viruses examples include Lister strain LC16mO strain, WR strain, and New York 'Board' ob 'health strain. These viruses can be attenuated in a variety of ways as needed, but the use of foul-box virus (FPV) is particularly preferred for increasing vaccine efficacy.
- FMV foul-box virus
- the supernatant can be stored as it is, or, if necessary, diluted or concentrated and frozen or lyophilized. When freeze-dried, it may be reconstituted with pharmacologically acceptable saline or distilled water before use.
- the vaccine of the present invention can be prepared by any method as long as the above-described recombinant DNA vector is expressed in an animal body and biosynthesizes an antigen protein encoded by a foreign gene. It may be administered by such a method.
- a vaccination can be made by scratching the skin and inoculating the target animal subcutaneously with a needle or other device. It is also possible to suspend the vaccine in the drinking water of the animal to be vaccinated or to orally administer the vaccine by mixing it with the solids of the feed.
- vaccines can be inhaled by aerosol or spray, intravenous inoculation, intramuscular inoculation, intraperitoneal inoculation, and the like.
- the amount of box virus is usually 10 3 to 10 6 pfu per bird (plaque forming unit) It is desirable that the amount of the recombinant DNA vector be 10 ng to 1 tg.
- I Li is preferably a Li 10 4 pfu per 1 birds.
- dilute with physiologically acceptable liquid such as saline and inoculate about 0.1 mL per bird.
- the vaccine of the present invention can be stored and used under the same conditions as ordinary poxvirus live vaccines. For example, if the vaccine of the present invention is lyophilized, it can be stored, handled, and transported at room temperature (about 20 to 22 ° C.) for a long time. In addition, the vaccine of the present invention can be stored by freezing the suspension at ⁇ 20 ° C. to ⁇ 70 ° C. Example
- fowlpox virus Vial One strain of fowlpox virus (Cul tures for Animal Hygiene) managed by the Japan Association of Veterinary Biologies (Cultures for Animal Hygiene); fowlpox A virus strain (strain number VA0101)) was transformed into chicken embryo fibroblasts cultured to subconfluence in a 1.5 cm diameter culture dish. 1 infected. After infection, and cultured for 1 week in 37 ° C in C0 2 incubator primary, disk Le - Remove the cells from the dish par, i collect peel cells, 000 x g, and centrifuged at 4 ° C 5 minutes.
- PBS physiological equilibration solution
- lysis buffer 1.25% Triton-X100, 250 mM 2-mercapto 0.8 mL of a PBS solution containing ethanol and 50 mM EDTA was added. After stirring with a vortex mixer, the mixture was left at room temperature for 5 minutes and centrifuged at 2,000 Xg for 5 minutes to remove cell debris.
- the supernatant was transferred to two Eppendorf tubes and centrifuged at 10,000 X g for 20 minutes at 4 ° C to collect a precipitate containing fowlpox virus.
- the separated aqueous layer was transferred to another Eppendorf tube by centrifugation at 20,000 Xg at 4 ° C for 2 minutes. To this, 16 L of 5M NaCl was added, and 1 mL of 100% ethanol cooled at -20 ° C was further added, and the mixture was left at -20 ° C for 30 minutes. Subsequently, DNA was precipitated by centrifugation at 4 ° C. and 20.000 ⁇ g for 10 minutes. The precipitate was washed with 70% ethanol and dried.
- This DNA was dissolved in an appropriate amount of a 10 mM Tris-HCl (pH 8.0) solution containing ImM EDTA (hereinafter referred to as a TE solution) and used.
- a 10 mM Tris-HCl (pH 8.0) solution containing ImM EDTA hereinafter referred to as a TE solution
- the above DNA was dissolved in a TE solution, and 0.6 g of the DNA was subjected to electrophoresis using a 0.8% agarose gel as a carrier, and electrophoresed at 100 V for 2 hours.
- the intensity of UV absorption (260 ⁇ ) of this 5 kbp DNA band on agarose gel was about half that of fowlpox virus genome DN ⁇ (about 300 kb). Therefore, despite the size of the genome being about 60: l (300kbp: 5kbp), the fact that the intensity of UV absorption is about half means that the number of copies of this 5kb DNA per cell is large. The copy number was estimated to be about 30 times that of fowlpox virus.
- the number of copies of this 5 kb DNA was 10,000 or more per cell, calculated from the amount of L //; 2din-cleaved DNA, the amount of sample DNA, and the number of cells from which the sample was prepared. Met.
- the plasmid vector used for subcloning was a plasmid pUCXG obtained by introducing one site into the 5 5 restriction site (hereinafter, the restriction site is called a site) of commercially available pUC18 plasmid using synthetic DNA. Using.
- the DNA solution (DNA concentration: 10 to 100 g / mL) prepared by the procedure of Example 1 was cleaved with 1 to 10 units / mL of restriction enzymes £ coRI, £ gJli, and J3 ⁇ 4al alone or in combination of two types.
- the double digestion of Bg I and? Al resulted in three fragments of 1.0 kb, 2.4 kb and 1.7 kb.
- Example 2 To subclone the region containing the ends, the 5 kb DNA (10 g) found in Example 1 was treated with DNA polymerase I (10 units / mL), cut with £ coRI, and cut into 1.3 kb DNA. A fragment and a 2.0 kb fragment were obtained. These two fragments, respectively Subcloning into pUC18XG plasmid double-cut with al and £ coRI yielded a plasmid into which the target DNA fragment had been inserted. These clones were named No. 20 and No. 22 (see Fig. 2).
- the .22 clone was cloned into 7 /// 2 (0.9410 that appeared by cutting at 1111), 0.5 kb, and 0.5 kb fragments from clones No. 73-1, No. 73-2, and No. 73-3, respectively. These were subcloned into PUC18 cut with.
- the 1.5 kb fragment that appeared when the No. 22 clone was double-cut with 1 and 2 was named No. 73-9, and was subcloned into PUC18XG double-cut with 3 ⁇ 4al and ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 4.
- nucleotide sequence of each subclone prepared in Example 2 was decoded using a nucleotide sequence analysis kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit) and an ABI fully automatic sequencer.
- Plasmid containing each subclone was used as a template, and P7 and P8 oligonucleotides commercially available from Toyobo Co., Ltd. were used as primers.
- the base sequence was similarly decoded by using a synthetic nucleotide created based on the sequence at the read position as a new primer.
- the sequences of these primers are shown in SEQ ID NOs: 6 and 7.
- the DNA had the following characteristics.
- the total length is 5,243 bp and the AT content is as high as 63.5 ° / 0 .
- a 217 bp inverted terminal repeat sequence is present at both ends, and the 30 bp repeated sequence represented by SEQ ID NO: 1 in these ITRs is repeated three times in tandem with no single base space.
- a consensus sequence TAAA T of the late transcription promoter of vaccinia virus exists upstream of the translation initiation codon of ⁇ RF.
- pPADOl plasmid containing the 5,243 bp full-length DNA was obtained by the procedure shown in FIG.
- the plasmid was constructed and named pPADOl. That is, No. 72-2 and No. 22 were double-cut with ⁇ 3 ⁇ 4al and a / dll respectively to recover DNA fragments of 5.lkb and 1.8kb, ligated, and ligated. 72-22 was built. .1 kb fragment obtained by cutting 20 with ⁇ 1 1 and 6.9 kb fragment obtained by cutting No. 72-22 with 711 and BAP (Bacterial Alkaline Phosphatase; manufactured by Takara Shuzo) The fragments were ligated to construct pPADOl.
- E. coli TG1 was transformed with PPDA01 according to a conventional method.
- Escherichia coli transformed with this pPADOl can be selected for its resistance to ampicillin, and can be easily prepared from Escherichia coli in the same manner as a commonly used plasmid such as PUC18. Furthermore, a linear DNA fragment of 5,243 bp can be cut out only by cutting pPADOl with the restriction enzyme ay / Ml.
- Escherichia coli transformed with pPADOl was cultured in LB medium containing ampicillin for 16 hours at 37 ° C for 16 hours, and pPADOl plasmid was prepared therefrom according to a conventional method. After cutting 5 g of this pPADOl brassmid with ⁇ , self-fry with T4 DNA ligase. A gated DNA was prepared in advance.
- the DNA vector of the present invention was not detected in CEF cells infected with the CEVA strain (approved by the US Department of Agriculture), one of the live fowlpox vaccine strains.
- the DNA fragmented from pPADOl and self-ligated was introduced by electroporation.
- MEM medium containing serum (5% CS) to stop trypsin digestion, and remove 50 mL of cells. Collected in a falcon tube. '1, 000 X g at centrifuged for 5 minutes, then discarding the centrifugal supernatant, Saline G buffer (8.0g NaCl, 0.4g KC1, 0.395g Na 2 HP0 4 ⁇ 12 ⁇ 2 0, 0.2g KH 2 P0 4, 0. lg MgCl 2 '6H 2 0, 0. lg CaCl 2 -6H 2 0, 1, the cells were washed with lg of glucose / L).
- the cells were suspended once again in 0.8 mL of Saline G, and 0.7 mL of the suspension was transferred to an election port cuvette (manufactured by Bio-Rad). To this, a previously prepared self-ligated DNA was added, and electrophoresis was performed under the conditions of 1.2 kV and 25 zF. Thereafter, the cells were allowed to stand at room temperature for 10 minutes, transferred to a 25 cm 2 flask, added with 5 mL of MEM medium, and cultured in a CO 2 incubator at 37 ° C. for 6 days.
- the culture was frozen and thawed with the flask. After three freeze-thaw cycles, the culture was transferred to a Falcon tube and centrifuged at 5,000 Xg for 5 minutes at 4 ° C to remove the cell debris and collect the supernatant. This solution contains the fowlpox virus CEVA strain and the replicated DNA vector.
- pPADO1 was double digested with the restriction enzymes £ co811 and Spel and blunt-ended with DNA polymerase I (10 units / mL) in reaction buffer. Thereafter, Escherichia coli TG1 was transformed by cyclization with T4 DNA ligase, and the end of the 7.5 kb plasmid fragment was cultured in an LB medium containing ampicillin.
- This plasmid is a plasmid containing a deletion of the DNA region from SEQ ID NO: 2 to positions 4,541 to 4,922, and was named pPADdl (4541-4922).
- pPADO 1 was double-cleaved with the restriction enzymes fcoS 11 and Spel, and a 7.5 kb plasmid fragment was recovered.
- the DNA sequence represented by SEQ ID NO: 3 was paired with a DNA fragment (SEQ ID NO: 4) forming a pair.
- SEQ ID NO: 4 was synthesized using T4DNA ligase and transformed into Escherichia coli.
- a plasmid was constructed in which the DNA region at positions 4,54 1 to 4,922 of SEQ ID NO: 2 of the obtained transformant was replaced with a synthetic adapter-1.
- This synthetic adapter has the base sequences of A3 ⁇ 4el and Bln Sal Xhod sites, which facilitate the introduction of foreign genes into these locations.
- This plasmid was named pP ADrepL (4541-4922).
- This transformant was treated with 0.03% of 5-bromo-4-cucto-3-indolyl- ⁇ -D-galactopyranoside, 0.03 mM of isopropyl- / 3-D-galactopyranoside, 40 g / mL.
- the cells were cultured on LB agar medium containing ampicillin for 15 hours at 37 ° C.
- white colonies were picked and cultured in LB liquid medium containing 40 g / mL ampicillin for 15 hours at 37 ° C.
- Transformants were recovered, plasmid was extracted according to the method of Birnboim and Dolly (Nucleic Acid Research, 7: 1513-, (1979)), and double digested with oRI and // ⁇ .
- the ⁇ -galactosidase gene (about 3.3 Kbp) was recovered from this double digested fragment by 0.6% agarose electrophoresis.
- 0.3 pUC19 was digested with ⁇ , extracted with phenol-chloroform (1: 1), and recovered by ethanol precipitation.
- the recovered PUC19 and the / 3-galactosidase gene were ligated with ligase to prepare recombinant plasmid PNZ66.
- the pNZ76 obtained as in (3-1) above was cut with ⁇ /; 7 II and Approximately 3kbp DNA fragment, which is linked to the 7.5K promoter gene of E. coli virus and the gene encoding / 3 galactosidase of E. coli immediately after, was excised and subjected to 0.8% agarose gel electrophoresis (100V, 40 minutes). Recovered from agarose gel. In addition, a 3.3 kb DNA fragment that appeared by cutting pPADOl with //// ⁇ I and a 4.5 kb DNA fragment that appeared by cutting pPADrepL (454-4922) with ZI were also recovered.
- the three recovered fragments were ligated with a T4 DNA ligase to transform E. coli TG1. From the ampicillin-resistant transformants that emerged by the transformation, a DNA fragment of about 3 kb containing the lacZ gene was inserted into the DNA region at positions 4,541 to 4,759 in SEQ ID NO: 2. Those having a plasmid of 10.8 kb were selected.
- This plasmid was named pPADin (4544544759) P7.5: lacZ.
- nucleotide sequence of 4,541 to 4,922 in SEQ ID NO: 32 was changed in exactly the same manner except that pPADrepL (4541-4922) constructed in (2) of Example 5 was used instead of pPADOl in Example 4. It was examined whether the recombinant DNA vector substituted with the bp multicloning site could not be introduced into cells.
- Vaccinia virus 7.5K promoter is located in the nucleotide sequence region of 4,541 to 4,759 of SEQ ID NO: 2.
- pPADin (4541-4759) P7.5 Escherichia coli TG1 transformed with lacZ was cultured in MEM at 37 ° C for 40 mL, and about 50 g of plasmid pPADin (4541-4759) P7.5: lacZ was obtained from the cells. It was prepared.
- CEF cells (1 ⁇ 10 7 cells) were collected by trypsinization, and suspended in 0.7 mL of Saline G as shown in Example 4.
- the culture solution was transferred to a 15 mL Falcon tube, and a preparation solution containing FPV from which cell debris had been removed and the recombinant DNA vector was collected by centrifugation at 5,000 X for 5 minutes.
- This solution was serially diluted four times in 10-fold MEM medium, and 1 mL of the solution was added to another CEF cell cultured at 37 ° C in a 9 cm diameter dish so that it became subconfluent. After discarding the supernatant later, a MEM medium containing 0.8% agar was overlaid and cultured for about 6 days.
- Buruogaru Halogenated indolyl- ⁇ -D-galactoside; GIBC0 BRL trade name and Bluo-ga further overlayed with 0.8% agar medium supplemented with, the 37 ° C C0 2 ink Yube - in ter After 3 hours, a number of blue plaques were found in the dish after three hours, indicating that -galactosidase was expressed.
- Example 6 The blue plaque obtained at the end of Example 6 was extracted with a pasteur pit, Suspend in 1 mL of MEM medium, serially dilute 10- to 1,000-fold, and add ME to a 9 cm-diameter dish.
- the cells were added to CEF cells (1 ⁇ 10 7 ) cultured in M medium, and after 1 hour, the supernatant was discarded. Thereafter, 0.8% agar medium was overlaid and cultured at 37 ° C for about 6 days.
- This suspension was added to CEF cells (1 ⁇ 10 6 ) cultured in MEM medium in a 1.5 cm dish. One hour later, the supernatant was discarded, and 20 mL of fresh MEM medium was added, followed by culturing for 1 week .
- Example 4 a preparation containing the fowlpox virus and the recombinant new DNA vector was recovered from the cultured cells by freeze-thawing. Then, the amount of fowlpox virus contained in this solution was measured.
- infected cells were collected from one dish, washed with PBS, and suspended in 0.5 mL of PBS. Later, the sample was frozen at -20 ° C.
- This reaction solution was incubated at 28 ° C and dissolved in an enzyme reaction solution to a concentration of 4 mg / mL.
- Substrate 0NPG (0-nitrophenyl / 3-D-galactopyranoside (Sigma; product number N1 127)) the enzymatic reaction was initiated by adding 0.2 mL, stop the enzymatic reaction in 1M K 2 C0 3 at appropriate time, the absorbance was measured at 420 nm.
- fNZ1029 is disclosed in Japanese Unexamined Patent Publication No. 168279 (US This is a recombinant fowlpox virus into which the / 3-galactosidase gene is inserted as disclosed in National Patent No. 5,387,519 (Fig. 6).
- 3-galactosidase (product number G6512) commercially available from Sigma was used as an enzyme standard.
- Nishigahara chicken pox virus strain (strain number VA0104) was obtained from the Cultures for Animal Hygiene strain managed by the Japanese Association of Veterinary Biologics. did.
- a 5 kb DNA was again obtained from infected cells by the same operation using this strain in place of the Sashimi strain of Example 1. This DNA was hybridized by Southern hybridization using a new DNA vector obtained from Sashimi strain as a probe. I did
- PNZ76 was digested with 5aH and electrophoresed on a 0.8% agarose gel to recover an approximately 2.9 kb fragment that did not contain the / 3-galactosidase gene.
- hybrid phage mplO-HN180 was double-digested with ⁇ 711 and electrophoresed on a 0.8% agarose gel to recover an approximately 1.8 kb NDV HN gene fragment.
- pPADOl was cleaved with ⁇ sfill, the cleaved end was blunted with SI nuclease, then the end was dephosphorylated with BAP, and phenol-treated. This treated pADOl was ligated with the previously recovered fragment of about 1.9 kb to transform E. coli TG1.
- plasmid having a total length of about 9.8 kb, in which a DNA fragment of about 1.9 kb containing the HN gene was inserted at position 377 of SEQ ID NO: 2 was selected. It was named pPADin (377) P7.5: HN.
- the plasmid pPAD in (377) P7.5 prepared in Example 9 was cultured at 37 ° C in 40 mL of an LB medium containing ampicillin at 37 ° C, and pPADin (377) P7.5: HN Was prepared. After cutting 5 ⁇ g of the plasmid with aI, DNA self-ligated with T4 DNA ligase was prepared in advance.
- the self-ligated DNA was dissolved in 50 L of HBS buffer (2.0 mM HEPES, 150 mM NaCl (pH 7.4)) after ethanol precipitation.
- solution A a solution obtained by diluting 10 L of the reagent DOSPER solution with 40 L of HBS buffer was used as solution B.
- solution A and solution B were mixed gently and allowed to stand at room temperature for 15 minutes. 100 L of this mixture was slightly dropped onto infected cells immersed in 0.5 mL of MEM medium culture solution. After 1 hour at 37 ° C, add 1.5 mL of culture solution and Cultured for days.
- the infected cells were collected together with the culture solution, and freeze-thawing was repeated three times, and then the supernatant was recovered by removing the cell debris by centrifugation at 5,000 xg for 5 minutes.
- This solution contains the fowlpox virus CEVA strain and the recombinant DNA vector.
- This solution was diluted 6 times 10 times, each concentration in the liquid lml were infected with CEF cells cultured in a 9 cm diameter dishes and overlaid with MEM medium lOmL C0 2 Inkyube containing 0.8% agar - C. in ter The cells were cultured at 37 ° C. About 6 days later, when plaques due to fowlpox virus were clearly visible, 24 plaque viruses were removed using pasteur pipets, and the viruses were infected with fresh CEF cells cultured on 24-well plates. I let it.
- the baked membrane was immersed in a pre-hybridization washing solution (5X SSC, 0.5% SDS, lmM EDTA) and washed at 42 ° C for 15 minutes.
- a pre-hybridization washing solution (5X SSC, 0.5% SDS, lmM EDTA)
- hybrida I See Chillon was allowed 1 hour (6X SSC, 0.5% SD S , 3% Sukimumiru h) and transferred to 68 ° C during the subsequent, HN gene radioisotopes monument - in 32 P -dCTP The solution was transferred to a hybridization solution to which a labeled DNA probe had been added, and left at 68 ° C. for 3 hours.
- the membrane was washed twice with 2 ⁇ SSC solution, once with 1 ⁇ SSC solution, once with 0.2 ⁇ SSC solution, air-dried, and subjected to autoradiography using X-ray film.
- the MEM medium containing 10 mL was overlaid and cultured at 37 ° C. in a 5% CO 2 incubator.
- plaques Once plaques have formed, remove the virus from the 24 blacks with Pasteur pits as before using Pasteur pits and remove the CEF cells from the 24-well plate. The vesicles were each infected. The same operation as above was repeated until all the spots of all the wells reacted with the DNA probe.
- a vaccine solution was prepared in the same manner as in the preparation of a fowlpox vaccine by culturing it in CEF cells.
- the vaccine solution thus prepared contained about 20 times as many recombinant DNA vectors as fowlpox virus vaccine strain DNA.
- Vaccinated solution prepared in Example 10 was diluted at a titer at 10 6 pfu appropriately by Uni becomes / ml PB S fowlpox virus, to the right wing membrane SPF chicken O.OlmL seven day old did.
- ND-HI antibody Hemagglutination-inhibiting antibody for Newcastle disease
- test serum was serially diluted 2-fold to 25; L, and 4 hemagglutinating units of NDV antigen (25 zL) were added thereto, mixed and allowed to stand for 10 minutes to sensitize. Thereafter, 50 L of a 0.5% chicken erythrocyte solution was added, and the mixture was allowed to stand at room temperature for 45 minutes, and red blood cell aggregation was observed.
- the ND-HI antibody titer was represented by the highest dilution of serum in which hemagglutination inhibition was observed.
- a novel DNA vector having a copy number several tens times that of a viral vector in animal cells, and a method for highly expressing a foreign gene using this vector are obtained. It can be used as a vector for use or as a vaccine.
- Sequence type nucleic acid
- Sequence type nucleic acid
- Sequence type nucleic acid
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
- Sequence type nucleic acid
- Sequence type other nucleic acid synthetic DNA
Abstract
A DNA vector of about 5 kb which originates in fowl pox virus; recombinant DNA vectors containing this DNA vector and at least one foreign gene under the regulation by a regulatory gene; and vaccines containing these vectors as the active ingredient. The above DNA vector of about 5 kb originating in fowl pox virus is a concomitant one which can grow not in non-pox virus-infected cells but exclusively in pox virus-infected cells. Because of having a large copy number per cell, this DNA vector makes it possible to provide vaccines with excellent preventive effects.
Description
明 細 書 Specification
新規 D N Aベクタ一、 及び組み換え新規 D N Aベクターを New DNA vector and recombinant new DNA vector
有効成分とするワクチン 産業上の利用分野 Vaccines as active ingredients Industrial applications
本発明は、 新規 D N Aベクターに関し、 さらに詳しくは動物細胞での高発現べ クタ一、 あるいはワクチンとして有用な新規 D N Aベクターに関する。 従来の技術 The present invention relates to a novel DNA vector, and more particularly, to a novel DNA vector useful as a vector having high expression in animal cells or a vaccine. Conventional technology
遺伝子工学においては、 外来遺伝子を細菌や細胞に導入して発現させることを 目的とする場合が多い。 この場合、 目的とする特定の遺伝子を含む D N A断片を 分離精製し、 それをそのまま細菌や細胞に入れて増やすことはできない。 D N A 断片は、 通常、 自ら複製することはできないし、 細胞に含まれる種々の D N A分 解酵素で分解される恐れがあるからである。 Gene engineering is often aimed at introducing foreign genes into bacteria and cells for expression. In this case, it is not possible to separate and purify the DNA fragment containing the specific gene of interest and put it directly into bacteria or cells to increase it. DNA fragments cannot normally be replicated by themselves and may be degraded by various DNA degrading enzymes contained in cells.
したがって、 遺伝子操作実験においては、 宿主細胞に移入でき、 且つ宿主細胞 中で自律的に増殖できる運搬体 （一般にこの運搬体はべクタ一と呼ばれる） に、 目的の外来遺伝子を共有結合で結びつけて一緒に行動させる。 ベクタ一は、 宿主 細胞との組み合わせに応じて様々なものが知られている。 例えば、 大腸菌や酵母 などの微生物を宿主とする場合では、 プラスミ ドと呼ばれる D N Aベクターがー 般に使用される。 この具体例としては、 PBR322や pUC 18など多くのものがあり、 『遺伝子操作実験法』 75頁〜 128頁 （高木康敬 著、 講談社サイェンティフイク） にも詳しく記載されている。 Therefore, in a genetic engineering experiment, a target foreign gene is covalently linked to a carrier that can be transferred to a host cell and that can multiply autonomously in the host cell (this carrier is generally called a vector). Let them act together. Various vectors are known depending on the combination with the host cell. For example, when a microorganism such as Escherichia coli or yeast is used as a host, a DNA vector called a plasmid is generally used. There are many specific examples of this, such as PBR322 and pUC18, which are also described in detail in “Gene Engineering Experiments”, pp. 75-128 (Yasutaka Takagi, Kodansha Scientifiq).
一方、 動物細胞などを宿主とする場合にも、 目的や宿主細胞に応じて様々なゥ ィルスがベクターとして用いられている。 例えば、 腫瘍ウィルスであるために最 近ではあまり使われていないが、 1970年代後半から使用されてきた SV40やポリォ —マウィルス、 あるいは 1980年代以降よく使われてきた、 ワクシニアウィルス、 鶏痘ウィルス、 バキュロウィルス、 レトロウイルスなどのウィルスベクターがあ る。 On the other hand, when an animal cell or the like is used as a host, various viruses are used as vectors depending on the purpose and the host cell. For example, SV40 and poliovirus, which have been used less recently because they are oncoviruses, but have been used since the late 1970s, or vaccinia virus, fowlpox virus, and baculo, which have been commonly used since the 1980s. There are viral vectors such as viruses and retroviruses.
しかしながら、 動物細胞を宿主とするウィルスベクターに共通する欠点は、 組
み換えウィルスを作製するのに手間がかかる点と必ずしも外来遺伝子の発現量が 十分でない点である。 発現量に関しては、 ウィルスベクタ一はプラスミ ドベクタ —に比べてコピー数 （宿主細胞当たりの複製されたべクタ一の割合） がかなり低 く、 強いプロモーターの利用で多少の改善はできるものの大幅な発現量の増加は 期待できない。 However, the disadvantages common to viral vectors that use animal cells as a host are: The point is that it takes time to produce the recombinant virus, and the expression level of the foreign gene is not always sufficient. Regarding the expression level, the viral vector has a much lower copy number (the ratio of the replicated vector per host cell) than the plasmid vector. The use of a strong promoter can improve the expression a little, but the expression level is large. The increase cannot be expected.
このため、 発現量の多いベクタ一が常にもとめられているのが現状であった。 発明の概要 For this reason, at present, vectors with high expression levels are always being sought. Summary of the Invention
本発明者らは、 前記の欠点を克服した動物細胞を宿主とする新しいベクタ一を 開発すべく鋭意研究した結果、 動物細胞において、 従来のウィルスベクターより も極めて高いコピー数を与える新規な D N Aベクタ一を見出し、 本発明を完成す るに到った。 また更に、 組み換え新規 D N Aベクタ一をワクチンとして利用する 方法を見出し、 本発明を完成するに到った。 The present inventors have made intensive studies to develop a new vector using an animal cell as a host which has overcome the above-mentioned drawbacks, and as a result, a novel DNA vector which gives an animal cell an extremely higher copy number than a conventional virus vector. They found one and completed the present invention. Furthermore, they have found a method of using the recombinant novel DNA vector as a vaccine, and have completed the present invention.
すなわち、 本発明の第一の態様は、 ボックスウィルス感染細胞のみで複製され る、 鶏痘ウィルス由来の約 5 kbの D N Aベクタ一である。 ここで、 上記 D N Aベ クタ一は、 配列番号 1記載の塩基配列を 2回以上繰返した配列を含む逆位末端反 復配列を含むことを特徴とする。 また、 上記 D N Aベクタ一は、 調節遺伝子と、 少なくとも 3以上の蛋白質のコ一ディング領域とを含むことを特徴とする。 That is, the first embodiment of the present invention is a fowlpox virus-derived DNA vector of about 5 kb, which is replicated only in box virus-infected cells. Here, the DNA vector is characterized in that it contains an inverted terminal repeat sequence containing a sequence obtained by repeating the nucleotide sequence of SEQ ID NO: 1 twice or more. Further, the DNA vector is characterized by containing a regulatory gene and at least three or more protein coding regions.
また、 配列番号 2記載の塩基配列からなる D N Aベクタ一である。 Also, it is a DNA vector consisting of the nucleotide sequence of SEQ ID NO: 2.
本発明の第二の態様は、 少なくとも 1以上の外来遺伝子を上記の D N Aベクタ —に組み込んでなる組み換え D N Aベクタ一である。 ここで、 上記少なくとも 1 以上の外来遺伝子は、 外来の調節遺伝子の制御下にあることを特徴とする。 また、 上記外来遺伝子の組み込み部位が、 非翻訳領域又は第 4番目のコ一ディシグ領域 内にあることを特徴とする。 A second aspect of the present invention is a recombinant DNA vector obtained by incorporating at least one or more foreign genes into the above DNA vector. Here, the at least one foreign gene is under the control of a foreign regulatory gene. Further, the integration site of the foreign gene is located in the untranslated region or the fourth codeic region.
上記外来遺伝子は、 病原体の抗原タンパク質をコ一ドする遺伝子であることが 好ましく、 上記病原体の抗原タンパク質をコードする遺伝子が、 日本脳炎ウィル スの Eタンパク質をコードする遺伝子、 ニューカッスル病ウィルスの H Nタンパ ク質をコードする遺伝子、 Fタンパク質をコードする遺伝子、 マレック病ウィル スの糖タンパク質 g Bをコードする遺伝子、 及び伝染性ファブリキウス嚢病ウイ
ルスの構造タンパク VP2をコードする遺伝子からなる群から選ばれる遺伝子であ ることが好ましい。 The foreign gene is preferably a gene encoding an antigenic protein of a pathogen. The gene encoding the antigenic protein of the pathogen may be a gene encoding the E protein of Japanese encephalitis virus, or an HN protein of the Newcastle disease virus. Gene encoding protein, gene encoding F protein, gene encoding glycoprotein gB of Marek's disease virus, and infectious bursal disease virus It is preferably a gene selected from the group consisting of the genes encoding the structural protein VP2 of luz.
また、 上記調節遺伝子が、 7.5kDaペプチドもしくは 11Kポリペプチドをコード するワクシニアウイルス遺伝子のプロモータ一またはこれらの改変体、 初期プロ モーターと後期プロモーターの両方の配列を有する合成プロモータ一、 および下 記塩基配列 （配列番号 5) Further, the regulatory gene may be a promoter of a vaccinia virus gene encoding a 7.5 kDa peptide or an 11K polypeptide or a variant thereof, a synthetic promoter having sequences of both an early promoter and a late promoter, and a base sequence described below. (SEQ ID NO: 5)
CTATTCTAATTTATTGCACTC-3' CTATTCTAATTTATTGCACTC-3 '
で表される DN A配列を有するプロモータ一からなる群から選ばれるプロモータ —であることが好ましい。 It is preferable that the promoter is selected from the group consisting of a promoter having a DNA sequence represented by
本発明の第三の態様は、 上記の組み換え DN Aベクターを有効成分とするワク チンである。 ここで、 本発明のワクチンは、 上記の組み換え DNAベクタ一と弱 毒ボックスウィルスとを含有していることが好ましい。 A third aspect of the present invention is a vaccine containing the above-mentioned recombinant DNA vector as an active ingredient. Here, the vaccine of the present invention preferably contains the above-mentioned recombinant DNA vector and an attenuated box virus.
本発明の第四の態様は、 外来遺伝子を含む組み換え DNAベクタ一を含むブラ スミ ドで大腸菌を形質転換し、 当該形質転換体を培養して組み換えベクターを増 幅する工程と、 前記組み換えべクタ一をボックスウィルス感染細胞に導入してさ らに増幅させる工程とを含む上記ワクチンの調製方法である。 A fourth aspect of the present invention is a method for transforming Escherichia coli with a plasmid containing a recombinant DNA vector containing a foreign gene, culturing the transformant to amplify the recombinant vector, And a step of introducing the vaccine into a box virus-infected cell to further amplify the vaccine.
ここで、 上記組み換え DNAベクターが、 外来遺伝子と配列番号 2記載の塩基 配列で表される DNAとを含有してなるものであることが好ましい。 Here, it is preferable that the recombinant DNA vector contains a foreign gene and DNA represented by the nucleotide sequence of SEQ ID NO: 2.
本発明の第五の態様は、 上述したワクチンの使用である。 A fifth aspect of the present invention is the use of the vaccine described above.
本発明の第六の態様は、 lOng l igの組み換え DNAベクターを、 オルソポッ クスウィルスおよび鶏痘ウイルスからなる群から選ばれるポックスウィルスと共 に感染させる、 感染症の予防及び Z又は治療方法である。 ここで、 1 X103〜 1 X 105pfuの弱毒ポックスウィルスと 10ng〜 1 β gの組み換え DNAベクタ一とを含 む上記のワクチンを、 経口投与、 皮内投与、 皮下投与、 静脈内投与、 筋肉内投与、 および腹腔内投与からなる群から選ばれる投与経路で投与することが好ましい。 かくして本発明によれば、 （ 1 ) ボックスウィルス感染細胞のみで複製するこ とができる新規 DNAベクターが提供され、 また （2) 当該 DNAベクタ一に外 来遺伝子を挿入した組み換え DNAベクタ一が提供され、 更に （3) 当該組み換
え DN Aベクタ一を有効成分とするワクチンが提供される。 図面の簡単な説明 A sixth aspect of the present invention is a method for preventing and / or treating infectious diseases, comprising infecting a recombinant DNA vector of lOng lig with a poxvirus selected from the group consisting of orthopoxvirus and fowlpox virus. . Here, the above vaccine containing 1 × 10 3 to 1 × 10 5 pfu of attenuated poxvirus and 10 ng to 1β g of the recombinant DNA vector is orally administered, intradermally administered, subcutaneously administered, intravenously administered, It is preferable to administer by an administration route selected from the group consisting of intramuscular administration and intraperitoneal administration. Thus, according to the present invention, (1) a novel DNA vector that can be replicated only in a box virus-infected cell is provided, and (2) a recombinant DNA vector in which a foreign gene is inserted into the DNA vector is provided. And (3) the recombination A vaccine comprising a DNA vector as an active ingredient is provided. BRIEF DESCRIPTION OF THE FIGURES
図 1に、 実施例で使用するべクタ一 pNZ66、 pNZ76、 及び pNZ76の構築を示す。 β anii Iで切断した pMAOO 1の 3.3kb断片を BSIIM Iで切断した pUC 19をライゲ一シヨンし て PNZ66を構築する。 これを T/'/JcIIで切断し、 p冊 P - 1から pal Iと £cd lとで切り 出し DNAポリメラーゼ処理した 7.5Kプロモータ一遺伝子とをライゲーションし、 PNZ76を構築する。 pNZ76をさらに dllで切断し、 mplO- HN18RFから得た^ a;rfil- /II断片とをライゲ一シヨンさせて PNZ87を構築する。 FIG. 1 shows the construction of the vectors pNZ66, pNZ76, and pNZ76 used in the examples. The 3.3 kb fragment of pMAOO1 cut with β anii I is ligated with pUC19 cut with BSIIM I to construct PNZ66. This is cut with T / '/ JcII, cut out from p-volume P-1 with pal I and £ cd l, and ligated with a DNA polymerase treated 7.5K promoter gene to construct PNZ76. pNZ76 is further digested with dll, and PNZ87 is constructed by ligating the ^ a; rfil- / II fragment obtained from mplO-HN18RF.
図 2に、 新規 DN Aベクタ一の制限酵素地図とサブクローニングされた各クロ ーンの位置関係を示す。 ここで、 鶏痘ウィルス泗水株から得た約 5kbの DN Aの 制限酵素切断部位と、 市販の pUC18プラスミ ドにサブクロ一ニングした各断片の 位置とを示す。 coRI切断断片の 0.6kb、 l. lkb、 711と^ al二重切断断片の 2.4k bの断片のサブクローンは、 それぞれ No.17、 No.16、 No.72- 2である。 DNAポリ メラ一ゼ I処理した後、 coRIで切断して出現した 1.3kbと 2.0kbの断片のサブク ローンが No .20および No.22である。 No.22のサブクローンを Ιί】·ηά 111で切断して出 現した 0.94kb、 0.5kb、 0.5kbのサブクローンが No.73-1、 No.73-2、 及び No.73 - 3 である。 .22のクロ一ンを 1と5 1とでニ重切断して出現した1.5¾の断片を 含むサブクローンが No.73-9である。 No.20のクローンを Si と^ ;χ1ΙΠ、 Stu\ヒ B gl\ Iとでそれぞれ二重切断して出現した 0.8kb、 0.9kbのサブクロ一ンが No.73-10、 No.73-12である。 Figure 2 shows the restriction map of the novel DNA vector and the positional relationship between the subcloned clones. Here, the restriction enzyme cleavage site of about 5 kb of DNA obtained from the fowlpox virus Sashimi strain and the position of each fragment subcloned into commercially available pUC18 plasmid are shown. The subclones of the 0.6 kb, l. lkb, 711 of the coRI digestion fragment and the 2.4 kb fragment of the ^ al double digestion fragment are No. 17, No. 16, and No. 72-2, respectively. No. 20 and No. 22 are the subclones of the 1.3 kb and 2.0 kb fragments that appeared after digestion with coRI after DNA polymerase I treatment. The 0.94 kb, 0.5 kb, and 0.5 kb subclones, which were obtained by cutting the No. 22 subclone with Ιί] η111, were used for No.73-1, No.73-2, and No.73-3. is there. No. 73-9 is a subclone containing a 1.5¾ fragment that appeared after double-cutting the .22 clone with 1 and 51. The 0.8kb and 0.9kb subclones that appeared after double-cutting the clone of No. 20 with Si and ^; χ1ΙΠ and Stu \ hi Bgl \ I were No. 73-10 and No. 73-12, respectively. It is.
図 3に、 pPADOlの構築方法を示す。 No.20、 No.22、 No.72-2の 3つのクロ一ン をそれぞれ含む PUC18XGプラスミ ドを使って、 図 2に示す手順で 5，243bp全長 DN Aを含むプラスミ ドを構築し、 このプラスミ ドを pPADOlと命名した。 Figure 3 shows the construction method of pPADOl. Using PUC18XG plasmid containing each of the three clones No. 20, No. 22, and No. 72-2, a plasmid containing a 5,243 bp full-length DNA was constructed according to the procedure shown in FIG. The plasmid was named pPADOl.
図 4に、 組み換え DNAベクターを含むプラスミ ド pPADin(4541— 4759)P7.5:1 acZの構築方法を示す。 pPADOlを coSlI/Spelで切断して pPADdI(454卜 4922)を構 築した。 また、 pPADOlを o81I/5"peIで切断し、 合成 DN Aリンカ一を用いて pPA DrepL(454卜 4922)を構築した。 pPADrepL(454卜 4922)を//«II で切断し、 4. FIG. 4 shows a method for constructing a plasmid pPADin (4541-4759) P7.5: 1 acZ containing a recombinant DNA vector. pPADOl was cut with coSlI / Spel to construct pPADdI (454-4922). In addition, pPADOl was cut with o81I / 5 "peI, and pPA DrepL (454-4922) was constructed using a synthetic DNA linker. PPADrepL (454-4922) was cut with //« II, and 4.
5kbの断片を得た。 pNZ76を //fldlll/Sa/Iで切断して得た 3kbの断片、 および pPADO
1を//// 7dIII/ 7; lで切断して得た 3.3kbの断片とともに、 pPADin(4541-4759)P7.5: lacZを構築した。 A 5 kb fragment was obtained. 3 kb fragment obtained by cutting pNZ76 with // fldlll / Sa / I, and pPADO PPADin (4541-4759) P7.5: lacZ was constructed with a 3.3 kb fragment obtained by cutting 1 with //// 7dIII / 7; l.
図 5に、 組み換え DNAベクタ一を含むプラスミ ド pPADin(377)P7.5:HNの構築 方法を示す。 PNZ87を
で切断し、 S1ヌクレアーゼで処理して得た 1. 9kbの断片と、 pPADOlを ssHIIで切断し、 S1ヌクレア一ゼと BAPで処理した断片と を用いて、 pPADin(377)P7.5:HNを構築した。 FIG. 5 shows a method for constructing a plasmid pPADin (377) P7.5: HN containing a recombinant DNA vector. PNZ87 Using a 1.9 kb fragment obtained by digesting with S1 nuclease and a fragment obtained by digesting pPADOl with ssHII and treating with S1 nuclease and BAP, pPADin (377) P7.5: HN Was built.
図 6に、実施例で使用する fNZ 1029を得るために使用する pNZ 1029の構築を示す。 ァビポックスウィルスを oRIで切断して得た 7.3kbの断片と、 fcd で切断し次 いでアル力リホスファタ一ゼで処理した pUC18とをライゲーションして pNZ133を 構築する。 これを oRVで部分消化し、 pNZ76から切り出した^; ¾ΠΠ - 断片 を DNAポリメラーゼで処理し、 ライゲ一ションして PNZ1029を構築する。 FIG. 6 shows the construction of pNZ1029 used to obtain fNZ1029 used in the examples. The 7.3 kb fragment obtained by digesting the abipox virus with oRI is ligated with pUC18 digested with fcd and treated with alkaline phosphatase to construct pNZ133. This is partially digested with oRV, and the; -¾ΠΠ fragment cut out from pNZ76 is treated with DNA polymerase and ligated to construct PNZ1029.
図 7に、 実施例で使用する PNZ2237の構築を示す。 アビボックスウィルスを £co RIで処理して得た 7.3kbの断片と、 £coRIとアルカリフォスファターゼで処理した pUC 18とをライゲーシヨンして pNZ 136を構築し、 Hind 111と £coRVとで部分消化し た断片を PVC18とライゲ一シヨンして PNZ136Sを得る。 これをさらに処理して pNZl 36SLを構築し Hind II Iと Badl Iとで処理し、 pNZ87等から得た HNを含む断片とライ ゲ一シヨンして PNZ2237を構築する。 発明の好適な実施態様 FIG. 7 shows the construction of PNZ2237 used in the examples. A 7.3 kb fragment obtained by treating Avibox virus with £ coRI and pUC18 treated with £ coRI and alkaline phosphatase were ligated to construct pNZ136, which was partially digested with Hind111 and £ coRV. The fragment is ligated with PVC18 to obtain PNZ136S. This is further processed to construct pNZl36SL, treated with HindIII and BadlI, and ligated with a fragment containing HN obtained from pNZ87 or the like to construct PNZ2237. Preferred embodiments of the invention
以下に、 本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
( 1 ) DNAベクター (1) DNA vector
本発明の DNAベクター （以下、 単に DNAベクターという） は、 鶏痘ウィル スに由来する約 5kbの DNAベクタ一であるが、 ボックスウィルス未感染細胞で は複製できず、 ボックスウイルス感染細胞では 0複製することができることから 随伴性 DN Aベクタ一である。 The DNA vector of the present invention (hereinafter, simply referred to as a DNA vector) is an approximately 5 kb DNA vector derived from fowlpox virus, but cannot replicate in box virus-uninfected cells, and does not replicate in box virus-infected cells. It is a concomitant DNA vector because it can
この DN Aベクタ一は、 直鎖状 DN Aであり、 その配列中に配列番号 1記載の 塩基配列を 2回以上含むものである。 This DNA vector is a linear DNA, and its sequence contains the nucleotide sequence of SEQ ID NO: 1 twice or more.
ここで本発明のべクタ一の末端構造は、 配列番号 1記載の塩基配列を一単位と し、 この配列を連続して 2回以上、 好ましくは 3回含む力 5'側と 3'側とでは塩
基配列が逆向きになっている、 逆位末端反復配列（Inverted Terminal Repeat; 以下、 I TRということがある）となっていることが好ましい。 Here, the terminal structure of the vector of the present invention is such that the base sequence described in SEQ ID NO: 1 is defined as one unit, and the sequence includes this sequence two or more times, preferably three times. Then salt It is preferable that the base sequence is an inverted terminal repeat sequence (Inverted Terminal Repeat; hereinafter, it may be referred to as ITR).
本発明の DNAベクターは、 より好ましくは、 配列番号 2記載の塩基配列を有 するものである。 すなわち、 本発明のアデニン及びチミジン含量 （AT含量） が 高く、 上記の逆位末端反復配列以外に、 少なくとも 3つのタンパク質コ一ディン グ領域 （Open Reading Frame:以下〇 R Fという） 、 及びプロモーターその他の 配列を含むものが好適である。 The DNA vector of the present invention more preferably has the nucleotide sequence of SEQ ID NO: 2. That is, the adenine and thymidine content (AT content) of the present invention is high, and in addition to the above-mentioned inverted terminal repeat sequence, at least three protein coding regions (Open Reading Frame: hereinafter referred to as RF), promoters and other Those containing sequences are preferred.
ここで、 AT含量は 60〜65%であることが好ましい。 本発明の DNAベクタ一 においては少なくとも 3つ以上の OR Fが存在し、 4つの OR Fを有することが 好ましく、 ORFが 4つ存在する場合には、 以下の塩基配列を有するものである ことが好ましい。 具体的には、 0RF1は、 配列番号 2の塩基配列の第 1，207〜671番 目、 0RF2は同じく第 1，257〜1，776番目、 0RF3は同じく第 1， 806〜4, 397番目、 0RF4 は同じく第 4， 991〜4， 422番目の塩基配列である。 ここで、 0RF1および 0RF4の翻訳 方向は逆向きである。 Here, the AT content is preferably 60 to 65%. The DNA vector of the present invention preferably has at least three or more ORFs, and preferably has four ORFs.If there are four ORFs, it may have the following nucleotide sequence. preferable. Specifically, 0RF1 is the 1,207-671st nucleotide sequence of SEQ ID NO: 2, 0RF2 is also the 1,257-7,776th nucleotide sequence, 0RF3 is the 1,806-6,397th nucleotide sequence, 0RF4 is the nucleotide sequence at positions 4,991 to 4,422. Here, the translation directions of 0RF1 and 0RF4 are opposite.
本発明の DNAベクターにおいては、 上記 4つの ORFのうち、 0RF1〜0RF3の いずれかの OR Fがー部でも欠損するとこのべクタ一 DN Aは増殖できなくなる が、 0RF4については一部欠損又はこの ORF自体が欠損しても DNAベクタ一の 増殖には影響しない。 In the DNA vector of the present invention, if any one of ORFs of 0RF1 to 0RF3 among the above four ORFs is deleted even in the-part, the vector cannot be proliferated. Deletion of the ORF itself does not affect the growth of the DNA vector.
また、 ORFの翻訳開始コドンの前には、 ORFが翻訳されて発現するために、 それぞれプロモーターが存在することが好ましい。 In addition, a promoter is preferably present before the translation initiation codon of the ORF in order to translate and express the ORF.
プロモーターその他の配列としては、 ワクシニアウィルスの 7.5kDaプロモータ 一、 ワクシニアウィルスの llkDaプロモーター、 Mossらの合成プロモータ一（Moss et al., J. Mol. Biol. , 215:749- 769(1989))やこれらの改変体などが挙げられ る。 The promoter and other sequences include the vaccinia virus 7.5 kDa promoter, the vaccinia virus llkDa promoter, the synthetic promoter from Moss et al. (Moss et al., J. Mol. Biol., 215: 749-769 (1989)) and These variants are exemplified.
上記のような構造を有する本発明のベクタ一は、 ボックスウィルス感染細胞中 で高い複製効率を有するものであり、 その複製効率は、 天然のボックスウィルス のコピー数の約 30倍以上であることが好ましい。 The vector of the present invention having the above structure has a high replication efficiency in a box virus-infected cell, and the replication efficiency is about 30 times or more the copy number of a natural box virus. preferable.
本発明の DN Aベクターにおいては、 上記の複製効率を維持できるものであれ ば、 その塩基配列中で、 1以上の塩基が自然発生的な突然変異等によりまたは変
異原物質や制限酵素を用いる公知の技術によリ人工的に、 置換 ·欠失 ·付加 ·挿 入などの変異が生じていてもよい。 In the DNA vector of the present invention, as long as the replication efficiency can be maintained, one or more bases in the base sequence are caused by a spontaneous mutation or the like. Mutations such as substitution / deletion / addition / insertion may be caused artificially by a known technique using a heterogenous substance or a restriction enzyme.
特に、 上述したように 0RF4(配列番号 2の第 4,991〜4，422番目の塩基配列） で は、 その一部欠損などが生じた場合でもベクターとしての機能は損なわれないた め、 0RF4中に欠損を生じさせたり、後述する外来遺伝子等を組み込むといった種々 の変異を生じさせることができる。 また、 非翻訳領域において上記のような変異 が生じても本発明のベクタ一としての機能は損なわれないため、 これらの領域に も同様の変異を生じさせることができる。非翻訳領域における変異部位としては、 例えば、 配列番号 2の第 377番目の制限酵素 ^ «II部位の変異などが挙げられる。 このような DN Aベクタ一は、 家畜衛生菌株である鶏痘ウィルス泗水株 （菌株 番号： VA0101/社団法人動物用生物学的製剤協会） 、 同じく鶏痘ウィルス Nishig ahara株 （菌株番号： VA0104,同協会）などの入手容易な鶏痘ウィルス株から取得 することができる。 In particular, as described above, 0RF4 (the nucleotide sequence at positions 4,991 to 4,422 of SEQ ID NO: 2) does not impair the function as a vector even if a partial deletion occurs. Various mutations such as deletion or integration of a foreign gene described below can be generated. Further, even if the above-mentioned mutations occur in the untranslated regions, the function of the vector of the present invention is not impaired, so that similar mutations can be caused in these regions. Examples of the mutation site in the untranslated region include a mutation at the 377th restriction enzyme II site of SEQ ID NO: 2. Such a DNA vector is a livestock hygiene strain of fowlpox virus Sashimi (strain number: VA0101 / Japan Association of Biologicals for Animals) and a fowlpox virus Nishig ahara strain (strain number: VA0104, same) It can be obtained from fowlpox virus strains that are readily available, such as the Japan Association for Aging.
すなわち、 これらの保存株を鶏胚繊維芽細胞 （CEF細胞） に常法に従って感 染させ、 約 37°Cで 1週間培養し、 その後、 感染細胞の細胞質に存在する DNAを 回収し、 ァガロースゲル電気泳動等によって分画し、 これによつて確認される鶏 痘ウィルスのゲノム DN A以外の約 5kbの DN A断片を取得すればよい。 That is, these preserved strains are infected with chicken embryo fibroblasts (CEF cells) according to a standard method, cultured at about 37 ° C for one week, and then the DNA present in the cytoplasm of the infected cells is recovered. The fraction may be obtained by electrophoresis or the like, and a DNA fragment of about 5 kb other than the fowlpox virus genomic DNA confirmed thereby may be obtained.
本発明の DN Aベクターは、 ボックスウィルス未感染細胞に導入しても複製さ れないが、 ボックスウィルス感染細胞に導入した場合には複製される、 随伴性の ものである。 The DNA vector of the present invention does not replicate when introduced into a box virus-uninfected cell, but replicates when introduced into a box virus-infected cell.
本発明の DN Aベクターとしては、 具体的には、 配列番号 2に記載の塩基配列 で表される DN Aを配列含むものが好ましい。 Specifically, the DNA vector of the present invention is preferably a vector containing the DNA sequence represented by the nucleotide sequence of SEQ ID NO: 2.
ここでいうボックスウィルスとしては、 ワクシニアウィルスなどに代表される オルソボックスウイルスゃ鶏痘ウィルスなどに代表されるアビボックスウイルス などが挙げられる。 本発明で用いるアビボックスウィルスの具体例としては、 鶏 痘ウィルスでは、 ATCC VR- 251、 ATCC VR- 250、 ATCC VR- 229、 ATCC VR- 249、 ATCC VR- 288、 Nishigahara株、 泗水株、 及び鶏痘生ワクチン株として市販されている CEVA株などのウィルスが例示される。 これらは、 寄託機関であるアメリカン - タ ィプ · カルチヤ一 · コレクション（American Type Culture Col lection)などの機
関から、 あるいは Intervet In などの製造元から市販ワクチンとして入手可能 である。 Examples of the box virus mentioned here include an orthobox virus represented by vaccinia virus and an avibox virus represented by fowlpox virus. Specific examples of the avibox virus used in the present invention include fowlpox virus, ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nishigahara strain, Sashimi strain, and Examples include viruses such as the CEVA strain which is commercially available as a live fowlpox vaccine strain. These include the depository institutions such as the American Type Culture Collection. It is available as a commercial vaccine from Seki or from manufacturers such as Intervet In.
これらのボックスウイルスを人工的に感染させた細胞、 またはこれらのウィル スに自然に感染した細胞をボックスウィルス感染細胞という。 Cells artificially infected with these box viruses or cells naturally infected with these viruses are called box virus infected cells.
ボックスウィルスが感染しやすい細胞としては、 以下のものが挙げられる。 例 えば、 アビボックスウィルスが感染しやすい例としては、 C E F細胞のような発 育鶏卵から調製される初代培養細胞や発育鶏卵の漿尿膜などが挙げられる。また、 ワクシニアウィルスが感染しゃすい細胞としては、 Vero細胞や RK13細胞などの動 物由来の株化培養細胞、 あるいは、 培養細胞ではなく動物個体の表皮細胞や筋肉 細胞などが挙げられる。 Cells that are susceptible to box virus infection include the following. For example, examples of aviboxvirus-susceptible infections include primary culture cells prepared from embryonated chicken eggs such as CEF cells and chorioallantoic membrane of embryonated chicken eggs. Vaccinia virus-infected cells include cultured cell lines derived from animals such as Vero cells and RK13 cells, and epidermal cells and muscle cells of animal individuals instead of cultured cells.
これらの細胞に前記ボックスウィルスを感染させる方法は、 通常のウィルスを 細胞に感染させる方法でよい。 具体的には、 例えば、 Eagl e' s MEM、 D- MEM等の培 地中で C E F細胞をサブコンフレントまで増殖させ、 培地を捨てて上記のボック スウィルスを m. 0. i . =0. 1〜10となるように感染させる。 The method of infecting these cells with the box virus may be a method of infecting cells with a normal virus. Specifically, for example, CEF cells are grown to subconfluence in a medium such as Eagle's MEM, D-MEM, and the like, and the medium is discarded and the box virus described above is m. Infect to be 1-10.
例えば、 本発明の D N Aベクターとして配列番号 2に表される D N A配列をを 用いる場合には、 C E F細胞を MEM培地中でサブコンフレントまで増殖させ、 こ こに上記のァビポックスウィルスを m. 0. i . =0. 5で感染させ、 感染数時間後に本発 明の D N Aベクタ一を培養細胞に加えて増殖させればよい。 For example, when the DNA sequence represented by SEQ ID NO: 2 is used as the DNA vector of the present invention, CEF cells are grown to subconfluence in a MEM medium, and the above avipox virus is m. Infection is carried out at 0. i. = 0.5, and several hours after infection, the DNA vector of the present invention may be added to cultured cells and propagated.
本発明の D N Aベクターは、 ボックスウイルス感染細胞内での複製効率が極め て高く、 当該 D N Aベクタ一に外来遺伝子を揷入すると外来遺伝子も極めて効率 よく複製されるという利点がある。 具体的には、 その複製効率は、 アビボックス ゥィルス泗水株ゲノムのコピ一数の約 30倍以上、 細胞あたリに換算して 10, 000コ ピ一以上である。 The DNA vector of the present invention has an extremely high replication efficiency in box virus-infected cells, and has the advantage that, when a foreign gene is inserted into the DNA vector, the foreign gene is also replicated very efficiently. More specifically, the replication efficiency is about 30 times or more the number of copies of the genome of the Avibox virus strain, and more than 10,000 copies per cell.
本発明の D N Aベクタ一を適当なベクタ一に常法に従って揷入して（Mo lecular Cloning; A Laboratory Manual ( 1982) , Cold Spring Harbor Laboratory 組み 換えべクタ一を得ることができ、 この組み換えべクタ一を用いれば、 ボックスゥ ィルス非感染細胞であっても当該 D N Aベクタ一 （遺伝子） を複製させることが 可能である。 The DNA vector of the present invention is inserted into an appropriate vector in a conventional manner (Molecular Cloning; A Laboratory Manual (1982) to obtain a Cold Spring Harbor Laboratory recombinant vector. If one is used, it is possible to replicate the DNA vector (gene) even in a box virus-uninfected cell.
本発明の D N Aベクターを組み込むベクタ一は、 目的に応じて任意に選択する
ことができる。 このようなベクターとしては、 例えば、 pBR322、 pBR325、 pUC7、 pUC8、 pUC18、 pUCBM- 20等のプラスミ ド、 M13ファージなどのファ一ジ、 pHC79等 のコスミ ドが挙げられる。 必要に応じて、 これらのベクタ一に _ガラクトシダ ーゼをコードする遺伝子などのマ一力一として用いられる遺伝子等を組み込んだ 改変べクタ一も用いることができる。 これらのベクタ一を適当な制限酵素で処理 し、 常法に従って本発明の DN Aベクタ一を組み込めばよい。 The vector into which the DNA vector of the present invention is incorporated is arbitrarily selected depending on the purpose. be able to. Examples of such vectors include plasmids such as pBR322, pBR325, pUC7, pUC8, pUC18, pUCBM-20, phages such as M13 phage, and cosmids such as pHC79. If necessary, a modified vector in which a gene such as a gene encoding ___________________________________________________________________________________________________________ either of which is incorporated in these vectors may be used. These vectors may be treated with an appropriate restriction enzyme, and the DNA vector of the present invention may be incorporated according to a conventional method.
本発明の DN Aベクタ一を組み込んだ組み換えべクタ一は、 TG1、 JM103などの 各種大腸菌その他の細菌を宿主として複製させることが可能である。 The recombinant vector into which the DNA vector of the present invention has been incorporated can replicate various Escherichia coli and other bacteria such as TG1 and JM103 as hosts.
(2) 組み換え DNAベクタ一 (2) Recombinant DNA vector
本発明の組み換え DNAベクターは、 上記 （ 1 ) で説明した DNAベクタ一、 外来遺伝子、 およびプロモーターその他の調節遺伝子等を含むものである。 The recombinant DNA vector of the present invention contains the DNA vector described in (1) above, a foreign gene, a promoter and other regulatory genes, and the like.
以下に、 本発明の実施例で使用した各べクタ一の構築について説明する。 (2 - 1 ) pNZ76の構築 Hereinafter, the construction of each vector used in the embodiment of the present invention will be described. (2-1) Construction of pNZ76
PNZ76は、 出開平第 1-157381号公報（米国特許第 5， 286, 639号）及び特開平第 1-16 8279号公報（米国特許第 5， 387， 519号） に記載したように構築する （図 1参照） 。 PNZ76 is constructed as described in Izukahei No. 1-157381 (US Pat. No. 5,286,639) and JP-A No. 1-168279 (US Pat. No. 5,387,519). (refer graph1) .
( 2 - 1 - 1 ) pUWP-Ιの作製 (2-1-1) Preparation of pUWP-Ι
ヮクシニアゥィルス 株の 7, 5KDaぺプチドをコードする DN Aのプロモーター を含む、 約 0.26kbの バ- 断片（Cell, 125:805- 813(1981))を pUC9の 5a7卜 5¾3 I部分に組み込んだプラスミ ド pUWP- 1が得られる。 An approximately 0.26 kb fragment (DNA, 125: 805-813 (1981)) containing the promoter of the DNA encoding the 7,5 kDa peptide of the vaccinia virus strain (Cell, 125: 805-813 (1981)) was added to the 5 53I portion of pUC9. The integrated plasmid pUWP-1 is obtained.
(2 - 1 - 2) pNZ66の構築 (2-1-2) Construction of pNZ66
pMAOOKShirakawa et al.， Gene, 28: 127- (1984))を a !Iで消化し、 フエノー ル： クロ口ホルムで抽出し、 エタノール沈殿にょリ開裂した pMAOOlを回収する。 5'-末端のリン酸をアルカリフォスファターゼ処理によって除去し、 DNAを再 びフエノール： クロ口ホルム抽出した後に、 エタノール沈殿によって、 約 3.3kb の^一ガラクトシダ一ゼ遺伝子（/acZ)を回収する。 pMAOOKShirakawa et al., Gene, 28: 127- (1984)) is digested with a! I, extracted with phenol: chloroform, and recovered pMAOOl that has been cleaved by ethanol precipitation. The 5'-terminal phosphate is removed by alkaline phosphatase treatment, and the DNA is again extracted with phenol: chloroform. Then, about 3.3 kb of the ^ -galactosidase gene (/ acZ) is recovered by ethanol precipitation.
一方、 PUC19を Sa !Iで消化し、 上記同様にフエノール： クロ口ホルム抽出し、 エタノール沈殿によリ回収する。 この PUC19からの回収断片と 7acZとをリガーゼ によってライゲーションさせ、 プラスミ ド pNZ66を得る。 On the other hand, PUC19 is digested with Sa! I, extracted with phenol: chloroform as described above, and recovered by ethanol precipitation. The recovered fragment from PUC19 and 7acZ are ligated with ligase to obtain plasmid pNZ66.
( 2 - 1 - 3 ) pNZ76の構築
pUWP- 1を Hpal Iと coRIとで消化し、 低融点ァガロース電気泳動にょリ 7.5Kプロ モーターを含む断片を分離する。 上記と同様にして、 付着末端を有する DNA断 片を回収する。 この DNA断片の付着末端を DNAポリメラーゼによって平滑末 端とする。 (2-1-3) Construction of pNZ76 Digest pUWP-1 with Hpal I and coRI and isolate the fragment containing the 7.5K promoter by low melting point agarose gel electrophoresis. In the same manner as described above, DNA fragments having cohesive ends are recovered. The cohesive end of this DNA fragment is made blunt by DNA polymerase.
PNZ66を Λ τκΙΙで消化し、 フエノール： クロ口ホルム抽出し、 エタノール沈殿 によって回収する。 この回収した DN Aと平滑末端とした 7.5Kプロモータ一を含 む上記の断片とをリガーゼによってライゲ一シヨンして、 PNZ76を得る。 Digest PNZ66 with ττκΙΙ, extract with phenol: chloroform, and recover by ethanol precipitation. The recovered DNA and the above-mentioned fragment containing the 7.5K promoter which has been blunt-ended are ligated with ligase to obtain PNZ76.
(2 - 2) pNZ87の構築 (2-2) Construction of pNZ87
( 2 - 2 - 1 ) mplO- mpaの構築 (2-2-1) Construction of mplO-mpa
M13- mplORFDNA (Amersham社製） を^; zillと^ alとで消化した後、 フエノール： クロ口ホルムで抽出し、 エタノール沈殿により、 開裂された M13-mpl0 RF DNAを 回収する。 After digestion of M13-mplORF DNA (manufactured by Amersham) with ^; zill and ^ al, extraction with phenol: cloth form is performed, and the cleaved M13-mpl0 RF DNA is recovered by ethanol precipitation.
一方、 下記の塩基配列 （配列番号 8) からなリ、 一部一本鎖を有する 40bpのォ リゴデォキシヌクレオチドからなるアダプタ一を Genet AIII (日本ゼオン製） を 用いて化学合成する。 On the other hand, an adapter consisting of the following nucleotide sequence (SEQ ID NO: 8) and consisting of a 40 bp oligodoxynucleotide partially having a single strand is chemically synthesized using Genet AIII (manufactured by Zeon Corporation).
CTAGATCTAAGGAGGAAAAAATTATGGTACCTCGAGCTCG CTAGATCTAAGGAGGAAAAAATTATGGTACCTCGAGCTCG
TAGATTCCTCCTTTTTTAATACCATGGAGCTCGAGCCTAG (配列番号 8 ) このアダプタ一には、 サイ ト、 サイ ト、 :1サイト、 A¾oIサイ ト、 B adllサイトの各切断部位が含まれている。 TAGATTCCTCCTTTTTTAATACCATGGAGCTCGAGCCTAG (SEQ ID NO: 8) This adapter includes site, site,: 1 site, A¾oI site, and Badll site cleavage sites.
このアダプタ一と、 上記開裂された M13-mpl0 RF DNAとを混合し、 リガ一ゼで ライゲーシヨンし、 常法（Methods in Enzymology, vol. 101)に従って、 コンビ テントな大腸菌に形質導入する。 この形質転換体を、 5-ブロモ -4-クロ口- 3-イン ドリル -/3 - D-ガラクトピラノシド、 ィソプロピル- 3-ィンドリル-) 3 - D-ガラクト ビラノシド含有 2 XYT寒天培地で培養し、 ファージ RF DNAを回収し、 Bg!llで 切断して、 ァガロース電気泳動によりアダプタ一 DN Aが揷入されたファージ mp 10 - mpaを得る。 This adapter and the cleaved M13-mpl0 RF DNA are mixed, ligated with a ligase, and transduced into Escherichia coli, which is convenient according to a conventional method (Methods in Enzymology, vol. 101). This transformant is cultured on 2 XYT agar medium containing 5-bromo-4-chloro-3-indolyl / 3 / 3-D-galactopyranoside and isopropyl-3-indolyl-) 3-D-galactoviranoside. Then, the phage RF DNA is recovered, digested with Bg! Ll, and phage mp10-mpa into which the adapter DNA is inserted is obtained by agarose electrophoresis.
( 2— 2 - 2 ) mplO- HN180の構築 (2-2-2) Construction of mplO-HN180
NDVの HN遺伝子を含むプラスミ ド XLIII- 10H(Virus Research, 7:241-255(198 7))を東京大学教養学部の川喜多正夫助教授よリ分与された。
XLin-lOHを Irallで消化し、 ァガロースゲル電気泳動より抽出し、 エタノール 沈殿により、 開裂された mplO- mpaを回収する。 Plasmid XLIII-10H containing the NDV HN gene (Virus Research, 7: 241-255 (1977)) was distributed by Assistant Professor Masao Kawakita of the University of Tokyo. Digest XLin-lOH with Irall, extract by agarose gel electrophoresis, and recover the cleaved mplO-mpa by ethanol precipitation.
上記 （ 2— 2— 1 ) で作製したハイブリッドファ一ジ mplO-mpaRF DNAを p lで 消化した後、 フエノール： クロ口ホルムで抽出し、 エタノール沈殿により回収す る。 After digesting the hybrid phage mplO-mpaRF DNA prepared in (2-2-1) with pl, extract with phenol: cloth form and recover by ethanol precipitation.
開裂された raplO-mpaRF DNAと、 上記 HN遺伝子 DN Aの ^l allとを混合し、 DN Aポリメラーゼで付着末端を平滑末端とし、 フエノール： クロ口ホルムで抽出し た後、 エタノール沈殿により回収する。 回収された DN Aをリガーゼでライゲ一 シヨンし、 上記と同様にコンビテントな大腸菌に形質導入し、 2 XYT寒天培地 で生育させる。 The cleaved raplO-mpaRF DNA is mixed with ^ l all of the above HN gene DNA, the cohesive ends are blunt-ended with DNA polymerase, and the mixture is extracted with phenol: black form and then recovered by ethanol precipitation. . The recovered DNA is ligated with ligase, transduced into Escherichia coli as described above, and grown on 2 XYT agar medium.
形成されたプラークからファージ RF DNAを回収し、 ^alと ftolとで切断し、 ァ ガロースゲル電気泳動により、 H N遺伝子断片約 1.8kbを含む mp 10-HN180を得る。 (2 - 3) pNZ87の構築 The phage RF DNA is recovered from the formed plaque, cut with ^ al and ftol, and mp10-HN180 containing about 1.8 kb of HN gene fragment is obtained by agarose gel electrophoresis. (2-3) Construction of pNZ87
PNZ76を Badi Iで消化し、 ァガロースゲル電気泳動により、 ^—ガラクトシダ一 ゼ遺伝子を含まない約 2.9kbの断片を回収する。 一方、 ハイブリッドファージ mpl 0- HN180を
とで消化した後、 ァガロースゲル電気泳動によリ約 1.8kb の HN遺伝子の DNA断片を回収する。 両者をリガーゼによりライゲーシヨンし、 コンビテントな大腸菌を形質転換し、 ビルンボイムとドーリ一の方法（Nuc leic A cid Research, 7:1513-(1979))に従ってプラスミ ドを抽出し、 ァガロースゲル電 気泳動によって HN遺伝子を含むプラスミ ド PNZ87を得る。 PNZ76 is digested with Badi I, and an approximately 2.9 kb fragment containing no ^ -galactosidase gene is recovered by agarose gel electrophoresis. On the other hand, the hybrid phage mpl 0-HN180 After that, a DNA fragment of about 1.8 kb of the HN gene is recovered by agarose gel electrophoresis. Both were ligated with ligase, transformed into E. coli, and plasmids were extracted according to the method of Birnboim and Dori (Nucleic Acid Research, 7: 1513- (1979)), and HN was extracted by agarose gel electrophoresis. The plasmid containing the gene, PNZ87, is obtained.
(3) 本発明の DN Aベクタ一からのサブクローンの構築 (3) Construction of a subclone from the DNA vector of the present invention
(3— 1 ) 本発明の DNAベクターの精製 (3-1) Purification of the DNA vector of the present invention
サブコンフレントまで培養した鶏胚繊維芽細胞 （以下、 CEFということがあ る） に、 アビボックスウィルスを感染させ、 一定時間増幅させた後に細胞をはが し、 遠心して集め、 リシスバッファーによって細胞を溶解させ、 再度遠心して沈 殿を得る。 フエノール： クロ口ホルム抽出し、 エタノール沈殿させて本発明の D N Aベクタ一を得る。 Chicken embryo fibroblasts (hereinafter sometimes referred to as CEF) cultured to subconfluence are infected with avibox virus, amplified for a certain period of time, peeled, collected by centrifugation, and lysed with lysis buffer. Dissolve and centrifuge again to obtain a precipitate. Phenol: Extracted with black-mouthed form and precipitated with ethanol to obtain the DNA vector of the present invention.
上記のようにして得た本発明の DN Aベクターを、 各種の制限酵素で切断し、 以下のサブクローンを得る （図 2及び図 3参照） 。
( 3— 2) サブクローン No.16、 No.17、 No.72- 2の構築 The DNA vector of the present invention obtained as described above is cleaved with various restriction enzymes to obtain the following subclones (see FIGS. 2 and 3). (3-2) Construction of subclone No.16, No.17, No.72-2
Eco Iによる切断断片及び Iと J^alとによる切断断片を得て、 pUC 18XGにラ ィゲ一シヨンすることにより、 No.16、 No.17、 No.72- 2が得られる （図 1 ) 。 No.16, No.17 and No.72-2 are obtained by obtaining fragments digested with Eco I and fragments digested with I and J ^ al and ligating them to pUC18XG (Fig. 1). ).
( 3— 3) サブクローン No.20、 No.22、 No.73-1、 No.73-2、 No.73-3、 No.73-9、 No.73- 10及び No.73 - 12の構築 (3-3) Subclone No.20, No.22, No.73-1, No.73-2, No.73-3, No.73-9, No.73-10 and No.73-12 Building
本発明の DN Aベクタ一を DN Aポリメラ一ゼで処理し、 coRIで切断した断 片を、 ¾/aIと £coRIとで処理した PUC18XGにライゲーシヨンして、 サブクロ一ン No. 20と No.22とが得られる。 The DNA vector of the present invention was treated with DNA polymerase, and the fragment cut with coRI was ligated to PUC18XG treated with ¾ / aI and £ coRI to obtain subclones No. 20 and No. 22 is obtained.
No.22を /«II IIで切断し、 /πΠΙΙで処理した pUC18にライゲ一シヨンすること により、 サブクロ一ン No.73-1、 No.73-2、 No.73- 3が得られる。 Subclones No.73-1, No.73-2 and No.73-3 are obtained by cutting No.22 with / «II II and ligating it to pUC18 treated with / πΠΙΙ.
？を^^と ^とで切断し、 pUC18にライゲ一シヨンすることにより、 サブ クローン No.73-9が得られる。 ? Is cut with ^^ and ^ and ligated to pUC18 to obtain subclone No. 73-9.
.20を5 1と Hind I IIとで切断し pUC 18XGとライゲ一シヨンするとサブクロ一 ン No.73- 10が、 また、 1^0,20を "1と^711とで切断し 11(：18乂6とラィゲーション するとサブクローン No.73-12が得られる。 When .20 was cut with 51 and Hind I II and ligated with pUC 18XG, subclone No. 73-10 was cut, and 1 ^ 0,20 was cut with "1 and ^ 711 and 11 (: Ligation with 18A6 gives subclone No. 73-12.
(4) 本発明の組み換えベクターの構築 (4) Construction of the recombinant vector of the present invention
上記 （ 1 ) 〜 （3 ) で構築したベクターおよびサブクローンを用いて、 本発明 の組み換えべクタ一を以下のように構築する （図 3〜図 5参照） 。 Using the vectors and subclones constructed in the above (1) to (3), the recombinant vector of the present invention is constructed as follows (see FIGS. 3 to 5).
(4一 1 ) pPADOlの構築 (4-1) Construction of pPADOl
サブクローン No.20を BgJl Iで切断した。 サブクローン No.22と No.72-2とを と £a Uとで切断してこれらの断片をライゲーシヨンし、 サブクロ一ン No.72-22 を得る。 ついで、 No.72- 22を Iで切断して BAPで処理した断片と、 サブクロー ン No.20の^^ II断片とをライゲ一シヨンすると、 pPADOlが得られる （図 2) 。 (4 - 2 ) pPADdl (4541-4922)の構築 Subclone No. 20 was cut with BgJlI. The subclones No.22 and No.72-2 are cut with and £ a U, and these fragments are ligated to obtain a subclone No.72-22. Next, pPADOl was obtained by ligating the fragment obtained by digesting No.72-22 with I and treating with BAP, and the ^^ II fragment of subclone No.20 (Fig. 2). (4-2) Construction of pPADdl (4541-4922)
上記 （4— 1 ) で得た pPADOlを co81Iと Spelとで切断し、 DNAポリメラ一ゼ で処理して経かつ末端とし、 自己ライゲーシヨンさせて大腸菌を形質転換し、 選 択すると pPADdl (454卜 4922)を得ることができる。 The pPADOl obtained in the above (4-1) was cut with co81I and Spel, treated with DNA polymerase to make the ends and ends, self-ligated to transform Escherichia coli, and selected to obtain pPADdl (454 to 4922). ) Can be obtained.
(4一 3 ) pPADrepl (4541-4922)の構築 (4-1-3) Construction of pPADrepl (4541-4922)
上記 （4— 1 ) で得た pPADOlを^: o81Iと《¾9elとで切断し、 配列番号 4の合成 D
NAリンカーとライゲ一シヨンして大腸菌を形質転換し、 選択すると pPADrepl(4 54卜 4922)を得ることができる。 PPADOl obtained in (4-1) above was cut with ^: o81I and << 9el to synthesize SEQ ID NO: 4. Escherichia coli is transformed by ligating with an NA linker and, when selected, pPADrepl (454-4922) can be obtained.
(4-4) pPADin(454卜 4922)p7.5:lacZの構築 (4-4) pPADin (454 4922) p7.5: Construction of lacZ
上記 （4— 1 ) で得た pPADOlを /τχΙΙΙΙと ;1とで切断し回収した断片と、 上 記 （4— 3) で得た pPADrepl(454 4922)を///； xlIIIと 1とで切断し回収した断 片と、 上記 （ 1 ) で構築した pNZ76を /; τάΙΙΙと とで切断した断片とをライゲ —シヨンして大腸菌を形質転換し、 選択すると、 pPADin(4541- 4922)p7.5:lacZを 構築した （図 4) 。 The fragment recovered by cutting pPADOl obtained in (4-1) above with / τχΙΙΙΙ and; 1 and the pPADrepl (454 4922) obtained in (4-3) above ///; xlIII and 1 The cut and recovered fragment and the fragment cut with pNZ76 constructed in (1) above and /; τ / were ligated and transformed into Escherichia coli, and when selected, pPADin (4541-4922) p7. 5: lacZ was constructed (Figure 4).
(4-4) pPADin(377)p7.5:HNの構築 (4-4) pPADin (377) p7.5: Construction of HN
上記 （2) で構築した pNZ87をガ 'τκΠΠと 1とで切断して S1ヌクレア一ゼで 処理して断片を回収した。 また、 上記 （3) で構築した pPADOlを 5ΗΙΙで切断し、 S 1ヌクレア一ゼと BAPとで処理して、 先に回収した pNZ87からの断片とライゲ一シ ヨンし、 大腸菌を形質転換して選択すると、 pPADin(377)p7.5:HNを得ることがで きる （図 5) 。 The pNZ87 constructed in (2) above was cleaved with the moieties' τκΠΠ and 1 and treated with S1 nuclease to recover the fragment. Also, the pPADOl constructed in (3) above is cut with 5ΗΙΙ, treated with S1 nuclease and BAP, ligated with the fragment from pNZ87 recovered earlier, and transformed into E. coli. If selected, pPADin (377) p7.5: HN can be obtained (Fig. 5).
以上のようにして得られたベクタ一に、 後述する外来遺伝子とプロモーターと を組み込むこともできる。 A foreign gene and a promoter, which will be described later, can also be incorporated into the vector obtained as described above.
(5) 発現量比較用 fNZ1029の構築 (5) Construction of fNZ1029 for expression level comparison
組み換えウィルス鶏痘ベクタ一と外来遺伝子の発現量を比較するために、 特開 平第卜 168279号公報に記載のようにして、 fNZ1029を構築する （図 6) 。 To compare the expression levels of the recombinant virus fowlpox vector and the foreign gene, fNZ1029 is constructed as described in JP-A-168279 (FIG. 6).
(5 - 1 ) pNZ133の作製 (5-1) Preparation of pNZ133
PUC18 (フアルマシア社製） を EcoRIと Hindlllで消化した後、 フエノール： ク ロロホルムで抽出し、 エタノール沈殿にょリ開裂した PUC18を回収した。 5'—末 端リン酸をアルカリフォスファタ一ゼ処理によって除去し、 DN Aを再びフエノ —ル： クロ口ホルム抽出後、 エタノール沈殿によって回収する。 開裂した ρϋΠ8 と精製アビボックスウィルス （ΝΡ株） DNAの EcoRI断片ををリガ一ゼによつ てライゲ一シヨンし、 コンビテントな大腸菌を形質転換し、 5—ブロモ—4—クロ 口一 3—インドリル一 /3— D—ガラクトビラノシド、 イソプロピル一 /9—D—ガラ クトピラノシド、 アンピシリン含有 L B寒天培地で培養する。 PUC18 (Pharmacia) was digested with EcoRI and Hindlll, and extracted with phenol: chloroform, and PUC18 cleaved by ethanol precipitation was recovered. The 5'-terminal phosphoric acid is removed by alkaline phosphatase treatment, and the DNA is recovered again by extraction with phenol: chloroform and ethanol precipitation. The cleaved ρϋΠ8 and the EcoRI fragment of the purified Aviboxvirus (strain) DNA were ligated with ligase, transformed into a competent Escherichia coli, and then treated with 5-bromo-4-chloro-3-. Incubate on LB agar medium containing indolyl 1 / 3-D-galactovyranoside, isopropyl 1 / 9-D-galactopyranoside and ampicillin.
寒天培地上に生育した白コロニーをアンピシリン含有 L B液体培地で培養し、
ビルンボイムとドーリ一の方法（Nucleic Acid Research, 7： 1513-( 1979) )でプラ スミ ドを抽出し、 ^1と //¾1111とで消化後、 ァガロースゲル電気泳動によって、 もとのアビボックスウィルス DN Aの CORI_7///K1III断片と同じ長さの断片を持 つハイプリッドプラスミ ドを検出し、 これを pNZ133とする。 The white colonies grown on the agar medium were cultured in LB liquid medium containing ampicillin, The plasmid was extracted by the method of Birnboim and Doli (Nucleic Acid Research, 7: 1513- (1979)), digested with ^ 1 and // ¾1111, and then subjected to agarose gel electrophoresis to obtain the original Avibox virus DN. A hybrid plasmid having a fragment of the same length as the CORI_7 /// K1III fragment of A is detected, and this is designated as pNZ133.
(5 - 2) pNZ1029の構築 (5-2) Construction of pNZ1029
PNZ76を / κ!ΙΠ、 alで消化した後、 低融点ァガロースゲル電気泳動で断片を 分離し、 ェチジゥムブロマイド染色で DNA断片を確認し、 ゲルを切り出してフ ェノール処理した後エタノール沈殿により DN A断片を回収する。 After digesting PNZ76 with / κ! ΙΠ and al, the fragments are separated by low-melting point agarose gel electrophoresis, DNA fragments are confirmed by ethidium bromide staining, the gel is cut out, phenol-treated, and DN precipitate by ethanol precipitation. Collect fragment A.
一方、 （ 5— 1 ) で構築した pNZ133を oRVで部分的に消化し、 フエノール： クロ口ホルム抽出し、 エタノール沈殿により回収した。 開裂された pNZ133の DN Aと前記の断片 （7.5Kプロモーター遺伝子と 3—ガラクトシダ一ゼ遺伝子の連結 断片） とを混合し、 DN Aポリメラ一ゼで付着末端を平滑末端とし、フエノール： クロ口ホルム抽出後、 エタノール沈殿により回収する。 On the other hand, pNZ133 constructed in (5-1) was partially digested with oRV, extracted with phenol: cloth form, and recovered by ethanol precipitation. The cleaved DNA of pNZ133 was mixed with the above fragment (ligated fragment of the 7.5K promoter gene and 3-galactosidase gene), the cohesive end was made blunt with DNA polymerase, and phenol: cloth form was used. After extraction, recover by ethanol precipitation.
回収した D N Aをリガーゼによってライゲ一シヨンし、 コンビテントな大腸菌 を形質転換し、 アンピシリンを含有する LB寒天培地で生育させた。 The recovered DNA was ligated with ligase, transformed into E. coli, and grown on LB agar medium containing ampicillin.
生育した大腸菌から、ビルンボイムとド一リーの方法（Nucleic Acid Research, 7:1513- (1979))でプラスミ ドを回収し、
で消化し、 ァガロースゲル電気泳 動で 一ガラクトシダ一ゼ遺伝子断片を含むハイプリッドプラスミ ドを選択し、 これを PNZ1029と命名した。 Plasmid was collected from the grown E. coli by the method of Birnboim and Doley (Nucleic Acid Research, 7: 1513- (1979)). Then, a hybrid plasmid containing a mono-galactosidase gene fragment was selected by agarose gel electrophoresis and named PNZ1029.
以上のようにして得た PNZ1029を用いて、 アビボックスウィルス （鳩痘中野株 (NP株ともいう） ） に 7.5Kプロモータ一遺伝子と /3—ガラクトシダ一ゼ遺伝子 とを含む DNA断片を、 常法により相同組み換えによって揷入し、 fNZ1029を得 る。 Using PNZ1029 obtained as described above, a DNA fragment containing a 7.5K promoter gene and a / 3-galactosidase gene in avibox virus (Hatopox Nakano strain (also referred to as NP strain)) was prepared by a conventional method. To obtain fNZ1029 by homologous recombination.
(6) pNZ2237の構築 (6) Construction of pNZ2237
PNZ2237の構築は、 特開平第卜 157381号公報に記載に従って、 以下のように行 う （図 7) 。 The construction of PNZ2237 is performed as follows according to the description in JP-A No. 157381 (FIG. 7).
(6 - 1 ) pNZ87- 22及び pNZ87- 37の構築 (6-1) Construction of pNZ87-22 and pNZ87-37
PNZ87を^ alと////] dillとで切断し、 1— ////τάΐ 11断片 （Α) を回収する。 同様 に ΡΝΖ87を 0alと rallとで切断し、 al— ^rall断片 （B) を回収する。 さらに、
PNZ87を/// /7dIIIと //rflllとで切断し、 /·τχ!Π I—///—rfl 11断片 （C) を得る。 PNZ87 is cut with ^ al and ////] dill, and 1 — /// τάΐ11 fragment (Α) is recovered. Similarly, ΡΝΖ87 is cleaved with 0al and rall, and the al— ^ rall fragment (B) is recovered. further, Cut PNZ87 with //// 7dIII and // rflll to obtain / · τχ! Π I — /// — rfl 11 fragment (C).
上記のようにして得た各断片に、 下記の配列からなる合成 DNA (配列番号 Each fragment obtained as described above was added to a synthetic DNA (SEQ ID NO:
9) 9)
AATCATTTATATTTTAAAAATG AATCATTTATATTTTAAAAATG
GTAAATATAAAATTTTTACCTG (配列番号 9 ) または下記の配列からなる合成 D N A (配列番号 10) GTAAATATAAAATTTTTACCTG (SEQ ID NO: 9) or a synthetic DNA consisting of the following sequence (SEQ ID NO: 10)
AATCATAAAGAACAGTACTCAATCAATAGCAATCATG AATCATAAAGAACAGTACTCAATCAATAGCAATCATG
GTATTTCTTGTCATGAGTTAGTTATCGTTAGTACCTG (配列番号 10) を加えて、 リガ一ゼによりライゲ一シヨンし、 ビルンボイムらの方法 （前出） に よってプラスミ ドを抽出すると、 2種類のプラスミ ド、 pNZ87— 22と pNZ87— 37と が得られる。 After adding GTATTTCTTGTCATGAGTTAGTTATCGTTAGTACCTG (SEQ ID NO: 10) and ligating with ligase and extracting plasmid by the method of Birnboim et al. can get.
(6 - 2) pNZ136Sの構築 (6-2) Construction of pNZ136S
(6— 2— 1 ) pUC18 (フアルマシア社製） を oRIと /; 2d IIIで消化した後、 フ ェノール： クロ口ホルム抽出し、 エタノール沈殿により開裂した pUC18を回収す る。 5 '—末端リン酸をアルカリフォスファターゼ処理によって除去し、 DNAを 再びフエノール： クロ口ホルム抽出した後、 エタノール沈殿によって回収する。 開裂した PUC18と精製アビボックスウィルス （NP株） DNAの fcd 断片をリガ —ゼでライゲ一シヨンし、 コンビテントな大腸菌を形質転換し、 5—プロモー 4— クロ口一 3—インドリル一 — D—ガラクトビラノシド、 イソプロピル一 -D- ガラクトビラメシド、 アンピシリン含有 L B寒天培地で培養する。 (6-2-1) After digesting pUC18 (Pharmacia) with oRI and /; 2dIII, extract phenol: cloth form and collect pUC18 cleaved by ethanol precipitation. The 5'-terminal phosphate is removed by alkaline phosphatase treatment, and the DNA is again extracted with phenol: cloth form and recovered by ethanol precipitation. The cleaved PUC18 and the fcd fragment of the purified Avibox virus (NP strain) DNA were ligated with ligase, transformed into E. coli, and then transformed with 5-promote 4-cloth 3-indolyl-D-. Culture on LB agar medium containing galactoviranoside, isopropyl-1-D-galactovirameside and ampicillin.
寒天培地上に生育した形質転換大腸菌のうち白コロニーをアンピシリン含有 L B液体培地で培養し、 ビルンボイムとドーリーの方法（前出）でプラスミ ドを抽出 し、 coRIと / /7dIIIで消化後、 ァガロースゲル電気泳動によって、 もとのアビポ ックスウィルス D N Aの AcoRI - Hindi II断片と同じ長さの断片を持つハイブリツ ドプラスミ ドを検出し、 これを pNZ136とする。 Among the transformed Escherichia coli grown on agar medium, white colonies were cultured in LB liquid medium containing ampicillin, plasmid was extracted by the method of Birnboim and Dawley (described above), digested with coRI and // 7dIII, and then agarose gel electrophoresed. By electrophoresis, a hybrid plasmid having a fragment of the same length as the AcoRI-Hindi II fragment of the original avipox virus DNA was detected and designated as pNZ136.
( 6 - 2 - 2 ) pNZ136Sの構築 (6-2-2) Construction of pNZ136S
上記 （6— 2— 1 ) のようにして得た PNZ136を// /τκΙΠΙで切断した後、 EcoRVで 部分消化し、 ァガロースゲル電気泳動によって £coRV - £coRV断片と £coRV— Hind I IIとが隣接した断片を回収する。
一方、 pUC18を/// / lIIIと oRIとで切断し、 フエノール： クロ口ホルムで抽出 し、 エタノール沈殿により開裂した pUC18をァガロースゲルより回収する。 EcdRV 一；/ /j7dIII断片を DNAポリメラーゼ処理し、 リガーゼによってライゲ一シヨン し、 コンビテントな大腸菌を形質転換する。 After cutting PNZ136 obtained as described above (6-2-1) with /// τκΙΠΙ, it was partially digested with EcoRV, and the £ coRV- £ coRV fragment and the £ coRV—Hind I II were separated by agarose gel electrophoresis. Recover adjacent fragments. On the other hand, pUC18 is cut with //// III and oRI, extracted with phenol: chloroform, and pUC18 cleaved by ethanol precipitation is recovered from agarose gel. The EcdRV-1; // j7dIII fragment is treated with DNA polymerase, ligated with ligase, and transformed into competent E. coli.
アンピシリン含有培地で選択すると、 £coRV— /; τάΙΙΙ を含む pNZ 6Sを得るこ とができる。 When selected on an ampicillin-containing medium, pNZ6S containing £ coRV-/; τάΙΙΙ can be obtained.
(6 - 3) pNZ2237の構築 (6-3) Construction of pNZ2237
上記 （6— 2—2) で得たプラスミ ド pNZ136Sを /τκΙΙΙΙとで a !Iとで切断し、 フエノール： クロ口ホルム抽出し、 エタノール沈殿により回収する。 The plasmid pNZ136S obtained in the above (6-2-2) is cut with a! I at / τκΙΙΙΙ, extracted with phenol: chloroform, and recovered by ethanol precipitation.
pNZ87、 pNZ87— 22及び pNZ87— 37をそれぞれ^ τχΙΠΙと a;rfllとで切断し、 ァガ ロースゲル電気泳動に供し、 ワクシニアウィルスの 7.5Kプロモータ一遺伝子と HN 遺伝子 DNAとを含む//// κΙΠΙ— a/rfll断片を回収する。 pNZ87, pNZ87-22 and pNZ87-37 were respectively cleaved with ^ τ; and a; rfll, subjected to agarose gel electrophoresis, and containing a vaccinia virus 7.5K promoter gene and HN gene DNA //// κΙΠΙ — Recover a / rfll fragments.
両者をリガーゼによってライゲ一ションし上述のように選択することにより、 PNZ2237を得ることができる。 By ligating both with ligase and selecting as described above, PNZ2237 can be obtained.
ここで、 本発明の組み換え DN Aベクターに組み込まれる外来遺伝子は特に限 定されない。 Here, the foreign gene to be incorporated into the recombinant DNA vector of the present invention is not particularly limited.
こうした外来遺伝子としては、 例えば、 各種の酵素タンパク質や抗原タンパク 質、 ぺプチドをコードする構造遺伝子や遺伝子発現を調節する調節遺伝子などが 好ましい例として挙げられる。 Preferred examples of such foreign genes include various enzyme proteins and antigenic proteins, structural genes encoding peptides, and regulatory genes that regulate gene expression.
より具体的な例としては、 酵素タンパク質としては、 /3—ガラクトシダーゼを コードする遺伝子、 ヒト組織プラスミノーゲン（tPA)などの酵素タンパク質をコ —ドする遺伝子などが挙げられる。 More specifically, examples of the enzyme protein include a gene encoding / 3-galactosidase and a gene encoding an enzyme protein such as human tissue plasminogen (tPA).
また、 抗原遺伝子としては、 哺乳類細胞、 哺乳類感染性病原体、 鳥類細胞、 鳥 類感染性病原体、 魚類細胞、 魚類病原体などに由来するものを挙げることができ る。 例えば、 日本脳炎ウィルスの E蛋白質をコードする遺伝子 （米国特許番号皇 5， 021,347号） 、 ニューカッスル病ウィルスの HNタンパク質をコードする遺伝 子（Millerら， J. Gen. Virol., §2， 1917-1927(1986))、 Fタンパク質をコードする遺 伝子（McGinnesら， Virus Res. , 5343- 5356(1986))、 マレック病ウィルスの糖タン パク質 g Bをコードする遺伝子（Rossら， J.Gen.Virol.， , 1789-1804(1988))、 伝
染性ファブリキウス嚢病ウイルスの構造タンパク V P 2をコードする遺伝子（Bay liss et al.， J. Gen. Virol. , , 1303- 1312(1990))等の感染防御に関与した抗原 をコードした遺伝子 （以下、 単に抗原遺伝子という） などの構造遺伝子が挙げら れる。 Examples of the antigen gene include those derived from mammalian cells, mammalian infectious pathogens, bird cells, bird infectious pathogens, fish cells, fish pathogens, and the like. For example, a gene encoding the E protein of Japanese encephalitis virus (US Pat. No. 5,021,347) and a gene encoding the HN protein of Newcastle disease virus (Miller et al., J. Gen. Virol., §2, 1917-). 1927 (1986)), a gene encoding the F protein (McGinnes et al., Virus Res., 5343-5356 (1986)), and a gene encoding the Marek's disease virus glycoprotein gB (Ross et al., J. Biol. Gen. Virol., 1789-1804 (1988)) Genes encoding antigens involved in the protection of infection, such as the gene encoding the structural protein VP2 of infectious bursal disease virus (Bay liss et al., J. Gen. Virol., 1303-1321 (1990)) Hereinafter, these genes are simply referred to as antigen genes).
調節遺伝子の具体例としては、 7.5kDaぺプチドゃ 11Kポリぺプチドなどをコ一 ドするワクシニアウィルス遺伝子のプロモータ一などが例示されるほか、 プロモ —ターとして機能する限りにおいては、 これらのプロモーターの一部が削除され た改変体等であってもよい。 また、 合成プロモータ一、 例えば、 初期プロモータ —と後期プロモーターの両方の配列を有する合成プロモータ一 （J. Mol. Biol. , 215， 749 - 769(1989)， ibid, 215, 77卜 781(1989))やその一部をプロモーター活 性が喪失しない範囲で、 削除、 塩基の修飾 ·置換等を行った改変体、 例えば、 塩 基配列が、 下記の配列 （配列番号 5) Specific examples of the regulatory gene include a promoter of a vaccinia virus gene that encodes a 7.5 kDa peptide, a 11K polypeptide, and the like. In addition, as long as it functions as a promoter, these promoters It may be a modified product in which a part is deleted. Also, synthetic promoters, for example, synthetic promoters having both the early and late promoter sequences (J. Mol. Biol., 215, 749-769 (1989), ibid, 215, 771 781 (1989)) ) Or some of them are modified to the extent that promoter activity is not lost, deletions, modification or substitution of bases, for example, the base sequence is as follows (SEQ ID NO: 5)
CTAATTTATTGCACTC-3' (配列番号 5 ) CTAATTTATTGCACTC-3 '(SEQ ID NO: 5)
で示されるもの等の遺伝子組み換え技術で使用する、 遺伝子の発現を調節する調 節遺伝子等が挙げられる。 And the like. Regulated genes that regulate gene expression used in gene recombination techniques such as
上述した外来遺伝子は、 単独でまたは必要に応じて 2種類以上を組み込むこと ができる。 本発明の DNAベクタ一に外来遺伝子を揷入する場合、 本発明の DN Aベクタ一がボックスウイルス感染細胞で複製できる性質を失わない限り、 これ らの遺伝子の揷入部位は特に限定されない。 しかし、 そのような領域としては、 非翻訳領域や 0RF4の領域が好ましく、 より好ましくは、 配列番号 2の第 395番目 の制限酵素 部位や第 4, 541〜4， 922番目の領域などを挙げることができる。 本発明の新規 DN Aベクターを含む組み換え DN Aベクタ一の作製方法は、 特 に限定されない。 一例としては、 本発明の DNAベクタ一をプラスミ ド中に組み 込み、 この DN Aベクター配列中の揷入可能部位に外来遺伝子を常法に従って組 み込んで組み換えプラスミ ドを作製する。 この組み換えプラスミ ドを大腸菌など の適当な細胞内に導入して当該組み換えプラスミ ドを増殖させ、 精製した後に、 適当な制限酵素、 例えば、 1により組み換えプラスミ ドから外来遺伝子を含 む組み換え D N Aベクタ一を切り離して、 組み換え D N Aべクタ一を得る方法が
挙げられる。 The above-described exogenous genes can be incorporated alone or, if necessary, in combination of two or more. When a foreign gene is inserted into the DNA vector of the present invention, the insertion site of these genes is not particularly limited, as long as the DNA vector of the present invention does not lose its ability to replicate in a box virus-infected cell. However, such a region is preferably a non-translated region or a region of 0RF4, more preferably a 395th restriction enzyme site of SEQ ID NO: 2 or a 4,541 to 4,922th region. Can be. The method for producing a recombinant DNA vector containing the novel DNA vector of the present invention is not particularly limited. As an example, a recombinant plasmid is prepared by incorporating the DNA vector of the present invention into a plasmid, and incorporating a foreign gene into an insertable site in the DNA vector sequence according to a conventional method. This recombinant plasmid is introduced into an appropriate cell such as E. coli, and the recombinant plasmid is propagated and purified. Then, a recombinant DNA vector containing a foreign gene from the recombinant plasmid with an appropriate restriction enzyme, for example, 1 is used. To get the recombinant DNA vector No.
元々大量に D N Aベクタ一を用意することができれば、 特に組み換えプラスミ ドを作製して増殖させる必要はない。 If a large amount of DNA vector can be prepared in the first place, there is no need to produce and propagate recombinant plasmids.
ボックスウィルスと本発明の組み換え D N Aベクターとを細胞に導入すること によって、 組み換え D N Aベクタ一に揷入された外来遺伝子を発現させることが 可能である。 By introducing a box virus and the recombinant DNA vector of the present invention into cells, it is possible to express a foreign gene inserted into the recombinant DNA vector.
組み換え D N Aベクタ一とボックスウィルスを細胞に導入する方法も、 特に限 定されない。 例えば、 まず、 上述のようにして得た組み換え D N Aベクタ一を適 当なベクタ一に組み込んだ組み換えべクタ一で大腸菌を形質転換して増殖させて この組み換えべクタ一を大量に調製する。 ついで、 予めボックスウィルスを感染 させた細胞にエレクトロポレーシヨン法、 リポフエクシヨン法などの方法で前記 組み換えベクターを直接又は組み換えベクターから常法に従って切り出し、 これ を導入してもよい。 この際、 細胞にボックスウィルスを感染させる方法も特に制 限されず、 所望の m. 0. i .でウィルスと細胞とを接触させるといった周知の方法で 行えばよい。 The method for introducing the recombinant DNA vector and the box virus into cells is not particularly limited. For example, first, Escherichia coli is transformed and grown in a recombinant vector in which the recombinant DNA vector obtained as described above is incorporated into an appropriate vector, and the recombinant vector is prepared in a large amount. Next, the recombinant vector may be directly or cut out from the recombinant vector by a conventional method into a cell previously infected with a box virus by an electroporation method, a lipofection method, etc., and introduced. At this time, the method of infecting the cell with the box virus is not particularly limited, and may be performed by a known method such as contacting the virus with the cell at a desired m.o.i.
( 3 ) 組み換え D N Aベクタ一を有効成分とするワクチン (3) Vaccine containing recombinant DNA vector as an active ingredient
本発明の組み換え D N Aベクターを有効成分とするワクチンは、 外来遺伝子と して抗原遺伝子を揷入した組み換え D N Aベクタ一をボックスウィルスの生ワク チン株を感染させた細胞に導入して、 そこから継代 ·純化を繰返すことによリ調 製される。 In the vaccine comprising the recombinant DNA vector of the present invention as an active ingredient, a recombinant DNA vector containing an antigen gene as a foreign gene is introduced into cells infected with a live box virus strain of a box virus, and transferred from the cell. Produced by repeating purification.
揷入される抗原遺伝子は上記 （2 ) で記載したように、 特に限定されない。 使 用するボックスウィルスも生ワクチン株として認められた株であれば特に限定さ れず、 例えば、 現在も広く使用されているアビボックスウィルスの鶏痘生ワクチ ン株などが好ましい例として挙げられる。 The antigen gene to be inserted is not particularly limited as described in (2) above. The box virus to be used is not particularly limited as long as it is a strain recognized as a live vaccine strain. For example, a fowlpox live vaccine strain of avibox virus, which is still widely used, is a preferred example.
継代 ·純化の際の選択方法は特に限定されるものではないが、 組み換え D N A ベクターに挿入した外来遺伝子をプローブとするハイブリダィゼーシヨン法でポ ジシグナルを示すかどうか、 又は、 外来遺伝子がコードしている抗原に対する抗 体を用いて、抗原が発現しているかどうかを確認する方法によることが好ましい。 そして、 ボックスウィルスのプラークを形成させて、 全てのプラークで組み換え
D N Aベクターに揷入した外来遺伝子の存在または抗原の発現を確認できるよう になるまで、 選択したプラークを継代して選択する純化作業を繰返すことがより 好ましい。 The selection method at the time of subculture / purification is not particularly limited.However, whether or not the foreign gene shows a posi signal by the hybridization method using the foreign gene inserted into the recombinant DNA vector as a probe, or whether the foreign gene It is preferable to use an antibody against the encoded antigen to confirm whether the antigen is expressed. Then, let the box virus plaques form and recombine in all the plaques It is more preferable to repeat the purification work of subculturing and selecting the selected plaques until the presence of the foreign gene inserted into the DNA vector or the expression of the antigen can be confirmed.
上述のようにして得られた組み換え D N Aベクタ一とボックスウィルスとを含 む調製液を、 通常のボックスウィルス生ワクチンを調製する方法と同じようにし て、 本発明のワクチン液を得ることができる。 この調製方法は一般的な方法であ り、 特に限定されない。 The vaccine solution of the present invention can be obtained by using the preparation solution containing the recombinant DNA vector and the box virus obtained as described above in the same manner as in a method for preparing a normal box virus live vaccine. This preparation method is a general method and is not particularly limited.
例えば、 本発明の組み換え D N Aベクタ一が増殖できる細胞にボックスウイル スと同時に感染させてこの細胞を培養し、 その細胞を回収して破砕した後に遠心 分離して、 高力価の組み換え D N Aベクタ一とボックスウィルスを含んだ遠心上 清と沈澱物とに分離する。 本質的に宿主細胞及びノ又はその破砕片を含まず、 細 胞培養培地、 組み換え D N Aベクタ一、 およびボックスウィルスとを含んだこの 遠心上清は、 本発明のワクチンとして使用できる。 For example, cells capable of proliferating the recombinant DNA vector of the present invention are simultaneously infected with a box virus, and the cells are cultured. The cells are collected, disrupted, and then centrifuged to obtain a high-titer recombinant DNA vector. And a centrifugal supernatant containing box virus and a precipitate. This centrifuged supernatant, which is essentially free of host cells and no or debris thereof, but contains the cell culture medium, the recombinant DNA vector, and the box virus, can be used as the vaccine of the present invention.
また、 上記のようにして調製した組み換え D N Aベクタ一とボックスウィルス とを含む調製液に、 弱毒ボックスウィルスを適宜加えてワクチンとして使用して もよい。 弱毒ボックスウィルスは特に限定されないが、 アビボックスウィルス、 オルソボックスウィルス等を好適に使用することができる。 ァビポックスウィル スの具体例としては、 鶏痘ウィルスでは、 ATCC VR-251、 ATCC VR- 250、 ATCC VR- 229、 ATCC VR- 249、 ATCC VR- 288、 N i sh i gahara株、 泗水株、 及び鶏痘ワクチン株 として市販されている CEVA株などのウィルスが例示される。 また、 ワクシニアゥ ィルスとしては、 リスター株 LC16mO株、 WR株、 ニューヨーク 'ボード 'ォブ 'へ ルス株などを挙げることができる。 これらのウィルスは、 必要に応じて種々の方 法で弱毒化することができるが、 特に、 ファウルボックスウィルス （FPV) を使 用すると、 ワクチンの有効性を高める上で好ましい。 Alternatively, an attenuated box virus may be appropriately added to a preparation containing the recombinant DNA vector and box virus prepared as described above, and used as a vaccine. The attenuated box virus is not particularly limited, but an avibox virus, an orthobox virus and the like can be preferably used. Specific examples of Avipoxvirus include fowlpox virus: ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nisshi gahara strain, Sashimi strain And viruses such as the CEVA strain which is commercially available as a fowlpox vaccine strain. Examples of vaccinia viruses include Lister strain LC16mO strain, WR strain, and New York 'Board' ob 'health strain. These viruses can be attenuated in a variety of ways as needed, but the use of foul-box virus (FPV) is particularly preferred for increasing vaccine efficacy.
さらに、 この上清をそのまま、 あるいは必要に応じて希釈または濃縮して凍結 保存するか、 凍結乾燥して保存することができる。 凍結乾燥した場合には、 薬理 学的に許容される生理食塩水、 蒸留水などで再構成して使用してもよい。 Further, the supernatant can be stored as it is, or, if necessary, diluted or concentrated and frozen or lyophilized. When freeze-dried, it may be reconstituted with pharmacologically acceptable saline or distilled water before use.
本発明のワクチンは、上述した組み換え D N Aべクターが動物体内で発現して、 外来遺伝子がコードする抗原蛋白質を生合成させるような方法であれば、 どのよ
うな方法で投与してもよい。 The vaccine of the present invention can be prepared by any method as long as the above-described recombinant DNA vector is expressed in an animal body and biosynthesizes an antigen protein encoded by a foreign gene. It may be administered by such a method.
例えば、 皮膚にひっかき傷をつけてそこにワクチンを接種したり、 注射針やそ の他の器具などによって、 接種対象動物の皮下に接種することができる。 また、 ワクチンを接種対象動物の飲み水に懸濁したり、 飼料の固形物に混入させて経口 投与することも可能である。 さらに、 エアロゾルやスプレーなどによりワクチン を吸入させる方法、 静脈内接種法、 筋肉中接種法、 腹腔内接種法などを用いるこ ともできる。 For example, a vaccination can be made by scratching the skin and inoculating the target animal subcutaneously with a needle or other device. It is also possible to suspend the vaccine in the drinking water of the animal to be vaccinated or to orally administer the vaccine by mixing it with the solids of the feed. In addition, vaccines can be inhaled by aerosol or spray, intravenous inoculation, intramuscular inoculation, intraperitoneal inoculation, and the like.
接種量は、 例えば、 組み換え D N Aベクタ一を、 それを増殖させるのに用いた ボックスウィルスと共にそのままワクチンとして使用する場合、 ボックスウィル ス量が 1羽あたり通常 103〜106pfu (プラーク形成単位） ノ組み換え D N Aべク タ一量で 10ng〜 1 t gとなるようにすることが望ましい。 よリ好ましくは 1羽あた リ 104pfuである。 注射する場合には、 生理食塩水などの生理学的に許容される液 体で希釈し、 1羽あたり 0. lmL程度を接種すればよい。 単離された組み換え D N Aベクタ一を、 適当なボックスウィルスと混在し、 ワクチンを調製する場合であ つても、 上述の場合と同様になるように調製する。 For example, if the recombinant DNA vector is used as a vaccine together with the box virus used to propagate it, the amount of box virus is usually 10 3 to 10 6 pfu per bird (plaque forming unit) It is desirable that the amount of the recombinant DNA vector be 10 ng to 1 tg. I Li is preferably a Li 10 4 pfu per 1 birds. For injection, dilute with physiologically acceptable liquid such as saline and inoculate about 0.1 mL per bird. Even if the isolated recombinant DNA vector is mixed with an appropriate box virus to prepare a vaccine, the vaccine is prepared in the same manner as described above.
本発明のワクチンは普通のポックスウィルス生ワクチンと同等の条件下で保存、 使用することが可能である。 例えば、 本発明のワクチンを凍結乾燥すれば、 室温 (20〜22°C程度） で長期保存、 取り扱い、 輸送することができる。 また、 本発明 のワクチンは、 懸濁液を- 20°C〜- 70°Cで凍結させて保存することも可能である。 実施例 The vaccine of the present invention can be stored and used under the same conditions as ordinary poxvirus live vaccines. For example, if the vaccine of the present invention is lyophilized, it can be stored, handled, and transported at room temperature (about 20 to 22 ° C.) for a long time. In addition, the vaccine of the present invention can be stored by freezing the suspension at −20 ° C. to −70 ° C. Example
以下に実施例を挙げて本発明を具体的に説明するが、 本発明はこれらの実施例 によって何ら限定されるものではない。 Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.
(実施例 1 ) D N Aベクタ一の取得およびコピー数の検討 (Example 1) Acquisition of DNA vector and study of copy number
( 1 ) D N Aベクターの作製 (1) Preparation of DNA vector
バイアル 1本の鶏痘ウィルス泗水株 （社団法人動物用生物学的製剤協会 (Japan ese Associ ation of Veterinary B i o logies)が管理している家畜衛生菌株（Cul tu res for Animal Hygi ene) ；鶏痘ウィルス泗水株（菌株番号 VA0101) )を、 直径 1. 5c mの培養デイツシュでサブコンフレントまで培養した鶏胚繊維芽細胞に、 バイァ
ル 1本感染させた。 感染後、 37°Cの C02インキュベータ一中で 1週間培養し、スク レ—パーでディッシュから細胞をはがし、 はがした細胞を集めて i, 000Xg、 4°C で 5分間遠心分離した。 Vial One strain of fowlpox virus (Cul tures for Animal Hygiene) managed by the Japan Association of Veterinary Biologies (Cultures for Animal Hygiene); fowlpox A virus strain (strain number VA0101)) was transformed into chicken embryo fibroblasts cultured to subconfluence in a 1.5 cm diameter culture dish. 1 infected. After infection, and cultured for 1 week in 37 ° C in C0 2 incubator primary, disk Le - Remove the cells from the dish par, i collect peel cells, 000 x g, and centrifuged at 4 ° C 5 minutes.
この感染細胞を生理的平衡溶液（以下、 P B Sという） 5mLで 2回洗浄した後、 1. 2mLの P B Sに再懸濁し、ここにリシスバッファ一（1.25%の Triton- X100、 250mM の 2-メルカプトエタノール、 50mMの EDTAを加えた P B S溶液） 0.8mLを加えた。 ボルテックスミキサーで攪拌した後に、 室温に 5分間放置し、 2，000Xgで 5分間 遠心分離し、 細胞残骸を取り除いた。 After washing the infected cells twice with 5 mL of physiological equilibration solution (hereinafter referred to as PBS), resuspend them in 1.2 mL of PBS, and add lysis buffer (1.25% Triton-X100, 250 mM 2-mercapto 0.8 mL of a PBS solution containing ethanol and 50 mM EDTA was added. After stirring with a vortex mixer, the mixture was left at room temperature for 5 minutes and centrifuged at 2,000 Xg for 5 minutes to remove cell debris.
この上澄をエツペンドルフチューブ 2本に移し、 4 °Cにて 10， 000 X gで 20分間 遠心し、 鶏痘ウィルスを含む沈殿物を回収した。 The supernatant was transferred to two Eppendorf tubes and centrifuged at 10,000 X g for 20 minutes at 4 ° C to collect a precipitate containing fowlpox virus.
この沈殿物に 1 g/mLの DNase、 1 μ g/mLの RNase A、 0.6mM NaClを含む 12.5mM トリス塩酸（PH7.5)を lmL加えて、 37°Cにて 30分間静置した。 その後、 0.5M EDTA を 25 L、 10% S D Sを 125 L、 滅菌蒸留水を 87 tL、 10mg/mLのプロテア一ゼ Kを 12.5^L、 2-メルカプトエタノールを 0.5/zL加えて、 55°Cで 30分間静置した。 その溶液をエツペンドルフチューブ 3本に 400 Lずつ分注し、 各エツペンドル フチューブをそれぞれ等量のフエノール： クロ口ホルム ：イソアミルアルコール (25:24:l(v/v/v))混合液で 2回抽出処理した。 To this precipitate was added 1 mL of 12.5 mM Tris-HCl (PH7.5) containing 1 g / mL DNase, 1 μg / mL RNase A, and 0.6 mM NaCl, and the mixture was allowed to stand at 37 ° C. for 30 minutes. Then add 25 L of 0.5 M EDTA, 125 L of 10% SDS, 87 tL of sterile distilled water, 12.5 ^ L of 10 mg / mL protease K, 0.5 / zL of 2-mercaptoethanol, and add 55 ° C. For 30 minutes. Dispense 400 L of the solution into three eppendorf tubes, and dispense each eppendorf tube with an equal volume of phenol: cloform: isoamyl alcohol (25: 24: l (v / v / v)). Extracted twice.
20，000Xgで、 4°Cにて 2分間の遠心により分離された水層を別のエツペンド ルフチューブに移した。 ここに、 5Mの NaClを 16 L加え、 さらに- 20°Cで冷やした 100%エタノールを lmL加えて- 20°Cに 30分間放置した。 ついで、 4°C、 20.000X gで 10分間遠心分離して DNAを沈殿させた。 この沈殿物を 70%エタノールで洗 浄後、 乾燥させた。 The separated aqueous layer was transferred to another Eppendorf tube by centrifugation at 20,000 Xg at 4 ° C for 2 minutes. To this, 16 L of 5M NaCl was added, and 1 mL of 100% ethanol cooled at -20 ° C was further added, and the mixture was left at -20 ° C for 30 minutes. Subsequently, DNA was precipitated by centrifugation at 4 ° C. and 20.000 × g for 10 minutes. The precipitate was washed with 70% ethanol and dried.
(2) コピー数の検討 (2) Examining the number of copies
この DNAを適当量の ImM EDTA含有 10mM トリス塩酸（pH8.0)溶液 （以下、 TE溶 液という） で溶解して使用した。 This DNA was dissolved in an appropriate amount of a 10 mM Tris-HCl (pH 8.0) solution containing ImM EDTA (hereinafter referred to as a TE solution) and used.
上記 DNAを TE溶液に溶かし、 その 0.6 g相当の DNAを、 0.8%ァガロース ゲルを担体とする電気泳動にかけ、 100V、 2時間の条件で電気泳動した。 The above DNA was dissolved in a TE solution, and 0.6 g of the DNA was subjected to electrophoresis using a 0.8% agarose gel as a carrier, and electrophoresed at 100 V for 2 hours.
その結果、 分子量マーカーである A/Z/xini切断 DNAの最大長のバンド（23kb P)より分子量の大きい位置に一本、 5kb付近に 1本の合計 2本のバンドが確認さ
れた。 23kbの大きいバンドは鶏痘ウィルスのゲノム DNAであるが、 5kbの小さ いバンドの DN Aは文献などでも今までに報告されていないものである。 As a result, two bands, one at a position with a higher molecular weight and one near 5 kb, were confirmed from the maximum length band (23 kb P) of the A / Z / xini-cut DNA, which is a molecular weight marker. Was. The large band of 23 kb is the genomic DNA of fowlpox virus, but the small band of 5 kb has not been reported in literature.
ァガロースゲル上におけるこの 5kbpの DN Aのバンドの U V吸収 （260ηπι) の 強さは、 鶏痘ウィルスゲノム DN Α (約 300kb) の約半分程度であった。 したが つて、 ゲノムの大きさが約 60:l(300kbp:5kbp)であるにもかかわらず、 UV吸収の 強度が約半分程度ということは、 この 5kbの DNAの細胞当たりのコピー数が多 いことを示し、 そのコピー数は鶏痘ウィルスの約 30倍と推計された。 The intensity of UV absorption (260ηπι) of this 5 kbp DNA band on agarose gel was about half that of fowlpox virus genome DNΑ (about 300 kb). Therefore, despite the size of the genome being about 60: l (300kbp: 5kbp), the fact that the intensity of UV absorption is about half means that the number of copies of this 5kb DNA per cell is large. The copy number was estimated to be about 30 times that of fowlpox virus.
この場合、 マ一力一に用いた； L//;2din切断 DNAの量及びサンプル DNAの 量とサンプルを調製した細胞数から計算すると、 この 5kbの DNAのコピー数は 細胞あたり 10， 000以上であった。 In this case, the number of copies of this 5 kb DNA was 10,000 or more per cell, calculated from the amount of L //; 2din-cleaved DNA, the amount of sample DNA, and the number of cells from which the sample was prepared. Met.
(実施例 2) DN Aベクターのサブクロ一ニング (Example 2) Subcloning of DNA vector
実施例 1で見出された 5kbの DNAを解析する第一歩として、 この DNAのサ ブクローニングと制限酵素地図の作成を行った。 尚、 サブクローニングに用いた プラスミ ドベクタ一は、 市販の pUC18プラスミ ドの 5 ΛΙ制限酵素切断部位（以下、 制限酵素切断部位をサイトという） に 1サイトを合成 DNAを使って導入し たプラスミ ド pUCXGを用いた。 As a first step in analyzing the 5 kb DNA found in Example 1, subcloning of this DNA and creation of a restriction map were performed. The plasmid vector used for subcloning was a plasmid pUCXG obtained by introducing one site into the 5 5 restriction site (hereinafter, the restriction site is called a site) of commercially available pUC18 plasmid using synthetic DNA. Using.
. 実施例 1の手順で調製した DNA溶液 （DNA濃度 10〜100 g/mL) を 1〜10 ユニット/ mLの制限酵素 £coRI、 £gJli、 J¾alを単独もしくは、 2種類組み合わせ て切断した。 coRI単独の切断で 0.6kb、 l.Okb, 1.2kb、 2.2kbの 4断片、 Bglllの 単独切断で 1.0kb、 4. lkbの 2断片、 alの単独切断で 3.5kbと 1.7kbの 2断片、 Bg Iと ?alの二重切断で 1.0kb、 2.4kb, 1.7kbの 3断片となった。 The DNA solution (DNA concentration: 10 to 100 g / mL) prepared by the procedure of Example 1 was cleaved with 1 to 10 units / mL of restriction enzymes £ coRI, £ gJli, and J¾al alone or in combination of two types. Four fragments of 0.6 kb, l.Okb, 1.2 kb, 2.2 kb by cleavage of coRI alone, 1.0 kb, four fragments of 4.1 kb by single cleavage of Bglll, two fragments of 3.5 kb and 1.7 kb by single cleavage of al, The double digestion of Bg I and? Al resulted in three fragments of 1.0 kb, 2.4 kb and 1.7 kb.
これらの DNAをァガロースゲルから回収して pUC18XGプラスミ ドにサブクロ —ニングしたところ、 £coRI切断断片の 0.6kb、 1. lkb, パ Iと J¾al二重切断断片 の 2.4kbの断片がサブクローニングでき、 これらサブクロ一ンをそれぞれ No.17、 No.16、 No.72- 2と命名した （図 2参照） 。 When these DNAs were recovered from agarose gel and subcloned into pUC18XG plasmid, 0.6 kb and 1.1 kb of the £ coRI digested fragment, and 2.4 kb of the double digested I and J¾al fragments could be subcloned. These were named No. 17, No. 16, and No. 72-2, respectively (see Fig. 2).
末端を含む領域をサブクローニングするため、 実施例 1で見出された 5kbの D NA (lO g) を DNAポリメラ一ゼ I (10ユニット/ mL) 処理した後、 £coRIで 切断して 1.3kbの断片と 2.0kbの断片とを得た。 これら 2つの断片を、 それぞれ
alと £coRIで二重切断した pUC18XGプラスミ ドにサブクロ一ニングしたところ、 目 的の DNA断片が揷入されたプラスミ ドを得ることができた。 それらの各クロ一 ンを No.20、 No.22と命名した （図 2参照） 。 To subclone the region containing the ends, the 5 kb DNA (10 g) found in Example 1 was treated with DNA polymerase I (10 units / mL), cut with £ coRI, and cut into 1.3 kb DNA. A fragment and a 2.0 kb fragment were obtained. These two fragments, respectively Subcloning into pUC18XG plasmid double-cut with al and £ coRI yielded a plasmid into which the target DNA fragment had been inserted. These clones were named No. 20 and No. 22 (see Fig. 2).
また、 .22のクローンを7///2(1111で切断して出現した0.9410)、 0.5kb、 0.5kbの 断片をそれぞれ No.73-1、 No.73-2、 No.73-3クローンと命名し、 これらを で切断した PUC18にサブクローニングした。 同じく No.22のクローンを 1と とで二重切断して出現した 1.5kbの断片を No.73-9と命名し、 ¾alと ·¾^Ιとで二重 切断した PUC18XGにサブクローニングした。 In addition, the .22 clone was cloned into 7 /// 2 (0.9410 that appeared by cutting at 1111), 0.5 kb, and 0.5 kb fragments from clones No. 73-1, No. 73-2, and No. 73-3, respectively. These were subcloned into PUC18 cut with. Similarly, the 1.5 kb fragment that appeared when the No. 22 clone was double-cut with 1 and 2 was named No. 73-9, and was subcloned into PUC18XG double-cut with ¾al and · ¾ ^ ¾.
更に、 ^.20のクロ一ンを " 1と7//2(1111、 Siulと^ "/IIとでそれぞれ二重切断 して出現した 0.8kb、 0.9¾の断片を^.73-10、 No.73-12と命名し、 それぞれの と /fldll I、 Smalと Bg l Iとで二重切断した pUC18XGにサブクローニングした。 実施例 2のサブクローンの位置関係と DNAベクターの制限酵素マップを図 2 にまとめて表示した。 Furthermore, the fragment of 0.8kb, 0.9¾ which appeared by double-cutting the clone of ^ .20 with "1 and 7 // 2 (1111, Siul and ^" / II respectively) was ^ .73-10, It was named No.73-12 and subcloned into pUC18XG which was double-cut with / fldll I, Smal and Bgl I. The positional relationship of the subclones of Example 2 and the restriction enzyme map of the DNA vector are shown. Displayed together in 2.
(実施例 3 ) DNAベクタ一の塩基配列解析 (Example 3) Base sequence analysis of DNA vector
実施例 2で作製した各サブクローンの塩基配列を、 塩基配列解析キット（ABI P RISM Dye Terminator Cycle Sequencing Ready Reaction Kit)と ABI全自動シー クェンサ一で解読した。 The nucleotide sequence of each subclone prepared in Example 2 was decoded using a nucleotide sequence analysis kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit) and an ABI fully automatic sequencer.
テンプレートとして各サブクローンの入ったプラスミ ドを、 プライマ一として 東洋紡 （株） から市販されている P 7、 P 8オリゴヌクレオチドを用いた。 Plasmid containing each subclone was used as a template, and P7 and P8 oligonucleotides commercially available from Toyobo Co., Ltd. were used as primers.
P 7及び P 8プライマ一では読めない領域は、 読み終えた箇所の配列を元に作 製した合成ヌクレオチドを新たなプライマ一にして、同じく塩基配列を解読した。 これらのプライマーの配列を配列番号 6〜 7に示す。 In the region that could not be read by the P7 and P8 primers, the base sequence was similarly decoded by using a synthetic nucleotide created based on the sequence at the read position as a new primer. The sequences of these primers are shown in SEQ ID NOs: 6 and 7.
一連の解析データを繋ぎあわせて解読したこの DN Aベクターの全塩基配列を 配列番号 2に記載する。 The entire nucleotide sequence of this DNA vector obtained by connecting a series of analysis data and decoding it is described in SEQ ID NO: 2.
シークェンスの結果、この DN Aには以下のような特徴があることがわかった。 As a result of the sequence, it was found that the DNA had the following characteristics.
①全長は 5， 243bpで AT含量が 63.5°/0と多い。 (1) The total length is 5,243 bp and the AT content is as high as 63.5 ° / 0 .
② 217bpの逆位末端反復配列が両末端に存在し、 それらの ITR内には配列番号 1で 示す 30bpの繰リ返し配列が 1塩基の空きもなくタンデムに 3回連続している。
③ 4つの蛋白翻訳領域が存在し、 これらの翻訳領域は、 それぞれ 179aa、 173aa、 864aa、 190aaのアミノ酸をコードしている。 また、 その〇 R Fの翻訳開始コドン の上流にはワクシニアウイルスの転写後期プロモーターのコンセンサス配列 TAAA Tが存在する。 (2) A 217 bp inverted terminal repeat sequence is present at both ends, and the 30 bp repeated sequence represented by SEQ ID NO: 1 in these ITRs is repeated three times in tandem with no single base space. (3) There are four protein translation regions, and these translation regions encode 179aa, 173aa, 864aa, and 190aa amino acids, respectively. In addition, a consensus sequence TAAA T of the late transcription promoter of vaccinia virus exists upstream of the translation initiation codon of 〇RF.
④配列番号 1の塩基配列を使って D N A配列のデータベース（GeneBank R92. 0)の ホモ口ジーサーチを行ったが、 類似したものは見つからなかった。 (4) A homology search was performed in the DNA sequence database (GeneBank R92.0) using the base sequence of SEQ ID NO: 1, but no similar sequence was found.
⑤ 4つの〇R Fがコードするそれぞれのァミノ酸配列でタンパク質データベース (PIR R46. 0、 SWISS-PROT R32. 0、 PROSITE R13. 0)のホモロジ一サーチをしたが、 類似性の高 、ものは見つからなかった。 ホ モ A homology search of the protein database (PIR R46.0, SWISS-PROT R32.0, PROSITE R13.0) was performed for each amino acid sequence encoded by the four 〇RFs. Did not.
このことより、 配列番号 2で示される D N Aは今までに報告されていない新規 な D N Aであることがわかった。 From this, it was found that the DNA represented by SEQ ID NO: 2 was a novel DNA that had not been reported so far.
(実施例 4 ) D N Aベクタ一を含むプラスミ ドの作製と細胞への導入（図 3参照） ( 1 ) D N Aベクターを含むプラスミ ドの作製 (Example 4) Preparation of plasmid containing DNA vector and introduction into cells (see Fig. 3) (1) Preparation of plasmid containing DNA vector
実施例 3で得た No. 20、 No. 22、 No. 72- 2の 3つのクローンをそれぞれ含む pUC18 XGプラスミ ドを使って、 図 3に示す手順で 5，243bp全長 D N Aを含むプラスミ ド を構築し、 このプラスミ ドを pPADOlと命名した。 すなわち、 No. 72- 2と No. 22とを ^¾alと a/dllとでそれぞれ二重切断して 5. lkbと 1. 8kbの D N A断片を回収し、 ラ ィゲ一ションして No. 72-22を構築した。 . 20を^^1 1で切断して得られる1. lkb の断片と、 No. 72-22を 711と BAP (Bacterial Alkal ine Phosphatase ;宝酒造製） とで切断して得られた 6. 9kbの断片をライゲ一ションし、 pPADOlを構築した。 Using the pUC18 XG plasmid containing the three clones No. 20, No. 22, and No. 72-2 obtained in Example 3, a plasmid containing the 5,243 bp full-length DNA was obtained by the procedure shown in FIG. The plasmid was constructed and named pPADOl. That is, No. 72-2 and No. 22 were double-cut with ^ ¾al and a / dll respectively to recover DNA fragments of 5.lkb and 1.8kb, ligated, and ligated. 72-22 was built. .1 kb fragment obtained by cutting 20 with ^^ 1 1 and 6.9 kb fragment obtained by cutting No. 72-22 with 711 and BAP (Bacterial Alkaline Phosphatase; manufactured by Takara Shuzo) The fragments were ligated to construct pPADOl.
PPDA01で常法に従って大腸菌 TG1を形質転換した。 E. coli TG1 was transformed with PPDA01 according to a conventional method.
この pPADOlで形質転換した大腸菌はアンピシリン耐性で選択することができ、 PUC18などの通常使用されるプラスミ ドと同様に大腸菌から容易に調製すること ができる。 その上、 この pPADOlを制限酵素 ay/Mlで切断するだけで 5, 243bpの直鎖 状 D N A断片を切り出すことができる。 Escherichia coli transformed with this pPADOl can be selected for its resistance to ampicillin, and can be easily prepared from Escherichia coli in the same manner as a commonly used plasmid such as PUC18. Furthermore, a linear DNA fragment of 5,243 bp can be cut out only by cutting pPADOl with the restriction enzyme ay / Ml.
pPADOlで形質転換した大腸菌をアンピシリン含有 L B培地中で 37°Cで 16時間、 培養して大量に増やし、 そこから常法に従って pPADOlプラスミ ドを調製した。 こ の pPADOlブラスミド 5 gを^ で切断した後、 T4D N Aリガ一ゼでセルフライ
ゲ一ションさせた DN Aを予め用意した。 Escherichia coli transformed with pPADOl was cultured in LB medium containing ampicillin for 16 hours at 37 ° C for 16 hours, and pPADOl plasmid was prepared therefrom according to a conventional method. After cutting 5 g of this pPADOl brassmid with ^, self-fry with T4 DNA ligase. A gated DNA was prepared in advance.
鶏痘生ワクチン株の一つである CEVA株 （米国農務省認可） を感染させた C EF 細胞からは本発明の D N Aベクタ一は検出されなかったので、 この CEVA株を感染 させた C E F細胞に、 上記の pPADOlから切り出しセルフライゲ一ションした DN Aをエレクトロボレ一ションで導入した。 The DNA vector of the present invention was not detected in CEF cells infected with the CEVA strain (approved by the US Department of Agriculture), one of the live fowlpox vaccine strains. The DNA fragmented from pPADOl and self-ligated was introduced by electroporation.
具体的には、 直径 1.5cmのディッシュに Eagle' s MEM培地中で培養した C E F細 胞に鶏痘ウィルス CEVA株を m.o. i. (感染価） = 1で感染させた。 感染 4時間後に P B Sで細胞を 2回洗浄し、 トリプシン処理をして細胞をディシュ底面からはが し、 血清 （5%CS) 入りの MEM培地を加えてトリプシン消化を止めるとともに、 細 胞を 50mL用ファルコンチューブに集めた。' 1， 000 X gで 5分間の遠心分離を行い、 遠心上澄を捨てたのち、 Saline G緩衝液 （8.0g NaCl、 0.4g KC1、 0.395g Na2HP0 4·12Η20、 0.2g KH2P04、 0. lg MgCl2'6H20、 0. lg CaCl2-6H20、 1, lg グルコース/ L) で細胞を洗浄した。 Specifically, CEF cells cultured in a 1.5 cm diameter dish in Eagle's MEM medium were infected with fowlpox virus CEVA strain at moi (infection titer) = 1. Four hours after infection, wash the cells twice with PBS, trypsinize the cells, detach the cells from the bottom of the dish, add MEM medium containing serum (5% CS) to stop trypsin digestion, and remove 50 mL of cells. Collected in a falcon tube. '1, 000 X g at centrifuged for 5 minutes, then discarding the centrifugal supernatant, Saline G buffer (8.0g NaCl, 0.4g KC1, 0.395g Na 2 HP0 4 · 12Η 2 0, 0.2g KH 2 P0 4, 0. lg MgCl 2 '6H 2 0, 0. lg CaCl 2 -6H 2 0, 1, the cells were washed with lg of glucose / L).
もう一度、 0.8mLの Saline Gで細胞を懸濁し、 この 0.7mLをエレクト口ポレーシ ヨンキュベット （バイオラッド社製） に移した。 ここに、 予め用意したセルフラ ィゲ一シヨンした DNAを加えて、 1.2kV、 25 zFの条件でエレクトロボレ一シ ヨンした。 その後、 室温に 10分静置して 25cm2フラスコに細胞を移し、 MEM培地を 5mL添加し、 37°Cの C02ィンキュベータ一中で 6日間培養した。 The cells were suspended once again in 0.8 mL of Saline G, and 0.7 mL of the suspension was transferred to an election port cuvette (manufactured by Bio-Rad). To this, a previously prepared self-ligated DNA was added, and electrophoresis was performed under the conditions of 1.2 kV and 25 zF. Thereafter, the cells were allowed to stand at room temperature for 10 minutes, transferred to a 25 cm 2 flask, added with 5 mL of MEM medium, and cultured in a CO 2 incubator at 37 ° C. for 6 days.
6曰後、 フラスコごと培養液を凍結融解した。 凍結融解を 3回繰リ返した後、 この培養液をファルコンチューブに移し、 4°C、 5，000Xgで 5分間遠心して細胞 の残骸を除いて上澄み液を回収した。 この液中には鶏痘ウィルス CEVA株と複製し た DNAベクタ一とが入っている。 After 6 the culture was frozen and thawed with the flask. After three freeze-thaw cycles, the culture was transferred to a Falcon tube and centrifuged at 5,000 Xg for 5 minutes at 4 ° C to remove the cell debris and collect the supernatant. This solution contains the fowlpox virus CEVA strain and the replicated DNA vector.
直径 9 cmデツイシュ中 MEM培地でサブコンフレントまで培養した別の C E F細 胞にこの液を 1/10量添加し、 1週間培養した。 1週間後に、 実施例 1で示した方 法によって感染細胞から D N Aを回収した。 0.8%ァガロースゲル電気泳動で見 てみると、 約 5 kbの DN Aが大量に確認された。 One-tenth volume of this solution was added to another CEF cell cultured to subconfluence in MEM medium in a dish with a diameter of 9 cm and cultured for one week. One week later, DNA was recovered from the infected cells by the method described in Example 1. When examined by 0.8% agarose gel electrophoresis, a large amount of about 5 kb of DNA was confirmed.
一方、 5kbの DNAを鶏痘ウィルス未感染細胞に導入した場合には、 DNAベ クタ一は検出されず、 この D N Aベクタ一が鶏痘ゥィルス感染細胞のみで複製す ることが示された。
(実施例 5 ) 組み換え DNAベクターの構築 On the other hand, when 5 kb DNA was introduced into fowlpox virus-uninfected cells, no DNA vector was detected, indicating that this DNA vector replicates only in fowlpox virus-infected cells. (Example 5) Construction of recombinant DNA vector
( 1 ) pPADdl(454卜 4922)の構築 (図 4参照) (1) Construction of pPADdl (454 4922) (See Fig. 4)
pPADO 1を制限酵素 £co811と Spelとで二重切断し、 反応緩衝液中で D N Aポリメ ラ一ゼ I (10ユニット/ mL) により平滑末端にした。 その後、 T4DNAリガ一ゼ で環状にして大腸菌 TG1を形質転換し、 7.5kbのプラスミ ド断片の末端をアンピシ リン含有 L B培地で培養した。 pPADO1 was double digested with the restriction enzymes £ co811 and Spel and blunt-ended with DNA polymerase I (10 units / mL) in reaction buffer. Thereafter, Escherichia coli TG1 was transformed by cyclization with T4 DNA ligase, and the end of the 7.5 kb plasmid fragment was cultured in an LB medium containing ampicillin.
出現したアンピシリン耐性コロニーをいくつか拾い、 常法に従ってそれらの形 質転換体からプラスミ ドを回収し、 全長が 7.5kbで、 a;zfllで切断した時 4.8kbの 断片が切リ出されるプラスミ ドを選択した。 このプラスミ ドは配列番号 2の 4, 54 1〜4，922番目までの DNA領域が欠損したものを含むプラスミ ドであり、 pPADdl (4541- 4922)と命名した。 Some of the emerged ampicillin-resistant colonies were picked up, and plasmids were recovered from the transformants according to a conventional method. The plasmid was 7.5 kb in total length, and a 4.8 kb fragment was cut out when cut with a; zfll. Was selected. This plasmid is a plasmid containing a deletion of the DNA region from SEQ ID NO: 2 to positions 4,541 to 4,922, and was named pPADdl (4541-4922).
(2) pPADrepL(4541- 4922)の構築 (図 4参照) (2) Construction of pPADrepL (4541--4922) (See Fig. 4)
pPADO 1を制限酵素 fcoS 11と Spelとで二重切断して 7.5kbのプラスミド断片を回 収し、 配列番号 3で示す DNA配列と、 これと対を形成する DNA断片 （配列番 号 4) とからなる合成 DN Aアダプタ一とを T4DN Aライゲースを使ってライゲ —シヨンして、 大腸菌を形質転換した。 得られた形質転換体の配列番号 2の 4,54 1〜4， 922番目までの DNA領域が合成アダプタ一と置換されたプラスミ ドを構築 した。 pPADO 1 was double-cleaved with the restriction enzymes fcoS 11 and Spel, and a 7.5 kb plasmid fragment was recovered. The DNA sequence represented by SEQ ID NO: 3 was paired with a DNA fragment (SEQ ID NO: 4) forming a pair. Was synthesized using T4DNA ligase and transformed into Escherichia coli. A plasmid was constructed in which the DNA region at positions 4,54 1 to 4,922 of SEQ ID NO: 2 of the obtained transformant was replaced with a synthetic adapter-1.
この合成アダプタ一には A¾el、 Bln Sal Xho dサイ トの塩基配列があ リ、 この場所に外来遺伝子を揷入しやすくしたものである。 このプラスミ ドを pP ADrepL (4541-4922)と命名した。 This synthetic adapter has the base sequences of A¾el and Bln Sal Xhod sites, which facilitate the introduction of foreign genes into these locations. This plasmid was named pP ADrepL (4541-4922).
( 3) pPADin(454卜 4759)P7.5:lacZの構築 (図 1及び 4参照） (3) Construction of pPADin (454 4759) P7.5: lacZ (See Figures 1 and 4)
( 3— 1 ) pNZ76の構築 （図 1参照） (3-1) Construction of pNZ76 (See Figure 1)
ワクシニアウィルス WR株の 7.5Kダルトンのぺプチドをコードする DN Aのプロ モータ一を含む約 0.26Kbpの 5a 〜^ al断片（Cell, 125:805-813(1983))を、 pUC9 の Sa A^I部分に組込み、 プラスミ ド pUWP-Ιを構築した。 An approximately 0.26 Kbp 5a to ^ al fragment (Cell, 125: 805-813 (1983)) containing the promoter of DNA encoding the 7.5 K dalton peptide of the vaccinia virus WR strain was obtained from SaA ^ of pUC9. A plasmid pUWP-II was constructed by incorporating it into the I part.
10 z gの pMA001(Shirakawaら、 Gene, 28: 127 -，（1984))を // MIで消化した。 2 gの PUC18(フアルマシア社製）を arfllで消化した後、 フエノール—クロ口ホル
ム （ 1 ： 1 ) で抽出し、 エタノール沈殿により、 開裂した pUC18を回収した。 5' - 末端リン酸をアルカリホスファタ一ゼで処理して除去し、 DN Aを再びフエノー ルークロロホルム （ 1 : 1 ) で抽出し、 エタノールで沈殿させて回収した。 10 zg of pMA001 (Shirakawa et al., Gene, 28: 127-, (1984)) was // digested with MI. After 2 g of P UC18 a (Pharmacia Co.) was digested with Arfll, phenol - black port Hol (1: 1), and cleaved pUC18 was recovered by ethanol precipitation. The 5'-terminal phosphate was removed by treatment with alkaline phosphatase, and the DNA was again extracted with phenol-chloroform (1: 1) and recovered by precipitation with ethanol.
開裂した 0.2 の pUC18と、
と// /τχΠΠとで消化した 1 gの精製 APV(NP 株） D N Aとをリガ一ゼによって連結し、 これによつてコンビテントな大腸菌 JM1 03を形質転換し、 形質転換体を得た。 0.2 pUC18 cleaved, Then, 1 g of purified APV (NP strain) DNA digested with and /// τ 連結 was ligated with ligase, thereby transforming competent Escherichia coli JM103 to obtain a transformant.
この形質転換体を、 0.03%の 5-ブロモ -4-ク口口- 3-ィンドリル- β - D-ガラクト ピラノシド、 0.03mMのイソプロピル- /3- D-ガラクトビラノシド、 40 g/mLのアン ピシリンを含む L B寒天培地で、 15時間、 37°Cにて培養した。 寒天培地上に生育 した形質体のうち、 白色コロニーをとり、 40 g/mLのアンピシリンを含む L B液 体培地で、 15時間、 37°Cにて培養した。 形質転換体を回収し、 ビルンボイムとド —リーの方法 （Nucleic Acid Research, 7:1513-, (1979)) に従ってプラスミ ド を抽出し、 oRIと //κΠΠとで二重消化した。 この二重消化断片を 0.6%ァガロ —ス電気泳動によって、 ^—ガラクトシダ一ゼ遺伝子 （約 3.3Kbp) を回収した。 一方、 0.3 の pUC19を τίΐΐで消化し、 フエノールークロロホルム （ 1 : 1 ) で抽出し、 エタノール沈殿により回収した。 この回収 PUC19と /3—ガラクトシダ —ゼ遺伝子とをリガ一ゼでライゲーシヨンし、 組換えプラスミ ド PNZ66を作製し た。 This transformant was treated with 0.03% of 5-bromo-4-cucto-3-indolyl-β-D-galactopyranoside, 0.03 mM of isopropyl- / 3-D-galactopyranoside, 40 g / mL. The cells were cultured on LB agar medium containing ampicillin for 15 hours at 37 ° C. Among the traits grown on the agar medium, white colonies were picked and cultured in LB liquid medium containing 40 g / mL ampicillin for 15 hours at 37 ° C. Transformants were recovered, plasmid was extracted according to the method of Birnboim and Dolly (Nucleic Acid Research, 7: 1513-, (1979)), and double digested with oRI and // κΠΠ. The ^ -galactosidase gene (about 3.3 Kbp) was recovered from this double digested fragment by 0.6% agarose electrophoresis. On the other hand, 0.3 pUC19 was digested with τίΐΐ, extracted with phenol-chloroform (1: 1), and recovered by ethanol precipitation. The recovered PUC19 and the / 3-galactosidase gene were ligated with ligase to prepare recombinant plasmid PNZ66.
一方、 40 gの pUWP-1を// pallと coRIとで二重消化し、 15%の低融点ァガ口一 ス電気泳動 （70V、 6時間） にかけて 7.5Kプロモーターを含む約 0.26kbpの断片を 分離し、 ァガロースゲルを細かく砕いて TE緩衝液（lOraMトリス塩酸、 lmM EDTA、 H 8.0) で DNAを回収した。 この DNA断片は付着末端を有するため、 DNA ポリメラーゼで平滑末端とし、 7.5Kプロモーター遺伝子を得た。 On the other hand, 40 g of pUWP-1 was double digested with // pall and coRI, and subjected to 15% low-melting point agarose gel electrophoresis (70 V, 6 hours) to obtain a fragment of about 0.26 kbp containing the 7.5K promoter. Was separated, and the agarose gel was crushed into small pieces, and the DNA was recovered using a TE buffer (lOraM Tris-HCl, lmM EDTA, H8.0). Since this DNA fragment had cohesive ends, it was made blunt with DNA polymerase to obtain a 7.5K promoter gene.
0.3；1^の 66を ^11で消化し、 フエノールークロロホルム （ 1 : 1 ) で抽 出し、 エタノール沈殿により回収した。 この///; κΙΙ消化 pNZ66 ( 一ガラクトシ ダーゼ遺伝子を含む） と上記のようにして得た 7.5Κプロモーター遺伝子とをリガ ーゼを用いてライゲーシヨンし、 プラスミ ド ΡΝΖ76を得た。 0.3; 1 ^ 66 was digested with ^ 11, extracted with phenol-chloroform (1: 1), and recovered by ethanol precipitation. This ///; κΙΙ digested pNZ66 (containing the galactosidase gene) and the 7.5Κ promoter gene obtained as described above were ligated using ligase to obtain plasmid ΡΝΖ76.
(3 - 2) pPADin(454卜 4759)P7.5:lacZの構築 (図 4参照) (3-2) pPADin (454 4759) P7.5: Construction of lacZ (see Figure 4)
上記 （3— 1 ) のようにして得た pNZ76を〃/;7 IIと とで切断し、 ワクシニ
ァウィルスの 7.5Kプロモータ一遺伝子と、 その直後に大腸菌の /3—ガラクトシダ —ゼをコードする遺伝子とが繋がつた約 3kbpの D N A断片を切り出し、 0.8 %ァ ガロースゲル電気泳動 （100V、 40分） した後にァガロースゲルから回収した。 また、 pPADOlを//// κΙΙΠΖ Iで切断して出現する 3.3kbの DNA断片、 及び pP ADrepL(454卜 4922)を Z Iで切断して出現する 4.5kbの DNA断片も同じく 回収した。 The pNZ76 obtained as in (3-1) above was cut with 〃 /; 7 II and Approximately 3kbp DNA fragment, which is linked to the 7.5K promoter gene of E. coli virus and the gene encoding / 3 galactosidase of E. coli immediately after, was excised and subjected to 0.8% agarose gel electrophoresis (100V, 40 minutes). Recovered from agarose gel. In addition, a 3.3 kb DNA fragment that appeared by cutting pPADOl with //// κΙΙΠΖI and a 4.5 kb DNA fragment that appeared by cutting pPADrepL (454-4922) with ZI were also recovered.
回収した 3断片を、 T4DNAライゲ一スでライゲーシヨンして大腸菌 TG1を形 質転換した。 形質転換により出現したアンピシリン耐性形質転換体の中から、 la cZ遺伝子を含む約 3 kbの D N A断片が、 配列番号 2の 4, 541番目〜4， 759番目の D N A領域に揷入された全長約 10.8kbのプラスミ ドを持つものを選択した。 The three recovered fragments were ligated with a T4 DNA ligase to transform E. coli TG1. From the ampicillin-resistant transformants that emerged by the transformation, a DNA fragment of about 3 kb containing the lacZ gene was inserted into the DNA region at positions 4,541 to 4,759 in SEQ ID NO: 2. Those having a plasmid of 10.8 kb were selected.
このプラスミ ドを pPADin(454卜 4759)P7.5： lacZと命名した。 This plasmid was named pPADin (4544544759) P7.5: lacZ.
(実施例 6 ) 組み換え D N Aベクタ一の細胞への導入と外来遺伝子の発現(Example 6) Introduction of a recombinant DNA vector into cells and expression of foreign genes
( 1 ) 配列番号 2の 4， 541〜4， 922の塩基配列が欠損した組み換え DNAベクタ一 の細胞への導入 (1) Introduction of a recombinant DNA vector lacking the nucleotide sequence of 4,541 to 4,922 of SEQ ID NO: 2 into cells
実施例 4の pPADOlの代わりに実施例 5の （ 1 ) で構築した pPADdl(454卜 4922) を用いること以外は全く同様にして、 配列番号 2の 4,541〜4,922番目の塩基配列 が欠損した組み換え D N Aベクタ一が細胞へ導入できないかどうかを調べた。 その結果、 鶏痘ウィルス感染細胞にエレクトロポレーシヨンで導入した場合、 4.8kbの D N Aベクタ一が複製されることが判明した。 In the same manner except that the pPADdl (454-4922) constructed in (1) of Example 5 was used instead of the pPADOl of Example 4, a recombinant DNA lacking the nucleotide sequence at positions 4,541 to 4,922 of SEQ ID NO: 2 It was examined whether the vector could not be introduced into cells. As a result, it was found that when introduced into fowlpox virus-infected cells by electroporation, a 4.8 kb DNA vector was replicated.
(2) 配列番号 2の 4， 541〜4， 922の塩基配列が 32bpのマルチクローニングサイト と置換されている組み換え DN Aベクターの細胞への導入 (2) Introduction of a recombinant DNA vector in which the nucleotide sequence of 4,541 to 4,922 of SEQ ID NO: 2 has been replaced with a 32 bp multicloning site
実施例 4の pPADOlの代わりに実施例 5の（ 2 )で構築した pPADrepL(4541- 4922) を用いること以外は全く同様にして、 配列番号 2の 4, 541〜4， 922の塩基配列が 32 bpのマルチクローニングサイ トと置換されている組み換え DNAベクタ一が細胞 へ導入できないかどうかを調べた。 The nucleotide sequence of 4,541 to 4,922 in SEQ ID NO: 32 was changed in exactly the same manner except that pPADrepL (4541-4922) constructed in (2) of Example 5 was used instead of pPADOl in Example 4. It was examined whether the recombinant DNA vector substituted with the bp multicloning site could not be introduced into cells.
その結果、 やはり鶏痘ウィルス感染細胞にエレクトロポレーシヨンで導入した 場合に、 4.8kbの DNAベクタ一が複製された。 As a result, a 4.8 kb DNA vector was also replicated when the cells were transfected into fowlpox virus-infected cells by electroporation.
( 3 ) 配列番号 2の 4， 541〜4, 759の塩基配列領域にワクシニアウイルス 7.5Kプロ
モ一ターと /3 _ガラクトシダ一ゼ遺伝子とが揷入された組み換え DN Aベクタ一 の細胞への導入と —ガラクトシダ一ゼの発現 (3) Vaccinia virus 7.5K promoter is located in the nucleotide sequence region of 4,541 to 4,759 of SEQ ID NO: 2. Introduction of a recombinant DNA vector into which a promoter and a / 3_galactosidase gene have been introduced and expression of galactosidase
(3— 1 ) 上記組み換え DNAベクターの細胞への導入 (3-1) Introduction of the above recombinant DNA vector into cells
pPADin(4541-4759)P7.5： lacZで形質転換した大腸菌 TG1を MEM中、 37°Cで 40mL 培養し、 その菌体からプラスミ ド pPADin(4541— 4759)P7.5： lacZを約 50 g調製 した。 pPADin (4541-4759) P7.5: Escherichia coli TG1 transformed with lacZ was cultured in MEM at 37 ° C for 40 mL, and about 50 g of plasmid pPADin (4541-4759) P7.5: lacZ was obtained from the cells. It was prepared.
鶏痘ウィルス CEVA株を m.o. i. =0.2で感染させて 4時間後の C E F細胞 （ 1 X 10 7細胞） をトリプシン処理で回収し、 実施例 4で示すように Saline Gで懸濁した 0. 7mLに pPADin(4541— 4759) P7.5： lacZをエレクトロボレ一シヨンで導入した。 その後、 室温に 10分静置して 25cm2フラスコに細胞を移し、 MEM培地を 5mL添加 した。 6日後、 フラスコごと培養液をドライアイス一エタノールと水浴とで凍結 融解させた。 Four hours after the fowlpox virus CEVA strain was infected at moi = 0.2, CEF cells (1 × 10 7 cells) were collected by trypsinization, and suspended in 0.7 mL of Saline G as shown in Example 4. pPADin (4541-4759) P7.5: lacZ was introduced by electroboration. Thereafter, the cells were allowed to stand at room temperature for 10 minutes, transferred to a 25 cm 2 flask, and 5 mL of MEM medium was added. Six days later, the culture solution together with the flask was freeze-thawed with dry ice / ethanol and a water bath.
凍結融解を 3回繰り返した後の培養液を 15mLのフアルコンチューブに移し、 5， 000 X で 5分間の遠心条件で細胞残骸を除いた FPVと組み換え DNAベクタ一と を含む調製液を回収した。 After the freeze-thawing was repeated three times, the culture solution was transferred to a 15 mL Falcon tube, and a preparation solution containing FPV from which cell debris had been removed and the recombinant DNA vector was collected by centrifugation at 5,000 X for 5 minutes.
(3 - 2) /3—ガラクトシダ一ゼの発現 Expression of (3-2) / 3-galactosidase
この液を MEM培地で 10倍ずつ 4回段階希釈して、 その lmLを直径 9 cmのディッ シュ中で 37°Cでサブコンフレントになるように培養した別の C E F細胞に添加し、 1時間後に上清を捨てた後、 0.8%の寒天を含む MEM培地を重層して約 6日間培養 した。 This solution was serially diluted four times in 10-fold MEM medium, and 1 mL of the solution was added to another CEF cell cultured at 37 ° C in a 9 cm diameter dish so that it became subconfluent. After discarding the supernatant later, a MEM medium containing 0.8% agar was overlaid and cultured for about 6 days.
500 g/mLのブルォガル（Halogenated indolyl-^ -D-galactoside； GIBC0 BRL社 の商品名 Bluo-ga を添加した 0.8%の寒天培地を更に重層し、 37°Cの C02インキ ュべ—ター中に静置したところ、 3時間後にはディッシュ中に青いプラークが多 数見出された。 これらのプラークは —ガラクトシダーゼが発現したことを示し ている。 500 g / mL of Buruogaru (Halogenated indolyl- ^ -D-galactoside; GIBC0 BRL trade name and Bluo-ga further overlayed with 0.8% agar medium supplemented with, the 37 ° C C0 2 ink Yube - in ter After 3 hours, a number of blue plaques were found in the dish after three hours, indicating that -galactosidase was expressed.
(実施例 7 ) 組み換え DNAベクタ一と組み換え鶏痘ウィルスベクタ一の外来遺 伝子との発現量の比較 (Example 7) Comparison of the expression levels of the recombinant DNA vector and the recombinant fowlpox virus vector with the foreign gene
実施例 6の最後の所で得られた青色プラークをパスツールピぺットで抜き取り、
1 mLの MEM培地に懸濁し、 10〜1， 000倍に段階希釈して直径 9 cmのディッシュで METhe blue plaque obtained at the end of Example 6 was extracted with a pasteur pit, Suspend in 1 mL of MEM medium, serially dilute 10- to 1,000-fold, and add ME to a 9 cm-diameter dish.
M培地にて培養された C E F細胞 （ 1 X 107個） に添加し、 1時間後に上清を捨て た。 この後に、 0.8%寒天培地を重層して 37°Cで約 6日間培養した。 The cells were added to CEF cells (1 × 10 7 ) cultured in M medium, and after 1 hour, the supernatant was discarded. Thereafter, 0.8% agar medium was overlaid and cultured at 37 ° C for about 6 days.
SOO g/mLのブルォガル（Halogenated indolyl- 3 -D-galactoside;GIBCO BRL社 の商品名 Bluo- gal)を添加した 0.8%寒天培地を更に重層し、 37°Cの C02インキュ ベータ—中に静置した。 約 3時間後には青色プラークが多数出現したので、 できるだけ他のブラークと 離れた青プラークを選択して、 それをパスツールピペットで抜き取り、 lmLの ME M培地に懸濁した。 SOO g / mL of Buruogaru (Halogenated indolyl- 3 -D-galactoside; GIBCO BRL trade name Bluo- gal) was further overlaid with 0.8% agar medium supplemented with, C0 2 incubator of 37 ° C beta - static in Was placed. After about 3 hours, many blue plaques appeared. Blue plaques were selected as far as possible from other plaques, extracted with a Pasteur pipette, and suspended in 1 mL of MEM medium.
この懸濁液を 1.5cmのディッシュ中 MEM培地で培養された C E F細胞 （ 1 X106 個） に添加し、 1時間後に上清を捨て、 20mLの新鮮な MEM培地を添加して 1週間 培養した。 This suspension was added to CEF cells (1 × 10 6 ) cultured in MEM medium in a 1.5 cm dish. One hour later, the supernatant was discarded, and 20 mL of fresh MEM medium was added, followed by culturing for 1 week .
実施例 4と同様に、 培養細胞から凍結融解によって鶏痘ウィルスと組み換え新 規 DNAベクターとを含む調製液を回収した。 そして、 この液に含まれる鶏痘ゥ ィルスのウイルス量を測定した。 As in Example 4, a preparation containing the fowlpox virus and the recombinant new DNA vector was recovered from the cultured cells by freeze-thawing. Then, the amount of fowlpox virus contained in this solution was measured.
培養した CE F細胞が入った直径 9 cmデッシュ 6枚に、 鶏痘ウィルスが m.o. i. = 1 となる様にこの液を適当に希釈して加えた。 添加後 0時間、 5時間、 10時間、 24時間、 48時間、 72時間後にそれぞれ 1枚のディッシュの感染細胞を集め、 PB Sで細胞を洗浄して、 0.5mLの PB Sに懸濁させた後、 このサンプルを—20°Cで 凍結した。 This solution was diluted appropriately and added to 6 plates of 9 cm in diameter containing the cultured CEF cells so that fowlpox virus could be m.o.i. = 1. At 0 hours, 5 hours, 10 hours, 24 hours, 48 hours, and 72 hours after the addition, infected cells were collected from one dish, washed with PBS, and suspended in 0.5 mL of PBS. Later, the sample was frozen at -20 ° C.
予定したサンプルが全て揃った後にサンプルを融解させ、 クロ口ホルムを 2滴 添加し、 その 10 tLを酵素反応溶液（60mM Na2HP04'7H20、 40mM NaH2P04-H20, lOmM KC1、 ImM MgS04'7H20、 50mM /3—メルカプトエタノール） 990^Lに加えた。 Samples were thawed after samples appointment has everything was added 2 drops of black hole Holm, the 10 tL enzyme reaction solution (60mM Na 2 HP0 4 '7H 2 0, 40mM NaH 2 P0 4 -H 2 0, lOmM KC1, ImM MgS0 4 '7H 2 0, 50mM / 3- mercaptoethanol) was added to 990 ^ L.
この反応液を 28°Cでィンキュベ一トし、 酵素反応液で 4mg/mLの濃度に溶かした 基質 0NPG (0-ニトロフエニル /3-D-ガラクトピラノシド（シグマ社製；製品番号 N1 127) を 0.2mL加えて酵素反応を開始し、 適当な時間で 1M K2C03で酵素反応を止め、 420nmの吸光度を測定した。 This reaction solution was incubated at 28 ° C and dissolved in an enzyme reaction solution to a concentration of 4 mg / mL. Substrate 0NPG (0-nitrophenyl / 3-D-galactopyranoside (Sigma; product number N1 127)) the enzymatic reaction was initiated by adding 0.2 mL, stop the enzymatic reaction in 1M K 2 C0 3 at appropriate time, the absorbance was measured at 420 nm.
また対照サンプルとして、 fNZ1029を同様に m.o. i.= 1で C E F細胞に感染させ、 /3—ガラクトシダーゼの発現量を調べた。 fNZ1029は特開平第卜 168279号公報（米
国特許第 5， 387， 519号） で開示されている /3—ガラクトシダ一ゼ遺伝子が挿入さ れた組み換え鶏痘ウィルスである （図 6) 。 As a control sample, fNZ1029 was similarly infected to CEF cells at moi = 1, and the expression level of / 3-galactosidase was examined. fNZ1029 is disclosed in Japanese Unexamined Patent Publication No. 168279 (US This is a recombinant fowlpox virus into which the / 3-galactosidase gene is inserted as disclosed in National Patent No. 5,387,519 (Fig. 6).
尚、 定量に当たってシグマ社より市販されている 3—ガラクトシダーゼ （製品 番号 G6512) を酵素標品とした。 For the determination, 3-galactosidase (product number G6512) commercially available from Sigma was used as an enzyme standard.
その結果を表 1に示すが、 明らかに組み換え新規 DN Aベクタ一による /3—ガ ラクトシダ一ゼ発現量の方が組み換え鶏痘ウイルスべクタ一よりも大きく、 数 10 倍以上となっている。 組み換えべクタ一及び組み換え鶏痘ゥィルスの揷入遺伝子 (/3—ガラクトシダ一ゼ遺伝子） やそのプロモータ一は同じであるので、 この発 現量の差はべクタ一の細胞内でのコピー数によるものと考えられる。 The results are shown in Table 1, and it is clear that the expression level of / 3-galactosidase by the recombinant new DNA vector is larger than that of the recombinant fowlpox virus vector by several tens of times. Because the transgene (/ 3 / 3-galactosidase gene) and the promoter of the recombinant vector and the recombinant fowlpox virus are the same, the difference in the expression level depends on the copy number of the vector in the cell. It is considered something.
CEF細胞に感染 ガラクトシダ一ゼ発現量 （Unit/106細胞） Infect CEF cells Galactosidase expression level (Unit / 10 6 cells)
後の時間（hr) PADin(4541-4759)P7.5:lacz fNZ1029 Later time (hr) PADin (4541-4759) P7.5: lacz fNZ1029
0 0 0 0
5 1,215 22 5 1,215 22
10 3,618 723 10 3,618 723
24 82, 268 4, 192 24 82, 268 4, 192
48 756,036 37,599 48 756,036 37,599
72 1， 684， 342 64， 250 72 1, 684, 342 64, 250
(実施例 8) (Example 8)
社団法人動物用生物学的製剤協会（Japanese Association of Veterinary Biol ogics)が管理している家畜衛生菌株（Cultures for Animal Hygiene)のうち、 鶏 痘ウィルス Nishigahara株 （菌株番号 VA0104)を同協会よリ入手した。 Nishigahara chicken pox virus strain (strain number VA0104) was obtained from the Cultures for Animal Hygiene strain managed by the Japanese Association of Veterinary Biologics. did.
実施例 1の泗水株の代わりにこの株を使用して同様の操作により感染細胞から やはり、 5kbのDNAが得られた。 この DN Aは泗水株から得られた新規 DN A ベクタ一をプローブとしてサザンハイプリダイゼ一シヨンを行うとハイプリダイ
ズした。 A 5 kb DNA was again obtained from infected cells by the same operation using this strain in place of the Sashimi strain of Example 1. This DNA was hybridized by Southern hybridization using a new DNA vector obtained from Sashimi strain as a probe. I did
この時、 対照として用いた鶏痘ウィルスの DNAは全くハイプリダイズしなか つたので、 この 5kbの DNAは、 鶏痘ウィルスゲノム由来の DNAではないこと が示された。 At this time, since the DNA of the fowlpox virus used as a control did not hybridize at all, it was shown that this 5 kb DNA was not DNA derived from the fowlpox virus genome.
また調べた限りにおいては、 Nishigahara株から調製された 5 kbの DN Aの制 限酵素パターンは図 1に示したものと同じであった。 In addition, as far as examined, the restriction enzyme pattern of the 5 kb DNA prepared from the Nishigahara strain was the same as that shown in FIG.
(実施例 9 ) 抗原遺伝子を揷入した組み換え D N Aベクタ一の作製 (Example 9) Preparation of recombinant DNA vector containing an antigen gene
( 1 ) PNZ87の構築 （図 1参照） (1) Construction of PNZ87 (See Figure 1)
PNZ76を 5a Hで消化し、 0.8%ァガロースゲルで電気泳動して、 /3—ガラクト シダ一ゼ遺伝子を含まない約 2.9kbの断片を回収した。 PNZ76 was digested with 5aH and electrophoresed on a 0.8% agarose gel to recover an approximately 2.9 kb fragment that did not contain the / 3-galactosidase gene.
一方、 ハイブリツドファ一ジ mplO- HN180を^ 711と とで二重消化し、 0.8% ァガロースゲルで電気泳動して、 約 1.8kbの NDVの HN遺伝子断片を回収した。 On the other hand, the hybrid phage mplO-HN180 was double-digested with ^ 711 and electrophoresed on a 0.8% agarose gel to recover an approximately 1.8 kb NDV HN gene fragment.
両者をリガーゼによって連結し、コンピテントな大腸菌 TG1株を形質転換した。 この形質転換体を、 0.03%の 5-ブロモ -4-クロ口- 3-ィンドリル-^- D-ガラクトビ ラノシド、 0.03mMのイソプロピル- /3-D-ガラクトビラノシド、 40/Ug/mLのアンピ シリンを含む L B寒天培地で、 15時間、 37°Cにて培養した。 寒天培地上に生育し た形質体のうち、 白色コロニーをとリ、 40 zg/mLのアンピシリンを含む L B液体 培地で、 時間、 37°Cにて培養した。 形質転換体を回収し、 ビルンボイムとド一 リーの方法 （Nucleic Acid Research, 7:1513-, (1979)) に従ってプラスミ ドを 抽出した。 このようにして HN遺伝子を含むプラスミ ド pNZ87を得た。 Both were ligated with ligase to transform competent E. coli TG1 strain. This transformant was treated with 0.03% of 5-bromo-4-chloro-3-indolyl-^-D-galactobilanoside, 0.03 mM of isopropyl- / 3-D-galactoviranoside, 40 / Ug / mL. The cells were cultured on LB agar medium containing ampicillin for 15 hours at 37 ° C. Among the transformants grown on the agar medium, white colonies were collected and cultured in LB liquid medium containing 40 zg / mL ampicillin at 37 ° C for an hour. Transformants were recovered, and plasmids were extracted according to the method of Birnboim and Dolly (Nucleic Acid Research, 7: 1513-, (1979)). Thus, plasmid pNZ87 containing the HN gene was obtained.
(2) 抗原遺伝子を挿入した組み換え DNAベクターの作製 （図 5参照） 上記のプラスミ ド pNZ87を制限酵素 '；7(1111と 3^1とでニ重切断し、 S 1ヌク レア—ゼでその切断末端を平滑にした後、 フエノール： クロ口ホルム ：イソアミ ルアルコール（体積比で 25:24:1)の混合液で 2回抽出処理して S1ヌクレアーゼを 失活させた。 ついで、 ァガロースゲル電気泳動 （0.8%ァガロースゲル、 100V、 4 0分） によって、 ワクシニウィルスの 7.5kDaプロモータ一とニューカッスル病ゥ ィルス （NDV) のへマグルチニン . ノィラミニダ一ゼ （HN) 遺伝子を含む約 1.9kbの断片を分離、 回収した。
また、 pPADOlを^ sfillで切断して、 切断末端を SIヌクレア一ゼにより平滑にし た後、 BAPで末端を脱リン酸化し、 フエノール処理した。 この処理 pADOlと先に回 収した約 1.9kbの断片とをライゲーシヨンして大腸菌 TG1を形質転換した。 (2) Preparation of recombinant DNA vector into which antigen gene was inserted (see Fig. 5) The above plasmid pNZ87 was double-digested with restriction enzyme '; 7 (1111 and 3 ^ 1, and then digested with S1 nuclease. After the blunt ends were blunted, S1 nuclease was inactivated by extracting twice with a mixture of phenol: cloth form: isoamyl alcohol (volume ratio: 25: 24: 1), followed by agarose gel electrophoresis. (0.8% agarose gel, 100 V, 40 min), a 1.9 kb fragment containing the vaccinia virus 7.5 kDa promoter and the Hemagglutinin.neuraminidase (HN) gene of Newcastle disease virus (NDV) was separated. Collected. Also, pPADOl was cleaved with ^ sfill, the cleaved end was blunted with SI nuclease, then the end was dephosphorylated with BAP, and phenol-treated. This treated pADOl was ligated with the previously recovered fragment of about 1.9 kb to transform E. coli TG1.
翌日、 アンピシリン耐性となった形質転換体のコロニーを選択し、 形質転換体 を常法に従って破砕して遠心し、 DNAを抽出した。 この DNAを
で切断 し、 上記のように 0.8%ァガロースゲル電気泳動 （100V、 40分） にかけて分離し た。 The next day, colonies of transformants that became ampicillin resistant were selected, and the transformants were crushed and centrifuged according to a conventional method to extract DNA. This DNA And separated by electrophoresis on a 0.8% agarose gel (100 V, 40 minutes) as described above.
以上の操作によって、 HN遺伝子を含む約 1.9kbの DNA断片が配列番号 2の第 3 77番目の位置に揷入されている全長約 9.8kbのプラスミドをもつものを選択した この目的のブラスミ ドを pPADin(377)P7.5:HNと命名した。 By the above procedure, a plasmid having a total length of about 9.8 kb, in which a DNA fragment of about 1.9 kb containing the HN gene was inserted at position 377 of SEQ ID NO: 2 was selected. It was named pPADin (377) P7.5: HN.
(実施例 10) 組み換え DNAベクタ一を有効成分とするワクチンの調製 (Example 10) Preparation of vaccine containing recombinant DNA vector as an active ingredient
実施例 9で作製したプラスミ ド pPAD i n (377) P7.5 : HNを持つ大腸菌をアンピシリ ン含有 L B培地 40mL中で、 37°Cにて培養し、 そこから pPADin(377)P7.5:HNを調製 した。 そのプラスミ ド 5 μ gを a Iで切断後、 T4DNAリガーゼでセルフライ ゲ一シヨンした DNAを予め用意した。 The plasmid pPAD in (377) P7.5 prepared in Example 9 was cultured at 37 ° C in 40 mL of an LB medium containing ampicillin at 37 ° C, and pPADin (377) P7.5: HN Was prepared. After cutting 5 μg of the plasmid with aI, DNA self-ligated with T4 DNA ligase was prepared in advance.
上記のセルフライゲ一シヨンした DNAをリポフエクチン試薬 （ベ一リンガ一 マンノヽィム社製、 商品名 Dosper Liposomal Transfection Reagent, Cat. No.178 The above-mentioned self-ligated DNA was used as a lipofectin reagent (manufactured by Boehringer Mannheim, trade name: Dosper Liposomal Transfection Reagent, Cat. No.178)
1995) を使って、 鶏痘生ワクチンとして使用されている CEVA株（米国農務省認可） を感染させた CEF細胞に導入した。 1995) was used to transfect the infected CEF cells with the CEVA strain (licensed by the USDA) used as a live fowlpox vaccine.
具体的には、 6ゥエルプレートにて MEM培地で培養した CE F細胞に鶏痘ウイ ルス CEVA株を m.o. i.=0.2で感染させ、 4時間後に MEM培地で 3回洗浄し、 0.5mLの Specifically, fowlpox virus CEVA strain was infected with m.o.i. = 0.2 to CEF cells cultured in MEM medium on a 6-well plate, washed 4 times with MEM medium 4 hours later, and 0.5 mL
MEM培地を加えた。 MEM medium was added.
セルフライゲ一シヨンした DNAは、 エタノール沈殿後、 HB S緩衝液（2.0mM HEPES, 150mM NaCl (pH7.4) )50 Lに溶解した。 The self-ligated DNA was dissolved in 50 L of HBS buffer (2.0 mM HEPES, 150 mM NaCl (pH 7.4)) after ethanol precipitation.
この溶液を溶液 Aとし、 試薬の DOSPER液 10 Lを HB S緩衝液 40^Lで希釈した 溶液を溶液 Bとした。 溶液 Aと溶液 Bを穏ゃかに混合して室温で 15分放置した。 この混合液 100 Lを、 0.5mLの MEM培地培養液に浸っている感染細胞にすこしず つ滴下した。 37°Cで 1時間置いた後、 1.5mLの培養液を追加して、 更に 37°Cで 6
日間培養した。 This solution was used as solution A, and a solution obtained by diluting 10 L of the reagent DOSPER solution with 40 L of HBS buffer was used as solution B. Solution A and solution B were mixed gently and allowed to stand at room temperature for 15 minutes. 100 L of this mixture was slightly dropped onto infected cells immersed in 0.5 mL of MEM medium culture solution. After 1 hour at 37 ° C, add 1.5 mL of culture solution and Cultured for days.
感染細胞を培養液ごと集めて、 凍結融解を 3回繰返した後、 5，000xgで 5分間 の遠心条件で細胞残骸を除いて上澄み液を回収した。 この液中には鶏痘ウィルス CEVA株と組み換え DNAベクタ一が含まれている。 The infected cells were collected together with the culture solution, and freeze-thawing was repeated three times, and then the supernatant was recovered by removing the cell debris by centrifugation at 5,000 xg for 5 minutes. This solution contains the fowlpox virus CEVA strain and the recombinant DNA vector.
この液を 6回 10倍希釈し、 各濃度の液 lmlを直径 9 cmディッシュで培養した C E F細胞に感染させ、 0.8%の寒天を含む MEM培地 lOmLを重層して C02ィンキュベ —ター中にて 37°Cで培養した。 約 6日後に、 鶏痘ウィルスによるプラークがはつ きり見えてきたら、 パスツールピぺットでプラークのウィルス 24個を抜きとリ、 そのウィルスを 24ゥエルプレートで培養した新鮮な C E F細胞に各々感染させた。 This solution was diluted 6 times 10 times, each concentration in the liquid lml were infected with CEF cells cultured in a 9 cm diameter dishes and overlaid with MEM medium lOmL C0 2 Inkyube containing 0.8% agar - C. in ter The cells were cultured at 37 ° C. About 6 days later, when plaques due to fowlpox virus were clearly visible, 24 plaque viruses were removed using pasteur pipets, and the viruses were infected with fresh CEF cells cultured on 24-well plates. I let it.
24ゥエルプレート中、 37°Cで約 1週間培養し、 各ゥエルの培養上清を保存して おき、 感染細胞に 0.2% S D Sの入った P B Sを 0.4mL加えて細胞を溶かし、 その 各ゥエルの溶解液 10 Lをニトロセルロースメンブレンにブロットした。 Incubate the cells in a 24-well plate at 37 ° C for about 1 week, store the culture supernatant of each well, add 0.4 mL of PBS containing 0.2% SDS to the infected cells, lyse the cells, and add 10 L of the lysate was blotted onto a nitrocellulose membrane.
その後、 アルカリ変性液 （1.5M NaCl, 0.5N NaOH) で DNAを変性させた後、 20X S SC液 （175.3gの NaCl、 88.2gのクェン酸ナトリウム（pH8.0)/L) で中和し てから風乾し、 80 °Cで 1時間放置してベ一キングした。 Then, after denaturing the DNA with an alkaline denaturing solution (1.5 M NaCl, 0.5 N NaOH), neutralize it with 20X SSC solution (175.3 g NaCl, 88.2 g sodium citrate (pH 8.0) / L). Then, it was air-dried and left at 80 ° C for 1 hour for baking.
ベーキングしたメンブレンはプレハイプリダイゼ一シヨン洗浄液 （5X S S C, 0.5% S D S , lmM EDTA) に浸して 42°Cで 15分間洗浄した。 The baked membrane was immersed in a pre-hybridization washing solution (5X SSC, 0.5% SDS, lmM EDTA) and washed at 42 ° C for 15 minutes.
その後、 ハイブリダィゼーシヨン液（6X S S C， 0.5%SD S、 3%スキムミル ク） 中に移して 68°Cで 1時間放置し、 その後、 HN遺伝子を放射性同位元素ひ-32 P -dCTPで標識した DNAプローブを加えたハイプリダイゼーシヨン液に移して、 6 8°C で 3時間置いた。 Thereafter, hybrida I See Chillon was allowed 1 hour (6X SSC, 0.5% SD S , 3% Sukimumiru h) and transferred to 68 ° C during the subsequent, HN gene radioisotopes monument - in 32 P -dCTP The solution was transferred to a hybridization solution to which a labeled DNA probe had been added, and left at 68 ° C. for 3 hours.
ついで、 メンブレンを 2 X S S C液で 2回、 1 X S S C液で 1回、 0.2x S S C液で 1回洗浄し、風乾し、 X線フィルムを用いてオートラジオグラフィ一した。 翌日、 X線フィルムを現像して、 黒くなつたスポットに対応するゥエルの保存 上清を、 再び 10倍ずつ段階希釈し、 9cmディッシュに培養した新鮮な CEF細胞 に感染させ、 0.8%の寒天を含む MEM培地 10mLを重層して、 5%C02インキュベータ —中にて 37°Cで培養した。 Then, the membrane was washed twice with 2 × SSC solution, once with 1 × SSC solution, once with 0.2 × SSC solution, air-dried, and subjected to autoradiography using X-ray film. On the next day, develop an X-ray film and invert the stored supernatant of the wells corresponding to the darkened spots again to 10-fold serial dilutions, infect fresh CEF cells cultured in a 9 cm dish, and remove 0.8% agar. The MEM medium containing 10 mL was overlaid and cultured at 37 ° C. in a 5% CO 2 incubator.
プラークが形成されたら、 パスツールピぺットで前と同じように 24個のブラ一 クからパスツールピぺットでウィルスを抜き取り、 24ゥエルプレートの C E F細
胞に各々感染させた。 前と同じ操作を繰返し、 すべてのゥエルのスポットがすべ て DN Aプローブと反応するまで行った。 Once plaques have formed, remove the virus from the 24 blacks with Pasteur pits as before using Pasteur pits and remove the CEF cells from the 24-well plate. The vesicles were each infected. The same operation as above was repeated until all the spots of all the wells reacted with the DNA probe.
すべてのプラークに組み換え DNAベクタ一を含むことが確認できたところで、 それを C E F細胞で培養して鶏痘ワクチンを作製するのと同じ方法でワクチン液 を調製した。 After confirming that all the plaques contained the recombinant DNA vector, a vaccine solution was prepared in the same manner as in the preparation of a fowlpox vaccine by culturing it in CEF cells.
こうして作製したワクチン液には、 組み換え DNAベクター数が鶏痘ウィルス ワクチン株の DNAより約 20倍多い割合で含まれていた。 The vaccine solution thus prepared contained about 20 times as many recombinant DNA vectors as fowlpox virus vaccine strain DNA.
(実施例 11) 組み換え DN Aベクタ一を有効成分とするワクチンによる鶏の免疫 の効果 (Example 11) Effect of immunization of chicken with vaccine containing recombinant DNA vector as an active ingredient
実施例 10で調製したワクチン液を、 鶏痘ウイルスの力価で 106pfu/mlになるよ うに適当に PB Sで希釈し、 O.OlmLを 7日齢の S P F鶏の右側翼膜に接種した。 免疫 2週間後に採血し、 血清中のニューカッスル病の赤血球凝集抑制抗体 （以下 ND-HI抗体と称す） を測定した。 Vaccinated solution prepared in Example 10, was diluted at a titer at 10 6 pfu appropriately by Uni becomes / ml PB S fowlpox virus, to the right wing membrane SPF chicken O.OlmL seven day old did. Two weeks after the immunization, blood was collected, and the serum of Hemagglutination-inhibiting antibody for Newcastle disease (hereinafter referred to as ND-HI antibody) in the serum was measured.
具体的には、 以下のように行った。被検血清を 2倍段階希釈した 25; Lの液に、 4赤血球凝集単位の NDV抗原 25 zLを加えて混和し、 10分間静置して感作した。 その後、 0.5%の鶏赤血球液 50 Lを加えて室温で 45分間静置し、 赤血球凝集を観 察した。 Specifically, the procedure was as follows. The test serum was serially diluted 2-fold to 25; L, and 4 hemagglutinating units of NDV antigen (25 zL) were added thereto, mixed and allowed to stand for 10 minutes to sensitize. Thereafter, 50 L of a 0.5% chicken erythrocyte solution was added, and the mixture was allowed to stand at room temperature for 45 minutes, and red blood cell aggregation was observed.
ND- HI抗体価は赤血球凝集抑制が認められた血清の最高希釈倍数で表した。 The ND-HI antibody titer was represented by the highest dilution of serum in which hemagglutination inhibition was observed.
また比較対照実験として、 米国特許第 5， 286， 639号の実施例 9で用いた PNZ2237 株 （図 7参照） でも同様に免疫し、 ND- HI抗体価を測定したところ、 本発明の組 み換え DN Aベクタ一を有効成分とするワクチンは従来の組み換え鶏痘ウィルス ワクチンよリも約 2倍高い HI抗体を誘導していることがわかった。 産業上の有用性 As a comparative experiment, the PNZ2237 strain (see FIG. 7) used in Example 9 of US Pat. No. 5,286,639 was similarly immunized, and the ND-HI antibody titer was measured. It was found that the vaccine containing the recombinant DNA vector as an active ingredient induced HI antibody about twice as high as that of the conventional recombinant fowlpox virus vaccine. Industrial utility
本発明によれば、 動物細胞において、 ウィルスベクターの数 10倍ものコピー数 となる新規な DN Aベクター、 及びこのべクタ一を用いて外来遺伝子を高発現さ せる方法が得られ、 動物細胞発現用ベクターとして、 あるいはワクチンとして利 用できる。
配列表 ADVANTAGE OF THE INVENTION According to the present invention, a novel DNA vector having a copy number several tens times that of a viral vector in animal cells, and a method for highly expressing a foreign gene using this vector are obtained. It can be used as a vector for use or as a vaccine. Sequence listing
配列番号： 1 SEQ ID NO: 1
配列の長さ ： 30塩基対 Sequence length: 30 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類： DNA Sequence type: DNA
配列 Array
ACTACCCACA AATCACCGCG TCTCCTCTTG 30 配列番号： 2 ACTACCCACA AATCACCGCG TCTCCTCTTG 30 SEQ ID NO: 2
配列の長さ ： 5243塩基対 Sequence length: 5243 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類： DNA Sequence type: DNA
配列 Array
GTGTTATAGT ATAAGTTCTC CAAAATAAAT AATTAATTCC TCACTACCCA CAAATCACCG 60 CGTCTCCTCT TGACTACCCA CAAATCACCG CGTCTCCTCT TGACTACCCA CAAATCACCG 120 CGTCTCCTCT TGAGTTCTGA CTCGTTTAAC GTCGCTTCAC GAGTCCTTGC AAAGAGGCCT 180 GCCGTCCTGC ACCGTCCAAT AACGCCTCAC CACCCCCGCT CAATTTTTTT CAAATAACGT 240 GCTAACGACT AGCGCACTAA CGGCATAACG CACTAACGAC TAGCGCACTA ACGGCATAAC 300 GCGCTAACGA CTAGCGCACT AACGGCATAA CGCGCTAACG ACTAGCGCAC TAACGGCATA 360 ACGCGTTAAC GACTAGCGCG CGAACGGCAT AACGCGCTAA TGACTCGGTC TCTTGAAAAG 420 AGTCCTGACT CGTTTAACGT CGCTTCACGA GTCCTTCGAT GTACGCGCTA ACGACTATAG 480 CACTAACGGC ATAACGCACG AACATCTTAC GTGTTACTAT ACATAGTAGA CAATATGATG 540 TCTTTGGCTA TTTGAGCTTC TGCTCTTAAT CTTCCGTTTC TTTCTTTTAT CAATATAGAT 600 CGATAGTATA AACATAGTAA TAATAGACCG ATTATCATTA TAGATAAAAT AACAATTATT 660
o o o o o o o o o o o oo oGTGTTATAGT ATAAGTTCTC CAAAATAAAT AATTAATTCC TCACTACCCA CAAATCACCG 60 CGTCTCCTCT TGACTACCCA CAAATCACCG CGTCTCCTCT TGACTACCCA CAAATCACCG 120 CGTCTCCTCT TGAGTTCTGA CTCGTTTAAC GTCGCTTCAC GAGTCCTTGC AAAGAGGCCT 180 GCCGTCCTGC ACCGTCCAAT AACGCCTCAC CACCCCCGCT CAATTTTTTT CAAATAACGT 240 GCTAACGACT AGCGCACTAA CGGCATAACG CACTAACGAC TAGCGCACTA ACGGCATAAC 300 GCGCTAACGA CTAGCGCACT AACGGCATAA CGCGCTAACG ACTAGCGCAC TAACGGCATA 360 ACGCGTTAAC GACTAGCGCG CGAACGGCAT AACGCGCTAA TGACTCGGTC TCTTGAAAAG 420 AGTCCTGACT CGTTTAACGT CGCTTCACGA GTCCTTCGAT GTACGCGCTA ACGACTATAG 480 CACTAACGGC ATAACGCACG AACATCTTAC GTGTTACTAT ACATAGTAGA CAATATGATG 540 TCTTTGGCTA TTTGAGCTTC TGCTCTTAAT CTTCCGTTTC TTTCTTA TATAG ATATAG ATTAGA TATAG ATATAG ATT ooooooooooo oo o
D C 1 00 to 00 D C 1 00 to 00
LO CO D LO CO D
1 1
09S
09S
S 06 S 06
菌 Fungus
JDVV VV3VV ν{ν1¾νΰν}ν JDVV VV3VV ν {ν1¾νΰν} ν
sVD 09 Iilv O13VU3.S 1 D0V111VV13DV V331VV〕 V 1。 V1V V13VV1VV11V3V VVD1_ sVD 09 Iilv O13VU3.S 1 D0V111VV13DV V331VV] V1. V1V V13VV1VV11V3V VVD1_
vvlivsv v- Qvv V 33VVV1VV533 VVVV3V 13V寒 z VV31VJL11
3G31V1V3VDVV L VSLLV3VDV33VV3Lvvlivsv v- Qvv V 33VVV1VV533 VVVV3V 13V Cold z VV31VJL11 3G31V1V3VDVV L VSLLV3VDV33VV3L
Vv {31 o3V3113VV VElL3V11VVVD o Vv (31 o3V3113VV VElL3V11VVVD o
CO O O O co CO O O O co
VDiVVDiVDiVVDi
3 Three
Vi: 09 V113Vl Vi: 09 V113Vl
1 1
VI VI
vs 0 vs 0
V3V V3V
3D3DVJS1 3D3DVJS1
«υ o «Υ o
οοε 31V οοε 31V
313V1V3 3333113 3 313V1V3 3333113 3
3V V Dvfwl11V1Vvll113 - V § V1LLV133I IIVIVDIIIV 133V1VJ 3V V Dvfwl11V1Vvll113-V § V1LLV133I IIVIVDIIIV 133V1VJ
3VV1LL 099 315iv V1V3VV133 VV113DVV sv3vjvVJvl IV 3VV1LL 099 315iv V1V3VV133 VV113DVV sv3vjvVJvl IV
ViiVvD339} OJ-V ililVM 311VVV.I3VV93J1一 ViiVvD339} OJ-V ililVM 311VVV.I3VV93J1
1 V3V ViiD 3VVDSD33V§1V1VVV1VD 。 vvv 1 V3V ViiD 3VVDSD33V§1V1VVV1VD. vvv
VVV VV3 V V1V3V SVVVVJ VVV VV3 V V1V3V SVVVVJ
Ls31V119 VV3V}3VVO 006ε 1V1VV11VV.I 03V3V DVVD1V13 fvv1V1 Ls31V119 VV3V} 3VVO 006ε 1V1VV11VV.I 03V3V DVVD1V13 fvv1V1
VJiνν1VDV96 r νν3 VVi33vlDv3 Vii 0 VI1V 31i一 VJiνν1VDV96 r νν3 VVi33vlDv3 Vii 0 VI1V 31i
303DV1V1 VU 0vvv JL33D3V1V3 303DV1V1 VU 0vvv JL33D3V1V3
l llDllDD3V3vvivll1VV D醫 vlvls DVVivl3vー l llDllDD3V3vvivll1VV D Doctor vlvls DVVivl3v ー
0
AAGAACATTC AGCGGCTTAT TTTAAAGGTT TGAAGTTTTA CTGCTTAGAT CAAAAAGTAG 42000 AAGAACATTC AGCGGCTTAT TTTAAAGGTT TGAAGTTTTA CTGCTTAGAT CAAAAAGTAG 4200
TAGCTAAAGG GATTAATAAA AAGATTCTAA AAGATATGAG TATGATGCAT ATTTTAGGAG 4260TAGCTAAAGG GATTAATAAA AAGATTCTAA AAGATATGAG TATGATGCAT ATTTTAGGAG 4260
AAGAAACGAT CGTTGAAAAC GAACGATGGA GCGTGGATGA AAAAGCTAGA ATCGTGATTA 4320AAGAAACGAT CGTTGAAAAC GAACGATGGA GCGTGGATGA AAAAGCTAGA ATCGTGATTA 4320
AAAATACAGA ATTTATTTAC GGAGATCGAT ACGGTAAAGG ATCTCACGTT AAAGAAGAAT 4380AAAATACAGA ATTTATTTAC GGAGATCGAT ACGGTAAAGG ATCTCACGTT AAAGAAGAAT 4380
ACGAAGCGTT AAACCGCTAA TTGAAGCGTT ACCGCTAATT AAAGCGTAGT CTTCCAAAAA 4440ACGAAGCGTT AAACCGCTAA TTGAAGCGTT ACCGCTAATT AAAGCGTAGT CTTCCAAAAA 4440
GGTTTAATCC AGTTAGCAGA TAAGCAAAAA GTATCTATAT TTGAATTATT ATGAGGCGGA 4500GGTTTAATCC AGTTAGCAGA TAAGCAAAAA GTATCTATAT TTGAATTATT ATGAGGCGGA 4500
AAAGGTTTTG GAAACGGTCC ATTATTACAA CTATCTCCCT GAGGATAAAC CTCTTGCCAT 4560AAAGGTTTTG GAAACGGTCC ATTATTACAA CTATCTCCCT GAGGATAAAC CTCTTGCCAT 4560
TTATCCTTCG TAACGTTCAC CGGAACCATC CAAAATTCAC CCGGTGGTAG CGCGTCTTCG 4620TTATCCTTCG TAACGTTCAC CGGAACCATC CAAAATTCAC CCGGTGGTAG CGCGTCTTCG 4620
TCGGGCCAGG AAAAGAAACC TATAGGATCG TGAGTTCTGT ATTTTTTCTC AAAATTTCTT 4680TCGGGCCAGG AAAAGAAACC TATAGGATCG TGAGTTCTGT ATTTTTTCTC AAAATTTCTT 4680
CGATTAATAC TCTCTTCACA CTGTGTTCTT AATTTACTAC CGGTTATGTT CATTCCGGTT 4740CGATTAATAC TCTCTTCACA CTGTGTTCTT AATTTACTAC CGGTTATGTT CATTCCGGTT 4740
TGTATCCACA TTCCGAAAGC TTGACAAGTA GCGGCGTTAA TACATTGGGT AGCGTTTAGA 4800TGTATCCACA TTCCGAAAGC TTGACAAGTA GCGGCGTTAA TACATTGGGT AGCGTTTAGA 4800
TACCCATCGA ATATTTCGGT AACGTTAGTA GCTTGGGGCA TCGCGACAGT AATTTTACTG 4860TACCCATCGA ATATTTCGGT AACGTTAGTA GCTTGGGGCA TCGCGACAGT AATTTTACTG 4860
TAATTACCGA TAGCCATAAA CGTACCAGTA AGACAGAATA AAATATTAGC CGGAATAGTA 4920TAATTACCGA TAGCCATAAA CGTACCAGTA AGACAGAATA AAATATTAGC CGGAATAGTA 4920
CTAGTATTTG TGATAATGGT AACGTTTACT TTAGTTATTG GAGGAAAAGT GGGTAGCGCT 4980CTAGTATTTG TGATAATGGT AACGTTTACT TTAGTTATTG GAGGAAAAGT GGGTAGCGCT 4980
ATTGCCGCCA TCTTACATAT TATATAAATT TATTATTTCG ATTTTGGGGG GTGGTGAGGC 5040ATTGCCGCCA TCTTACATAT TATATAAATT TATTATTTCG ATTTTGGGGG GTGGTGAGGC 5040
GTTATTGGAC GGTGCAGGAC GGCAGGCCTC TTTGCAAGGA CTCGTGAAGC GACGTTAAAC 5100GTTATTGGAC GGTGCAGGAC GGCAGGCCTC TTTGCAAGGA CTCGTGAAGC GACGTTAAAC 5100
GAGTCAGAAC TCAAGAGGAG ACGCGGTGAT TTGTGGGTAG TCAAGAGGAG ACGCGGTGAT 5160GAGTCAGAAC TCAAGAGGAG ACGCGGTGAT TTGTGGGTAG TCAAGAGGAG ACGCGGTGAT 5160
TTGTGGGTAG TCAAGAGGAG ACGCGGTGAT TTGTGGGTAG TGAGGAATTA ATTATTTATT 5220TTGTGGGTAG TCAAGAGGAG ACGCGGTGAT TTGTGGGTAG TGAGGAATTA ATTATTTATT 5220
TTGGAGAACT TATACTATAA CAC 5243 配列番号： 3 TTGGAGAACT TATACTATAA CAC 5243 SEQ ID NO: 3
配列の長さ ： 31塩基対 Sequence length: 31 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類： DNA他の核酸 合成 DNA Sequence type: DNA Other nucleic acids Synthetic DNA
配列 Array
TGAGGCTAGC CTAGGTCGAC TCGAGAGCTC A 31
配列番号： 4 TGAGGCTAGC CTAGGTCGAC TCGAGAGCTC A 31 SEQ ID NO: 4
配列の長さ ： 32塩基対 Sequence length: 32 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
CTAGTGAGCT CTCGAGTCGA CCTAGGCTAG CC 32 配列番号： 5 CTAGTGAGCT CTCGAGTCGA CCTAGGCTAG CC 32 SEQ ID NO: 5
配列の長さ ： 84塩基対 Sequence length: 84 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
TTTTTTTTTT TTTGGCATAT AAATAATAAA TACAATAATT AATTACGCGT AAAAATTGAA 60 AAACTATTCT AATTTATTGC ACTC 84 配列番号： 6 TTTTTTTTTT TTTGGCATAT AAATAATAAA TACAATAATT AATTACGCGT AAAAATTGAA 60 AAACTATTCT AATTTATTGC ACTC 84 SEQ ID NO: 6
配列の長さ ： 18塩基対 Sequence length: 18 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
CTCTGACTAC TCGGTCGC 18 配列番号： 7
配列の長さ ： 18塩基対 CTCTGACTAC TCGGTCGC 18 SEQ ID NO: 7 Sequence length: 18 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
GGCCCGACGA AGACGCGC 18 配列番号： 8 GGCCCGACGA AGACGCGC 18 SEQ ID NO: 8
配列の長さ ： 41塩基対 Sequence length: 41 base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
CTAGATCTA AGGAGGAAAA AATTATGGTA CCTCGAGCTC G 41 TAGAT TCCTCCTTTT TTAATACCAT GGAGCTCGAG CCTAG 配列番号： 9 CTAGATCTA AGGAGGAAAA AATTATGGTA CCTCGAGCTC G 41 TAGAT TCCTCCTTTT TTAATACCAT GGAGCTCGAG CCTAG SEQ ID NO: 9
配列の長さ ：塩基対 Sequence length: base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
AATCATTTAT ATTTTAAAAA TG 22 GTAAAT ATAAAATTTT TACCTG
配列番号： 10 AATCATTTAT ATTTTAAAAA TG 22 GTAAAT ATAAAATTTT TACCTG SEQ ID NO: 10
配列の長さ ：塩基対 Sequence length: base pairs
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
AATCATAAAG AACAGTACTC AATCAATAGC AATCATG 37 GTATTTC TTGTCATGAG TTAGTTATCG TTAGTACCTG
AATCATAAAG AACAGTACTC AATCAATAGC AATCATG 37 GTATTTC TTGTCATGAG TTAGTTATCG TTAGTACCTG
Claims
1.ボックスウィルス感染細胞のみで複製される、 鶏痘ウィルス由来の約 5 kbの D N Aベクター。 1. An approximately 5 kb DNA vector derived from fowlpox virus that is replicated only in box virus infected cells.
2.配列番号 1記載の塩基配列を 2回以上繰返した配列を含む逆位末端反復配列を 含む請求項 1記載の D N Aベクター。 2. The DNA vector according to claim 1, comprising an inverted terminal repeat sequence comprising a sequence obtained by repeating the base sequence described in SEQ ID NO: 1 twice or more.
3.調節遺伝子と、 少なくとも 3以上の蛋白質のコーディング領域とを含む請求項 1または 2記載の D N Aベクタ一。 3. The DNA vector according to claim 1, comprising a regulatory gene and at least three or more protein coding regions.
4.配列番号 2記載の塩基配列からなる D N Aベクター。 4. A DNA vector consisting of the nucleotide sequence of SEQ ID NO: 2.
5.少なくとも 1以上の外来遺伝子を請求項 1記載のベクターに組み込んでなる組 み換え D N Aベクター。 5. A recombinant DNA vector obtained by incorporating at least one or more foreign genes into the vector according to claim 1.
6.前記少なくとも 1以上の外来遺伝子が外来の調節遺伝子の制御下にある請求項 5記載の組み換え D N Aベクタ一。 6. The recombinant DNA vector according to claim 5, wherein said at least one foreign gene is under the control of a foreign regulatory gene.
7.前記外来遺伝子の組み込み部位が、 非翻訳領域又は第 4番目のコ一ディング領 域内にある請求項 5に記載の D N Aベクタ一。 7. The DNA vector according to claim 5, wherein the integration site of the foreign gene is in an untranslated region or a fourth coding region.
8.前記外来遺伝子が、 病原体の抗原蛋白質をコードする遺伝子である請求項 5〜 8. The foreign gene is a gene encoding an antigenic protein of a pathogen.
7のいずれかに記載の組み換え D N Aベクター。 7. The recombinant DNA vector according to any one of 7 above.
9.前記病原体の抗原蛋白質をコードする遺伝子が、 日本脳炎ウィルスの E蛋白質 をコードする遺伝子、 ニューカッスル病ウィルスの HNタンパク質をコードする 遺伝子、 Fタンパク質をコードする遺伝子、 マレック病ウィルスの糖タンパク 質 gBをコードする遺伝子、 及び伝染性ファプリキウス嚢病ウィルスの構造タン パク VP2をコードする遺伝子からなる群から選ばれる遺伝子である請求項 5〜 8のいずれかに記載の組み換え D N Aベクター。 9. The gene encoding the antigenic protein of the pathogen is the gene encoding the E protein of Japanese encephalitis virus, the gene encoding the HN protein of Newcastle disease virus, the gene encoding the F protein, the glycoprotein gB of Marek's disease virus The recombinant DNA vector according to any one of claims 5 to 8, which is a gene selected from the group consisting of: a gene coding for: and a gene coding for the infectious bursal disease virus VP2 structural protein.
10.前記調節遺伝子が、 7. 5kDaぺプチドもしくは 1 1Kポリペプチドをコードするヮ クシニアウィルス遺伝子のプロモーターまたはこれらの改変体、 初期プロモー ターと後期プロモータ一の両方の配列を有する合成プロモータ一、 および下記 塩基配列 （配列番号 5 ) 10. The regulatory gene is a promoter of a 7.5 kDa peptide or 11K polypeptide, or a variant thereof, a synthetic promoter having a sequence of both an early promoter and a late promoter, And the following nucleotide sequence (SEQ ID NO: 5)
5' -TTTTTTTTTTTTTGGCATATAAATAATAAATACAATAATTAATTACGCGTAAAAATTGAAAAA CTATTCTAATTTATTGCACTC-3'
で表される DN A配列を有するプロモーターからなる群から選ばれるプロモ一 ターである請求項 5又は 6に記載の DNAベクタ一。 5 '-TTTTTTTTTTTTTGGCATATAAATAATAAATACAATAATTAATTACGCGTAAAAATTGAAAAA CTATTCTAATTTATTGCACTC-3' 7. The DNA vector according to claim 5, which is a promoter selected from the group consisting of a promoter having a DNA sequence represented by:
11.請求項 5〜 10のいずれかに記載の組み換え DN Aベクタ一を有効成分とする ワクチン。 11. A vaccine comprising the recombinant DNA vector according to any one of claims 5 to 10 as an active ingredient.
12.請求項 5〜 10のいずれかに記載の組み換え DN Aベクタ一と弱毒ボックスゥ ィルスと含有するワクチン。 12. A vaccine containing the recombinant DNA vector according to any one of claims 5 to 10 and an attenuated box virus.
13.外来遺伝子を含む組み換え DN Aベクタ一を含むプラスミ ドで大腸菌を形質 転換し、 当該形質転換体を培養して組み換えベクターを増幅する工程と、 前記 組み換えべクタ一をボックスウイルス感染細胞に導入してさらに増幅させるェ 程とを含む請求項 11又は 12記載のワクチンの調製方法。 13. a step of transforming Escherichia coli with a plasmid containing a recombinant DNA vector containing a foreign gene, culturing the transformant to amplify the recombinant vector, and introducing the recombinant vector into a box virus-infected cell 13. The method for preparing a vaccine according to claim 11, further comprising a step of further amplifying the vaccine.
14. 前記組み換え DNAベクタ一が、 外来遺伝子と配列番号 2記載の塩基配列で 表される DN Aとを含有してなるものである請求項 11又は 12に記載のワクチン の調製方法。 14. The method for preparing a vaccine according to claim 11, wherein the recombinant DNA vector comprises a foreign gene and DNA represented by the nucleotide sequence of SEQ ID NO: 2.
15.請求項 11又は 12に記載のワクチンの使用。 15. Use of the vaccine according to claim 11 or 12.
16.10ng〜 1 f gの組み換え DNAベクタ一を、 オルソボックスウイルスおよび鶏 痘ウィルスからなる群から選ばれるボックスウィルスと共に感染させる、 感染 症の予防及び 又は治療方法。 16. A method for preventing and / or treating an infectious disease, comprising infecting a recombinant DNA vector of 10 ng to 1 fg with a box virus selected from the group consisting of an orthobox virus and a fowlpox virus.
17. 1 X103〜 1 xl05pfuの弱毒化ボックスウィルスと 10ng〜l gの組み換え DN Aベクタ一とを含む請求項 12に記載のワクチンを、 経口投与、 皮内投与、 皮下 投与、 静脈内投与、 筋肉内投与、 および腹腔内投与からなる群から選ばれる投 与経路で投与することからなる感染症の予防および または治療方法。
17. The vaccine of claim 12 comprising a 1 X10 3 of ~ 1 x10 5 pfu attenuated boxes virus and 10ng~lg recombinant DN A vector mono-, oral administration, intradermal administration, subcutaneous administration, intravenous administration A method for preventing and / or treating an infectious disease, which comprises administering by an administration route selected from the group consisting of intramuscular administration, intramuscular administration and intraperitoneal administration.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP54142298A JP3924328B2 (en) | 1997-03-28 | 1998-03-26 | Novel DNA vector and vaccine comprising recombinant new DNA vector as active ingredients |
| AU65182/98A AU6518298A (en) | 1997-03-28 | 1998-03-26 | Novel dna vector and vaccines containing novel recombinant dna vectors as the active ingredient |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9487597 | 1997-03-28 | ||
| JP9/94875 | 1997-03-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1998044093A1 true WO1998044093A1 (en) | 1998-10-08 |
Family
ID=14122232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP1998/001358 WO1998044093A1 (en) | 1997-03-28 | 1998-03-26 | Novel dna vector and vaccines containing novel recombinant dna vectors as the active ingredient |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP3924328B2 (en) |
| AU (1) | AU6518298A (en) |
| WO (1) | WO1998044093A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05192165A (en) * | 1991-08-26 | 1993-08-03 | Immuno Ag | Recombinant fowl pox virus |
| WO1996040880A1 (en) * | 1995-06-07 | 1996-12-19 | Syntro Corporation | Recombinant fowlpox viruses and uses thereof |
-
1998
- 1998-03-26 AU AU65182/98A patent/AU6518298A/en not_active Abandoned
- 1998-03-26 JP JP54142298A patent/JP3924328B2/en not_active Expired - Lifetime
- 1998-03-26 WO PCT/JP1998/001358 patent/WO1998044093A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05192165A (en) * | 1991-08-26 | 1993-08-03 | Immuno Ag | Recombinant fowl pox virus |
| WO1996040880A1 (en) * | 1995-06-07 | 1996-12-19 | Syntro Corporation | Recombinant fowlpox viruses and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU6518298A (en) | 1998-10-22 |
| JP3924328B2 (en) | 2007-06-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104364381B (en) | Multivalent recombinant avian herpesviruses and vaccines for immunizing birds | |
| JP3194751B2 (en) | Recombinant herpes virus of turkey and live vector vaccine derived from the virus | |
| EP0308220B1 (en) | Recombinant avipoxvirus | |
| JP3159476B2 (en) | Recombinant Marek's disease virus | |
| JP3587065B2 (en) | Recombinant avian infectious herpes virus and recombinant vaccine using the same | |
| US20090274724A1 (en) | Novel recombinant and mutant adenoviruses | |
| US7638132B2 (en) | Highly safe smallpox vaccine virus and vaccinia virus vector | |
| US6913751B2 (en) | Recombinant avian herpesvirus useful in vaccine production | |
| JP2000507092A (en) | Parapoxviruses containing foreign DNA, their production and their use in vaccines | |
| US5369025A (en) | Recombinant fowlpox vaccine for protection against Marek's disease | |
| US20100008948A1 (en) | Recombinant herpesvirus useful in vaccine production | |
| US5231023A (en) | Recombinant Marek's disease virus | |
| EP0404576A2 (en) | Recombinant avipoxvirus | |
| US5279965A (en) | Recombinant infectious laryngotracheitis virus | |
| US5286639A (en) | Recombinant avipoxvirus | |
| JP3924328B2 (en) | Novel DNA vector and vaccine comprising recombinant new DNA vector as active ingredients | |
| JP2638622B2 (en) | Recombinant avipox virus | |
| CN117025675B (en) | Method for improving exogenous gene expression quantity of Admax recombinant adenovirus packaging system and application | |
| JP3675569B2 (en) | Recombinant virus and vaccine comprising the same | |
| JP3458880B2 (en) | Recombinant, polyvalent recombinant, and method for producing the same | |
| JP2638677B2 (en) | Promoter, plasmid containing the promoter and recombinant avipox virus | |
| JPH1028586A (en) | Non-essential region of avipoxvirus, recombinant avipoxvirus and vaccine using this region | |
| JPH06253852A (en) | Ccv vaccine | |
| JP2002045184A (en) | Non-essential dna region for proliferation of virus, and recombinant canary pox virus utilizing the same | |
| JPH06141853A (en) | Recombinant avian infectious laryngotrachitis inducing virus and its producing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP US |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| WWE | Wipo information: entry into national phase |
Ref document number: 09381911 Country of ref document: US |
|
| 122 | Ep: pct application non-entry in european phase | ||
| NENP | Non-entry into the national phase |
Ref country code: CA |