WO2002034041A1 - Animal non humain a proteine-marqueur de la senescence 30 defectueuse, anticorps et son procede de production - Google Patents
Animal non humain a proteine-marqueur de la senescence 30 defectueuse, anticorps et son procede de production Download PDFInfo
- Publication number
- WO2002034041A1 WO2002034041A1 PCT/JP2001/009243 JP0109243W WO0234041A1 WO 2002034041 A1 WO2002034041 A1 WO 2002034041A1 JP 0109243 W JP0109243 W JP 0109243W WO 0234041 A1 WO0234041 A1 WO 0234041A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- smp30
- antibody
- gene
- deficient
- human animal
- Prior art date
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 65
- 108090000623 proteins and genes Proteins 0.000 title claims description 159
- 102000004169 proteins and genes Human genes 0.000 title claims description 41
- 239000003550 marker Substances 0.000 title description 6
- 230000009758 senescence Effects 0.000 title description 2
- 101000582767 Homo sapiens Regucalcin Proteins 0.000 claims abstract description 46
- 102100030262 Regucalcin Human genes 0.000 claims abstract description 33
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 22
- 230000002759 chromosomal effect Effects 0.000 claims abstract description 14
- 238000012217 deletion Methods 0.000 claims abstract description 12
- 230000037430 deletion Effects 0.000 claims abstract description 12
- 230000002950 deficient Effects 0.000 claims description 97
- 241000282414 Homo sapiens Species 0.000 claims description 55
- 241000699670 Mus sp. Species 0.000 claims description 39
- 239000012634 fragment Substances 0.000 claims description 36
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 31
- 238000004519 manufacturing process Methods 0.000 claims description 29
- 102000036639 antigens Human genes 0.000 claims description 26
- 108091007433 antigens Proteins 0.000 claims description 26
- 150000001413 amino acids Chemical class 0.000 claims description 24
- 239000000427 antigen Substances 0.000 claims description 24
- 241000700159 Rattus Species 0.000 claims description 23
- 230000003053 immunization Effects 0.000 claims description 21
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 17
- 241000282472 Canis lupus familiaris Species 0.000 claims description 13
- 230000014509 gene expression Effects 0.000 claims description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 13
- 241000282412 Homo Species 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 11
- 241000124008 Mammalia Species 0.000 claims description 10
- 238000003780 insertion Methods 0.000 claims description 10
- 230000037431 insertion Effects 0.000 claims description 10
- 229930193140 Neomycin Natural products 0.000 claims description 8
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 8
- 230000007812 deficiency Effects 0.000 claims description 8
- 210000004408 hybridoma Anatomy 0.000 claims description 8
- 238000002649 immunization Methods 0.000 claims description 8
- 229960004927 neomycin Drugs 0.000 claims description 8
- 241000283984 Rodentia Species 0.000 claims description 7
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 claims description 7
- 102000048028 human RGN Human genes 0.000 claims description 7
- 241000272875 Ardeidae Species 0.000 claims description 6
- 206010059866 Drug resistance Diseases 0.000 claims description 6
- 101100412696 Rattus norvegicus Rgn gene Proteins 0.000 claims description 5
- 238000007792 addition Methods 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 241000283086 Equidae Species 0.000 claims description 2
- 241001137251 Corvidae Species 0.000 claims 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 claims 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 claims 1
- 230000000474 nursing effect Effects 0.000 claims 1
- 230000007547 defect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 59
- 230000006801 homologous recombination Effects 0.000 description 20
- 238000002744 homologous recombination Methods 0.000 description 20
- 230000006870 function Effects 0.000 description 17
- 125000003275 alpha amino acid group Chemical group 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 14
- 241000894007 species Species 0.000 description 12
- 210000000349 chromosome Anatomy 0.000 description 11
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 239000002299 complementary DNA Chemical group 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000009257 reactivity Effects 0.000 description 8
- 210000000056 organ Anatomy 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 241000271566 Aves Species 0.000 description 5
- 230000032683 aging Effects 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 102100036250 GPI mannosyltransferase 4 Human genes 0.000 description 4
- 101001074618 Homo sapiens GPI mannosyltransferase 4 Proteins 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 238000002105 Southern blotting Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 230000016784 immunoglobulin production Effects 0.000 description 4
- 238000011813 knockout mouse model Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 101150110188 30 gene Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000003915 cell function Effects 0.000 description 3
- 235000013330 chicken meat Nutrition 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003494 hepatocyte Anatomy 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- 101100412692 Mus musculus Rgn gene Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 210000001766 X chromosome Anatomy 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000004094 calcium homeostasis Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000013611 chromosomal DNA Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 241000269420 Bufonidae Species 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- 101710205660 Calcium-transporting ATPase Proteins 0.000 description 1
- 101710134161 Calcium-transporting ATPase sarcoplasmic/endoplasmic reticulum type Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 102100028314 Filaggrin Human genes 0.000 description 1
- 101710088660 Filaggrin Proteins 0.000 description 1
- -1 G418 Natural products 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 241000283203 Otariidae Species 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 108050007056 Regucalcin Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000002823 anti-activator Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011491 glass wool Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 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
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Definitions
- the present invention provides an animal of a novel strain deficient in the function of senescence marker protein 30 (hereinafter referred to as "SMP30"), a method for producing the same, and an antibody produced using the animal according to the present invention. About the production method.
- SMP30 senescence marker protein 30
- genes and gene product-deficient mice can be produced in ES cells (Embryonic Stem Cells) by using homologous recombination of genes (Koller et al.). Natl. Acad. Sci. USA, Vol. 86, pp. 8927-8931, 1989).
- a gene encoding a target protein is disrupted. Specifically, the target gene to be destroyed is cloned, and an evening getter vector to be inserted into the gene is created. This vector is introduced into mouse ES cells and homologous recombination takes place.
- the recombinant ES cells are adsorbed to the 8-cell embryo of the fertilized egg, transplanted into the fallopian tubes of a pseudopregnant in advance, and born. D from the tail of the mouse born later
- the NA is extracted, and recombination of the target gene is confirmed by PCR (Polymerase Chain Reaction) or Southern hybridization. This mouse is further bred to establish a pure knockout mouse strain.
- the disrupted gene is not necessarily limited to the gene encoding the target protein. For a particular gene, one of a series of steps from its transcription to translation, or any of the steps if post-translational processing is required for the gene product (protein) to function properly. Modification of a gene that inhibits the protein also results in deletion of the protein. In this case, depending on which process is inhibited, the protein exhibits various phenotypes such as no production of the protein or no function even if produced.
- the gene disrupted by the foreign gene is usually incorporated into all cells, including germ cells, and is accurately transmitted to offspring according to Mendel's law. That is, newly created knockout mice can be established as one new strain, and as long as their reproductive ability is not impaired, they can be continuously supplied as experimental animals with the same genetic qualities by repeated mating. It is possible. In addition, by connecting a gene regulatory region such as a promoter to enhance the gene to be introduced, tissue-specific expression can be controlled. On the other hand, the production and use of antibodies that are indispensable for biological analysis has a long history, and methods for producing monoclonal antibodies have been reported in 1975.
- SMP30 was reported in 1992 as a protein in rat hepatocytes that decreases with age (Fujita et al., Biochim Biophys Acta, Vol. 1 116, 122-128, 1992), and its amino acid sequence and cDNA sequence are also human. (Fujita et al., Biochim Biophys Acta, Vol. 1263, 249-252, 1995), rat (Fujita et al., Biochim Biophys Acta â Vol. 132, 297-305, 1992), mouse (Fujita et al., Biochim Biophys â , Vol.
- Intracellular calcium is an essential ion for various cell functions, but an excessive increase significantly reduces cell functions including cell membrane damage. Therefore, maintenance of intracellular calcium homeostasis is essential for maintaining cell function.
- SMP30 is a new type of calcium-binding protein that is known to have a biological function of maintaining intracellular calcium homeostasis by activating the cell membrane calcium pump.
- the details and mechanisms of the function of SMP30 in species including humans have not been fully elucidated, and there are many points that need to be elucidated, particularly in relation to diseases and involvement in various disease states. I have.
- it is essential to provide an animal model deficient in SMP30.
- the present inventor prepared an anti-human SMP30 polyclonal antibody by immunizing a egret with human SMP30 (Japanese Patent Application Laid-Open No. 7-97399).
- an object of the present invention is to provide a non-human animal deficient in SMP30.
- Another object of the present invention is to provide a highly reactive anti-SMP30 antibody that could not be produced by conventional techniques, and to provide means for producing the same. Disclosure of the invention
- the inventors of the present application failed to produce a highly reactive anti-human SMP30 monoclonal antibody / anti-human SMP30 monoclonal antibody in the prior art.
- the cause was thought to be that the amino acid sequence of SMP30 had a high degree of homology between different species. In other words, healthy animal individuals do not produce antibodies against their own proteins (immunologic tolerance). Therefore, immunization with a protein from a heterologous animal that is highly homologous to the self protein of the animal does not produce an antibody against a portion homologous to the self protein due to tolerance. Acted as an immunogen, which led to the idea that the resulting polyclonal antibody had low reactivity and the probability of obtaining a monoclonal antibody was extremely low.
- the first aspect of the SMP30-deficient non-human animal of the present invention is a non-human animal deficient in SMP30 function.
- the deficiency of the function of the SMP30 causes deletion of at least a part of the DNA sequence or insertion of another sequence in the chromosomal SMP30 gene. Or replacing at least a portion of the DNA sequence with another sequence, or a combination thereof. SMP30 deficient non-human animal.
- a third aspect of the SMP30-deficient non-human animal of the present invention is a gene encoding a gene product in which the deficiency of SMP30 function is involved in the process of chromosomal SMP30 gene expression or the process of SMP30 processing. Deleting at least a part of the DNA sequence, inserting another sequence, or replacing at least a part of the DNA sequence with another sequence, or a combination thereof. SMP30 deficient non-human animal.
- a fourth aspect of the SMP30-deficient non-human animal of the present invention is an SMP30-deficient non-human animal, wherein the other sequence is a drug resistance gene.
- a fifth aspect of the SMP30-deficient non-human animal of the present invention is characterized in that the drug resistance gene is a neomycin resistance gene inserted into an exon of a chromosomal SMP30 gene, Animal.
- a sixth aspect of the SMP30 deficient non-human animal of the present invention is an SMP30 deficient non-human animal, wherein the SMP30 deficient non-human animal is a mammal.
- a seventh aspect of the SMP30-deficient non-human animal of the present invention is an SMP30-deficient non-human animal, wherein the SMP30-deficient non-human animal is a rodent.
- An eighth aspect of the SMP30-deficient non-human animal of the present invention is an SMP30-deficient non-human animal, wherein the SMP30-deficient non-human animal is mouse 1 .
- the present invention further provides an anti-SMP30 antibody having a high antibody titer obtained by immunizing a non-human animal deficient in SMP30. That is, a first embodiment of the antibody of the present invention is an antibody which is obtained by immunizing a non-human animal deficient in SMP30 with SMP30 and has SMP30 or a partial peptide thereof as an antigen.
- a second aspect of the antibody of the present invention is an antibody obtained by immunizing any of the above-described SMP30-deficient non-human animals and having SMP30 or a partial peptide thereof as an antigen.
- a third aspect of the antibody of the present invention is an antibody characterized by using vertebrate SMP30 or a partial peptide thereof or vertebrate partial SMP30 or a partial peptide thereof as a common antigen. is there.
- a fourth embodiment of the antibody of the present invention is an antibody, wherein the vertebrate is a mammal.
- the fifth embodiment of the antibody of the present invention is characterized in that the antibody is immunized with SMP30 of any one of mice, rats, humans, rabbits, rabbits, dogs, dogs, and horses or a partial peptide thereof.
- the obtained antibody is used for mouse, rat, human, rabbit, egret, rabbit, dog, dog, and all or all of them, SMP30 or a part thereof. Is a common antigen.
- a sixth embodiment of the antibody of the present invention is a mouse, rat, human, rabbit, mouse, dog, dog, bush, and kael obtained by immunizing rat SMP30 with SMP30-deficient mice.
- An antibody characterized in that SMP30 or a partial peptide thereof is used as a common antigen.
- a seventh aspect of the antibody of the present invention is an antibody characterized in that the anti-SMP30 antibody is obtained by immunization with SMP30 modified by addition, substitution, deletion, or insertion of amino acids. is there.
- An eighth aspect of the antibody of the present invention is an antibody, wherein the above antibody is a polyclonal antibody.
- a ninth aspect of the antibody of the present invention is an antibody, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof.
- fragment refers to an antigen-binding fragment, and does not include a fragment that does not bind to an antigen (eg, Fc fragment).
- the present invention also provides a method for producing an SMP30-deficient non-human animal.
- the first embodiment of the method for producing an SMP30-deficient non-human animal of the present invention comprises deleting at least a part of the DNA sequence of the chromosomal SMP30 gene, inserting another sequence, or at least A method for producing an SMP30-deficient animal, comprising producing a mutant SMP30 gene whose function has been lost by replacing a part of the DNA sequence with another sequence, or by combining these sequences. It is.
- the second embodiment of the method for producing an SMP30-deficient non-human animal of the present invention comprises at least a part of a gene encoding a gene product involved in the process of expression of the chromosomal SMP30 gene or the process of SMP30 processing.
- the present invention relies on an antibody method in which homology between species is not high or the antibody cannot be produced with high antibody titer. That is, the first aspect of the antibody spotting method according to the present invention is the production of an anti-SMP30 antibody using SMP30 or a part thereof as an antigen, based on immunization of a non-human animal deficient in SMP30.
- the above-mentioned anti-SMP3 antibody comprises any one of the above-mentioned SMP30-deficient non-human animals using any of the above-mentioned SMP30-deficient non-human animals.
- This is a method for producing antibodies.
- the third! â of the antibody production method of the present invention is that the antibody produced by the above-described method is polyclonal. This is a method for producing an anti-SMP30 antibody that is a null antibody or a monoclonal antibody or its original fragment.
- the antibody of the present invention, the anti-activator method of the present invention, Iitani, Jit is a monoclonal antibody or a monoclonal antibody thereof, wherein the antibody obtained by the above-described method shows human SMP30 or SJiT. This is a method that assumes that it is a sex fragment.
- the first aspect of the hybridoma immunization method of the present invention comprises an anti-SMP30 antibody hybridoma which immunizes an SMP30 deficient non-human animal and makes the SMP30 or its 3 â 4 a â .
- the second aspect of the hybridoma production S3 â 4 method according to the present invention is that the anti-SMP30 antibody, which is described by the above-mentioned dipridoma, is any of the antibodies of the above-mentioned B®.
- the present invention is not limited to SMP30, but also provides an HIS-like method for antibody leakage that leaks proteins showing high homology between species.
- Production of Antibodies of the Present Invention! â â â â ya's Fiber is an antibody or a fiber thereof containing an amino acid having a homology of 40% or more between amino acids between vertebral shelves. It is illegal, however, to immunize an animal deficient in the primordium to produce an antibody or a fangogenic fragment thereof against the primordium.
- the present invention is particularly effective when the amino acid homology between spinal â products is 50% or more: â
- another method for producing the antibody of the present invention is a method for preparing an antibody of the invention between mammals.
- a method for preparing an antibody or its binding I 'live fragment, which observes amino acids having amino acid homology of 50% or more, and immunizing a three-membered animal of The present invention relates to a method for producing an antibody against â or its source 1 â 1 'raw fragment.
- the present invention is particularly effective when the amino acid homology between flL animals is 60% or more. L If the homology between animals is 60%, the homology between the animals is higher than that of the burp (Xya has a higher homology). This is an extremely effective means for producing an antibody of the cytoplasm preserved in the above.
- FIG. 1 is a schematic diagram of the SMP30 gene cloned from a 129ZSv mouse.
- Fig. 2 shows the promoter containing the promoter used in the construction of the targeting vector.
- Figure 3 is a conceptual diagram of the preparation of the evening-getting vector.
- FIG. 4 is a schematic diagram of a PCR system for detecting homologous recombination.
- FIG. 5 shows the genotypes and phenotypes of the first to third generations of SMP30 gene-deficient mice.
- the cDNA of the SMP30 gene has been cloned from humans, mice, rats, rabbits, rabbits, sea lions, chickens, chickens, etc., and the cDNA sequence and the amino acid sequence of the encoded protein (SMP30) have been reported. (Fujita et al. Or Misawa et al., Mentioned above). In mice, the structure of the chromosomal SMP30 gene has also been reported (Fujita et al., Biochim Biophys Vol. 1308, 49-57, 1996).
- SMP30s consist of 299 amino acids and show a high homology of about 70% to 92%, especially 89% in mice and humans and 94% in mice and rats (Fujita et al., Biochim Biophys. Vol. 1308, 49-57, 1996).
- SMP30 or the SMP30 gene encoding the same may be a previously reported amino acid sequence of SMP30 in humans, mice, rats, rabbits, rabbits, mice, chickens, etc., a cDNA sequence of the SMP30 gene, or a dienomic. As long as they have homology to the DNA sequence, those other than those already reported and those other than those derived from animals are also included.
- SMP30 or the SMP30 gene encoding it include, for example, (1) NCBI accession number Q64374 (European Molecular Biolaby Laboratory (EMB) L) a protein having the amino acid sequence of the mouse described in (SEQ ID No. 1) or a gene encoding the same; (2) a mouse having the previously reported NCBI accession number Q64374 (EMBL); A protein having an amino acid in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence (SEQ ID No. 1), or a gene encoding the same.
- the addition, deletion, or substitution of amino acids is usually 1 to about 90, preferably 1 to about 60, more preferably 1 to about 30, still more preferably 1 to about 15, and still more preferably One to several.
- SMP 30 in the present invention is usually 70% or more, preferably 80% or more, more preferably 70% or more of the previously reported mouse amino acid sequence described in NCB I access number Q64374 (EMBL) (SEQ ID No. 1). Has an amino acid sequence homology of 90% or more, more preferably 95% or more.
- the homology of amino acid sequences can be easily calculated using well-known computer software such as FASTA, and such software is also used by INN Yuichi Net.
- the SMP30 gene cDNA in the present invention is usually 60% or more, preferably 70% or more, more preferably 70% or more, with the cDNA sequence described in NCB I access number U28937 (DDBJ) (SEQ ID No: 2). It has 80% or more, more preferably 90% or more nucleotide sequence homology.
- SMP30 deficiency Means a non-human animal that does not produce SMP30 due to a mutation introduced into the chromosome, or that produces a mutant SMP30 with a defective function.
- SMP30 deficiency is most commonly caused by having a mutant SMP30 gene whose function is deficient, and methods have been established for genetic engineering. In the present invention, the insertion of another sequence (sequence different from the original sequence of the gene)
- An SMP30-deficient animal having a mutant SMP30 gene according to the present invention is given as an example of the implementation.
- SMP 30 deficiency is not necessarily limited to mutations in the chromosomal SMP 30 gene.
- a mutant SMP30 gene with a defective function includes a gene in which gene expression is suppressed or gene product activity is at least partially lost as compared to a normal gene without mutation.
- a typical example is a gene in which gene expression is completely suppressed or gene product activity is completely lost.
- the mutant SMP30 gene is present in a homozygous state on the chromosome (homodeficient animal) or in a heterozygous state in all of the constituent cells. (Hetero-deficient animals). Also, a chimeric animal in which the mutant SMP30 gene is present in a homozygous or heterozygous state in only a part of its constituent cells is included.
- Non-human animals include, for example, mammals such as mice, rats, egrets, and bushes, birds such as birds, and amphibians such as potatoes. Among them, rodents such as mouse and rat are preferred because they have short life cycle and are easy to breed, and are advantageous in that they have established technology for producing genetically deficient animals. .
- the SMP30-deficient animals of the present invention are most commonly used for chromosomal SMP3 It can be produced by artificially introducing a mutation into the 0 gene and deleting its function. Specifically, the function of the SMP30 gene is deleted by deleting at least a portion of the DNA sequence of the SMP30 gene on the chromosome, inserting another sequence, or substituting another sequence. Can be deleted.
- the site where the deletion or other sequence is inserted (substituted) (target site) may be any of the promoter region, untranslated region, and translated region in the SMP30 gene. Depending on which of these parts is targeted, the transcript (mRNA) of the SMP30 gene can no longer be produced or the transcript can be destabilized.
- SMP30 protein
- fragments thereof can also produce inactivated SMP30 (protein) and fragments thereof.
- the promoter region or the translation region is preferable to use the promoter region or the translation region as a target.
- Mutation introduction into the SMP30 gene on the chromosome as described above can be performed by homologous recombination of the gene. Specifically, for example, it can be implemented as follows.
- an evening getter vector to be used for homologous recombination is prepared.
- the evening getting vector contains all or a part of the chromosome-derived SMP30 gene to be replaced by homologous recombination and a marker gene.
- the upstream and downstream of the marker gene are surrounded by a nucleotide sequence site homologous to the insertion site of the SMP30 gene.
- All or part of the SMP30 gene can be obtained by isolating the SMP30 gene region from chromosomal DNA of an animal species in which the SMP30 gene is to be deleted.
- SMP can be performed from the chromosome DNA (DNA) library of the animal species by PCR using the polymerase chain reaction (PCR), colony hybridization, plaque hybridization, or a combination thereof. 30 gene clones can be obtained. In the evening-getting vector, insert the marker gene. This is the vector This is for selecting only the introduced cells when-is introduced into the cells.
- Such marker genes include, for example, the neomycin resistance gene (neo), the diphtheria toxin A fragment gene (DT-A), the iglomycin resistance gene, the tetracycline resistance gene, the streptomycin resistance gene, and the herpes swirl thymidine kinase gene (HSV Genes used for normal drug resistance selection, such as -tk), can be used.
- neomycin resistance gene neo
- DT-A diphtheria toxin A fragment gene
- iglomycin resistance gene the tetracycline resistance gene
- streptomycin resistance gene the herpes swirl thymidine kinase gene
- HSV Genes used for normal drug resistance selection such as -tk
- cells into which the neomycin resistance gene has been introduced show resistance to neomycin such as G418, so that by adding neomycin during cell culture, only cells containing the vector can be selected.
- a repo overnight gene may be
- the repo overnight gene may be a commonly used gene, such as the lacZ (Escherichia coli-galactosidase) gene, the CAT (chloramphenic acid acetylacetyltransferase) gene, the GUS (? -Gluconidase) gene, the luciferase gene, and the equorin gene.
- the taumarin gene can be used.
- the evening targeting vector for homologous recombination is constructed as follows. That is, the fragment obtained by cleaving the chromosome-derived SMP30 isolated as described above with an appropriate restriction enzyme is ligated in a suitable order to a chimeric gene, a reporter gene, a DT-A cassette, and the like. Let it. At this time, if necessary, a synthesized linker: DNA may be used. In addition, a part of the fragment of the isolated chromosome-derived SMP30 gene can be amplified by removing PCI as necessary.
- Targeting vector for homologous recombination Can be easily prepared by ordinary DNA recombination techniques. Next, the homologous recombination target vector is linearized and introduced into appropriate cells.
- ES cells embryonic stem cells
- ES cells embryonic stem cells
- the cells into which the targeting vector has been incorporated can be selected by the expression of the gene in the vector.
- the marker is a drug resistance gene
- it can be selected by culturing in the presence of the drug for an appropriate period.
- Homologous recombination in the selected cells can be confirmed by the PCR method or Southern hybridization.
- the ratio of homologous recombination is higher than usual, but in any case, it is necessary to confirm the site of recombination when confirming the insertion.
- Cells eg, ES cells
- ES cells eg, ES cells
- agglutination method allowed to develop to the blastocyst stage, and then transplanted into the pup of a pseudopregnant animal.
- chimeric offspring e.g, ES cells
- Offspring of these chimeric animals whose germ cells are derived from ES cells can be crossed with wild type to obtain heterozygous SMP30 gene-deficient animals.
- the obtained SMP30 hetero-deficient animals can be crossed to homozygous deletion.
- the genotype of these animals can be confirmed by analyzing the chromosomal DNA obtained from a part of the body of the animal (for example, the tip of the tail) by a PCR method, a Southern hybridization method, or the like.
- the animal of the present invention can be bred by the same method as a normal animal.
- SMP30 deficient non-human animals can be used to elucidate diseases caused by SMP30 deficiency and their therapeutic methods.
- an antigen-binding fragment refers to a fragment that does not bind to an antigen (eg, an Fc fragment).
- rodents such as mice and rats are generally used for the production of antibodies, but as described above, the amino acid sequence of SMP30 is used in humans, mice, rats, and rabbits. And maintain very high homology (Fujita 3 â 4, Mech Aging Dev. Vol.
- Antibody can be prepared by Whether an antibody recognizes multiple antigens as a common antigen depends on the species
- SMP30 which is a cornerstone of the antigen, has been reported as the SMP30 amino acid sequence of humans, mice, rats, rabbits, rabbits, birds, etc. As long as they have homology, they include sequences other than those already reported, those not derived from these animals, and those modified by addition, substitution, deletion, or insertion of an amino acid.
- SMP30 for example,
- amino acid sequence of the rat described in the NCB I access number D 31662 previously reported; (2) one or several amino acids in the amino acid sequence of the rat described in the previously reported NCB I access number D 31662 Proteins having amino acids with added, substituted, deleted or inserted residues are included.
- the addition, substitution, deletion, or insertion of amino acids is usually 1 to about 90, preferably 1 to about 60, more preferably 1 to about 30, more preferably 1 to about 15, More preferably, the number is one to several.
- SMP30 used as an antigen in the present invention may be any of the previously reported sequences, such as the above-described sequence of the rat, force (NCB I accession number AB 033368), mouse (Q 64374 or D 86217), mouse (AB 035446), It has homology of 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more with the amino acid sequence of human (D31815) or the like. The proportion of what is called âhigh homologyâ is probably left to the researcher's subjective opinion, but proteins that are more than 70% homologous at the amino acid level are generally âevolved very wellâ.
- the homology is more than 60% between mammals and more than 50% between vertebrates, it is probable that antibody production is probabilistically difficult.For experiments, polyclonal antibodies must be used, and those with low sensitivity must be used. Absent. Therefore, not only in SMP30 but also in the production of antibodies of highly homologous proteins, it is necessary to create a defective channel and use it as an antibody. This is because, since the protein is completely difficult for deficiency, the portion having protein homologue can also be used as a bitmap. In the present invention, the amino acid homology between male and female is more than 40%.
- amino acids having a vertebral vertebrae with an affinity of 50% W_h are more advantageous, and those having a homology of 'I and a life of 60% or more are particularly powerful.
- the present invention is more effective when the homology of amino acids during sleep is 50% & â , and when the homology of amino acids is 60% â â .
- â in which the homology of amino acids among mammals is 70% or more is particularly nuclear. That is, if the homology between objects is 40% or more, or the homology between mammals is 50% or more, homology is higher in rodents actually used, and most of the ages are antibodies.
- the present invention provides an extremely effective means for producing an antibody of a protein that is conserved between species.
- This antibody is expected to have higher versatility than the conventional one. That is, if the prepared antibody is an antibody having an amino acid sequence portion conserved between different species as an epitope, only one kind of the antibody will react with any kind of the same kind of protein. This is of course applicable to polyclonal antibodies reacting with multiple shrimp totopes, but it is possible to select antibody-producing cells having such a wide reactivity in producing monoclonal antibodies. . In addition, since the antigen is completely foreign to the immunized animal, it is expected that highly sensitive polyclonal antibodies will be produced. That is, every part of the protein introduced into the body as an antigen is effective as an epitope, and a myriad of combinations of antibodies that react with them show stronger reactions.
- mice the SMP30 gene is located on the X chromosome, so males of first-generation SMP30-deficient mice have a genotype (Y /-) and females have a heterogeneous (+/-) genotype in most cases. Becomes This first-generation (Y / â) male has an SMP30 deficient phenotype, but recognizes exogenous SMP30 as a heterologous protein because it has acquired tolerance in the mother's womb. do not do. In the next generation of mice, both males (Y /-) and homozygous females (1-/-) have a heterozygous mother (+ Z-).
- the antibody thus obtained is used, for example, for measuring the amount of SMP30 in body fluids and tissues.
- SMP30 is present in all body fluids and tissues such as blood, urine, stomach fluid, gastric fluid, bile, and cerebrospinal fluid, and the amount increases or decreases due to various organ disorders. These can be measured using an anti-SMP30 antibody.
- any of general measuring methods such as ELISA and sandwich method may be used.
- the developed measurement method is effective across species as described above, and can be used not only for humans, but also for livestock, pets, experimental animals, and the like.
- the above-mentioned antibodies can be used immunohistologically in pathological tissue diagnosis according to a standard method.
- SMP30 is expressed in almost all organs, but has strong expression especially in hepatocytes and renal tubular epithelial cells, which are major target organs for drug disorders. Since these target cells are vulnerable to drug damage in SMP-deficient animals, It is possible to detect side effects on organs with high sensitivity. Animals deficient in SMP30 are effective as highly sensitive detection models for side effects in drug development. In animal experiments, it is known that oxidative stress reduces the expression of SMP30. In addition, the expression of SMP30 decreases with aging, and as a result, the amount of SMP30 protein in blood also decreases (Fujita et al., Biochim Biophys Acta, Vol. 1308, 49-57, 1996). It is presumed that cell aging is enhanced by stress. For this reason, for example, by measuring the amount of SMP30 protein in blood, a decrease in SMP30 expression can be used as a parameter for the degree of aging.
- NCB I access number X
- pGM6 SMP 30 (NCBI accession number; U32170 (DDB J)) of dienomic DNA derived from C57BL / 6 mouse (Fujita et al., Biochim Biophys Vol. 1308, 49-57, 1996) was used as a mirror type to Set the primer (SEQ ID No: 3) in the direction 3 from C1aI of the third exon, and set the antisense primer (SEQ ID No: 4) in the direction 5 about 2 Kbp downstream of it. A 2 Kbp fragment (short arm) was prepared by PCI removal ( Figure 1).
- ES cells Mouse embryonic stem cells (ES cells) (1 [2 cell lines, â chromosome) (Yagi et al., Anal Biochem, Vol. 214, â 0-76, 1993) are cultured by an electroporation method. The evening getter, which had been linearized by the Not I treatment described above, was introduced into the ES cells by the racion method. After the electroporation, the cells were selectively cultured in a medium containing G418 to obtain 502 neomycin-resistant clones (evening-ES cells into which the targeting vector was introduced). The presence or absence of homologous recombination was confirmed for these 502 clones by the Southern hybridization method.
- mice Male and female ICR mice were mated to collect 8-cell embryos from pregnant mice, plugs were confirmed, goblet cysts were removed from the uterus of female mice that appeared to be pregnant, and 8-cell embryos were obtained. Then, ES cells (# 41) in which homologous recombination of the SMP30 gene had occurred were adsorbed to the embryo surface by the aggregation method. After culturing for an additional day, those that developed in the goblet blastocysts were transplanted into the uterus of the foster mother. The genotype of the born chimeric mouse was It was confirmed by the hybridization and the PCR method.
- DNA extracted from mouse liver was digested with the restriction enzyme EcoRI, and a 7-Kbp fragment was identified.
- TS3 SEQ ID No: 5
- TA4 SEQ ID No: 6
- a band of 323 bp due to the combination of NE01 (SEQ ID No: 7) and TA4 and a band of 1363 bp due to the combination of TS3 and TA4 were observed.
- the expression of the SMP30 gene in the liver of a mouse in which homologous recombination occurred in the SMP30 gene and a mouse in which the homologous recombination did not occur was analyzed by Western blotting.
- Western plot was performed using a polyclonal antibody prepared by immunizing rabbits with rat SMP30, a band was observed at a site of about 30 KDa corresponding to the molecular weight of SMP30 in wild-type mice. None was found in the deficient mice. From these results, it was confirmed that SMP30-deficient mice were established by homologous recombination of the SMP30 gene.
- Example 4 It was confirmed whether highly reactive anti-SMP30 polyclonal antibodies could be obtained using SMP30-deficient mice in which the effectiveness of anti-SMP30 antibody production in SMP30-deficient mice was confirmed.
- Polyclonal antibody production is a widely and generally known technique in the field of biology.
- the mice used were M1 of the first generation; F1 and M2 and F2 born of their parents, and M3 of these parents.
- the genotype was Ml (Y / Y / â), F 1 (+/-), M2 (Y / â), F 2 (one / one), M3 (Y / â) (SMP 30 is located on the X chromosome, The mouse genotype is Y /-).
- the myeloma cells PX 63 Ag 8. 653 American Type Cu 1 ture Co l lect ion (Rockvi l le, MD, USA) and type hand than, the fusion as day 5-1 Ox 10 7 cells are obtained 1 Culture was started a week ago. On the day, the cells were centrifuged at 1200 rpm for 5 minutes at 4 ° C, Eagle MEM was added to the precipitate, the cells were resuspended and centrifuged, and 20 ml of Eagle MEM was added and suspended, and the number of cells was counted.
- the spleen removed from the immunized mouse was scraped out of cells on a stainless steel mesh, washed with an Eagle MEM (Nissui), and the cells were dropped on a culture dish.
- the cell suspension was filtered through glass wool, and the filtrate was centrifuged at 4 ° C and 1000 rpm for 5 minutes to collect the cells, and 40 ml of Eagle's MEM was added to the cells. Counted the number.
- the myeloma cells and spleen cells were mixed at a ratio of 1: 2 and centrifuged at 1000 rpm at 4 ° C for 5 minutes. The supernatant was removed, 1 ml of PEG-Eagle MEM was gradually added to loosen the cells, and then Eagle MEM 3 Oml at 37 ° C was gradually added. After centrifugation (800 rpm, room temperature, 5 minutes), remove the supernatant, add 1 Oml of 10% FCS + RPMI-1640 at 37 ° C, leave it at 37 ° C for 60 minutes, close the lid, and loosen it on the top and bottom. Shake to loosen the precipitate. 4% of 10% FCS + RPMI-1640 was added, and the mixture was dispensed in 25 ml portions, and 10% FCS + RPMI-1640 was added to make 5 Oml.
- HAT On the day after the fusion, 2% HAT was prepared by adding 10% FCS + RPMI-164 (H;: 50 â HAT (SI GMA)), and 10 Ol was added to each well, followed by 100 â 1 every 2 or 3 days. The soil was changed, and 10 days after the fusion, the cells were cultured in 2xHT / l 0% FCS + RPMI-1640.
- mice severely immunodeficient mice. Two weeks later, ascites was collected and 30% sulfur After immunoprecipitation and dialysis with PBS, the reactivity with SMP30 in the liver cells of mice, rats, humans, rabbits, and rabbits was confirmed by Western plotting. Was shown, suggesting that it also reacts to sites that are highly conserved among species.
- Antibodies obtained using normal mice generally use a site different from the SMP30 amino acid sequence of their own, so the monoclonal antibody used was an SMP30-deficient mouse. Therefore, it was presumed that the antibody was obtained.
- the animal in which the function of SMP30 of the present invention is deficient is useful for analyzing the biological function of SMP30, relating to disease, and searching for a treatment method.
- SMP30 is reduced in systemic organs with aging, it is also useful for elucidating the mechanism of age-related diseases. Also, since hepatocytes are fragile, they can be used as a side effect screening for new drugs. Furthermore, anti-SMP30 antibodies produced using SMP30-deficient animals are more reactive than wild-type antibodies, are effective across species, and have been developed to measure SMP3 â . Can be widely applied.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
æ现æž
ã»ããã»ã³ã¹ãäžã«ãŒãããã€ã³ 3 0æ¬ æéããåç©ã æäœããã³ãã®äœè£œæ¹æ³ æè¡åé
æ¬çºæã¯ã»ãã
ã»ã³ã¹ãäžã«ãŒãããã£ã³ 3 0 (Senescence Marker Protein 30, ä»¥äž ãS M P 3 0ã ãšããïŒ ã®æ©èœãæ¬ æããæ°èŠãªç³»çµ±ã®åç©ãšãã®äœåºæ³ã åã³æ¬çºæã«ããåç©ãçšããŠäœè£œããæäœãšãã®äœåºæ³ã«é¢ããã åŸæ¥ã®æè¡
çºçå·¥åŠçæè¡ã®çºå±ãšååçç©åŠã®ç¥èã®èç©ã«äŒŽããéºäŒåã人çºçã«æ äœãã åç©åäœãžå°å
¥ããŠã çš®ã
ã®éºäŒåæ¹å€åç©ã®äœè£œãè©Šã¿ãããŠããã ã ããã¯ããããéºäŒåçµæã ïŒãã©ã³ã¹ãžã±ããã¯ïŒ åç©ãšåŒã°ãã å»åŠ .çç©â åŠé åã§æ§ã
ãªå¿çšãæåŸ
ããã ç¹ã«ã éºäŒåæäœã«ããç¹å®ã®èçœè³ªãæ¬ æã ããåç©ã¢ãã«ã¯ã ã¯ããŒãã³ã°ãããéºäŒåã®ã çäœå
ã§ã®ççåŠçæ©èœãã çŸæ£ãšã®é¢é£æ§ã®è§£æã«ãããŠã æã匷åãªæ段ã®ã²ãšã€ãšãããŠããã
äŸãã°ããŠã¹ã§ã¯ E S现è ïŒEmbryonic Stem CellïŒèæ§å¹¹çŽ°èïŒ ã«ãããŠã éº äŒåã®çžåççµæããå©çšããææ³ã«ããã éºäŒåããã³éºäŒåç£ç©æ¬ æããŠã¹ (ããããããã¯ã¢ãŠãããŠã¹ïŒ ã®äœè£œãå¯èœã§ãã ïŒKollerãã Proc. Natl . Acad. Sci . USAã 第 8 6å·ã 8 9 2 7ã8 9 3 1é ã 1 9 8 9å¹ŽïŒ ã äžè¬ã«ã ç¹å®ã®éºäŒåç£ç©ãæ¬ æãããæ¹æ³ãšããŠã¯ã .ç®çãšããèçœè³ªãã³ äžãããéºäŒåãç Žå£ããããšãè¡ãããã å
·äœçã«ã¯ç Žå£ããæšçéºäŒåã®ã¯ ããŒãã³ã°ãè¡ãã ãã®éºäŒåå
éšã«æ¿å
¥ããå€äžã²ããã£ã³ã°ãã¯ã¿äžãäœè£œ ããã ãã®ã¹ã¯ã¿äžã¯ããŠã¹ E S现èã«å°å
¥ããã çžåççµæããè¡ãããã çµ æããçãã E S现èãå粟åµã® 8现èæèã«åžçããã ãããããåœåŠåš ãã ãŠããã仮芪ã®åµç®¡ã«ç§»æ€ãã åºçãããã ãã®åŸåºçããããŠã¹ã®å°Ÿéšãã D
N Aãæœåºãã P C Ræ³ïŒPolymerase Chain Reaction)ãããã¯ãµã¶ã³ãã€ã ãªãã€ãŒäžã·ãšã³ã«ããæšçéºäŒåã®çµæãã確èªããã ãã®ããŠã¹ãããã«é 亀é
ãã çŽç³»ã®ããã¯ã¢ãŠãããŠã¹ç³»ãšããŠç¢ºç«ããã
ç Žå£ãããéºäŒåã¯å¿
ãããç®çãšããèçœè³ªãã³ãŒãããéºäŒåã«éå®ãã ãªãã ããç¹å®éºäŒåã«ã€ããŠã ãã®è»¢åãã翻蚳ã«ãããäžé£ã®éçšã®ããã ãã ãããã¯ã ãã®éºäŒåç£ç© ïŒèçœè³ªïŒ ãæ£åžžã«æ©èœããããã«ã 翻蚳åŸã®ã ãã»ã
ã·ã³ã°ãå¿
èŠã§ãããªãã ãã®éçšã®ãããããé»å®³ãããããªéºäŒåã® æ¹å€ãã åœè©²èçœè³ªã®æ¬ æãšãªãã ãã®å Žåã©ã®éçšãé»å®³ããããã«ãã£ãŠã åœè©²èçœè³ªããŸã£ããç£çãããªãã£ããã ãããã¯ç£çãããŠãæ©èœããªãç ã®æ§ã
ãªè¡šçŸåã瀺ãã ããããŠäœè£œãããããã¯ã¢ãŠãããŠã¹ã«ã¯éåžžçæ®çŽ°èãå«ããå
šãŠã®çŽ°è ã«å€æ¥éºäŒåã«ããç Žå£ãããéºäŒåãçµã¿èŸŒãŸããŠããã ã¡ã³ãã«ã®æ³åã«åŸ ã€ãŠæ£ç¢ºã«åå«ã«äŒéãããã å³ã¡æ°èŠã«äœæãããããã¯ã¢ãŠãããŠã¹ã¯ã 1 ã€ã®æ°ãã系統ãšããŠæš¹ç«ããããšãã§ãã ç¹æ®èœåãæãªãããªãéãã 亀é
ãç¹°ãè¿ãããšã«ãã£ãŠåãéºäŒçè³è³ªãæã£ãå®éšåç©ãšããŠäŸçµŠãç¶ããã ãšãå¯èœã§ããã ãŸãå°å
¥ããéºäŒåã®äžæµã«ããã¢ãŒå€äžãã§ã³ãã³ãµãŒãªã© ã®éºäŒåå¶åŸ¡é åãã€ãªããããšã«ããçµç¹ç¹ç°çã«çºçŸãå¶åŸ¡ããããšãå¯èœã§ ããã äžæ¹ã çç©åŠçåæã«æ¬ ããããšã®ã§ããªãæäœã®äœæãšå©çšã¯æŽå²ãé·ãã ç¹ã«ã¢ãã¯ããŒãã«æäœã®äœè£œæ³ã¯ãã§ã« 1 9 7 5幎ã«å ±åãããŠããã 以æ¥ã ãã®æ¹æ³ã¯ã¢ãã¯ãäžãã«æäœã®æçæ§ãšãããŸã£ãŠç 究è
ã«åºãäŒæãã ä»æ¥ ã®ååçç©åŠã®åºç€ãç¯ãäžç¿Œãšãªã£ããšãã£ãŠãéèšã§ã¯ãªãã äŸãã°ååç ç©åŠã®å®éšæç§æžãšããŠäžçã§æãã²ããèªãŸããŠãã Methods in Enzymology ã§ã¯ãã§ã« 1981幎ã«ã¢ãã¯ããŒãã«æäœã®äœè£œæ¹æ³ã確ç«ãããæ¹æ³ãšããŠèšèŒ ãããŠããã ãããããã§ã¯ã¢ãã¯ããŒãã«æäœç£çãã€ã㪠ãäžã现èã®ç¢ºç« ã¯æ±ºããŠç°¡åã§ã¯ãªãããšãèšããã ã§ããã°äœçš®é¡ãã®åç©ãçšããããšãå§ ããŠãã ïŒMethods in Enzymology 73å· ç¬¬ 1ç« Production of Antibodies 1981 幎 Academic Press) 0
æäœãæãšããŠååãªåå¿æ§ãæããªãå Žåãããããšã¯åºãç¥ãããŠããã ããããã®åå ã®ã²ãšã€ãã ç®çãšããæäœã®æåãã å
ç«ãããåŽã®åçš®ã¿ã³ ãã¯è³ªãšçžåæ§ãé«ãããšã¯ã»ãšãã©èŠéãããŠããã äžèšã® Methods in Enzymolog ã§ãåç©ã«ãã£ãŠæäœã®æ§è³ªã«å·®ãåºãããšãèªãã€ã€ã æåãšãª ãã¿ã³ãã¯è³ªã®çžåæ§ã«ã€ããŠã®èšåã¯äžåãªãã ãã®ããšã¯ãããªã¯ããŒãã«æäœã®äœè£œã«ãããŠãåæ§ã§ã ãå®éšçç©åŠè¬åº§ã ã® ã14å·å
ç«çç©åŠã ïŒäžžåã æå 57å¹ŽïŒ ã§ã¯ ãçµéšãã "æåã®çš®é¡ãšå ç©çš®ã®çµåã" ãè¯ãæäœãäœãããã«å¿
èŠã§ããã ãšããŠãããã ç®çãšãã æäœã®æåãšã宿䞻ã®åçš®ã¿ã³ãã¯è³ªãšã®éã®çžåæ§ã«ã¯äžå觊ããããŠããªãã ãããäžè¬ã«ã¯ã çç©ã«ãšã£ãŠéèŠãŸãã¯å¿
é ãªã¿ã³ãã¯è³ªã¯é²åã®éçšã§ãã ãä¿åããã çš®éã®çžåæ§ãé«ãããšãç¥ãããŠããã å³ã¡ã çç©ã«ãšã£ãŠéèŠ ãªã¿ã³ãã¯è³ªã§ããã»ã©ã æäœãäœè£œãã«ããåŸåã«ããã SMP 30ã¯ãã®å
ž åçãªäŸã§ããã
SMP 30ã¯å 霢ã«äŒŽãæžå°ããã©ããè现èå
ã®èçœè³ªãšã㊠1992å¹Žã« å ±åããïŒFujitaãã Biochim Biophys Acta,第 1 116å·ã 122ã 128ã 1992å¹ŽïŒ ã ãã®ã¢ããé
žé
åã cDNAé
åããã ïŒFujitaãã Biochim Biophys Acta, 第 1263å·»ã 249ã 252ã 1995å¹ŽïŒ ã ã©ã
ã ïŒFujita ãã Biochim Biophys Acta㜠第 1 132å·ã 297ã 305ã 1992å¹ŽïŒ ã ããŠã¹ ïŒFujitaãã Biochim Biophys㜠第 1308å·ã 49ã57ã 1996å¹ŽïŒ ãã ã©ãããã ã¥ã·ããã®ããªã ããã¬ãšã«ïŒt 0 ad) (Misawaãã Int J Mol Medã 第 6å·ã 19 1ã196ã 2000å¹ŽïŒ ã«ãããŠãã§ã«æ±ºå®ãããŠããã ãŸãããŠã¹ã«ã€ããŠã¯ãžãšããã
㯠DNAé
åãšæ§é ãå ±åããã ïŒFujitaãã Biochim Biophys, 第 1308å·ã 49ã57ã 1996å¹ŽïŒ ã ïŒ Fuj i t a - Ma ruy am aã以å€ã®ç 究ã°ã«ãŒããé
ããŠåäžã®ç©è³ªã®ã¯ãäžãã³ã°ãè¡ ã r e gu c a 1 c i nãšç§°ããŠãããã 以äžã SMP 30ãšããå称ã§çµ±äžã ã ïŒãŸãã SMP 30ãã³ãŒãããæè²äœäžã®éºäŒåãä»¥äž SMP 30éºäŒåãš ããïŒ ã
æ¬é¡çºæè
ã¯ã å
ã«ãã SMP 30ã§ã¥ãµã®ãå
ç«ããããšã«ããã æãã S MP 30ããªã¯ããŒãã«æäœã®äœè£œã«æåãã ç¹èš±åºé¡ãã ïŒç¹éå¹³ 7-97399å·å
¬ å ±ïŒ ã
å 霢ã«äŒŽãåèåšã§ã¯å€æ§ãªç
å€ãçºçããã å
çãããã¯å€çåºæ¿ã«ããè åšãæ§æãã现èã¯é害ãåããã ãã®éã«éèŠãªçŽ°èå
äºè±¡ã¯çŽ°èå
ã«ã«ã·ã¥ ã ã®äžæã§ããã 现èå
ã«ã«ã·ãŠã ã¯çš®ã
ã®çŽ°èæ©èœã«å¿
é ãªã€ãªã³ã§ã¯ããã éå°ãªäžæã¯çŽ°èèé害ãå§ããšããŠçŽ°èæ©èœãèããäœäžãããã åŸã£ãŠçŽ°è æ©èœã®ç¶æã«ãããŠçŽ°èå
ã«ã«ã·ãŠã ã®æåžžæ§ç¶æã¯å¿
é ã§ããã '
SMP 30ã¯æ°ããã¿ã€ãã®ã«ã«ã·ãŠã çµåèçœè³ªã§ããã 现èèã«ã«ã·ãŠã ãã³ãã掻æ§åããããšã«ãã现èå
ã«ã«ã·ãŠã ã®æåžžæ§ãä¿ã€çç©æ©èœãæ〠ãŠããããšãç¥ãããŠããã ããããªããã ãããå«ããçç©çš®ã«ããã SMP 30ã®åãã®è©³çŽ° ·æ©åºã¯ååã«è§£æãããŠãããã ç¹ã«çŸæ£ãšã®é¢é£ã åçš®ç
æ
ãžã®é¢äžçã 解æãã¹ãç¹ãæ°å€ãæ®ãããŠããã ãããã®åé¡ã解決ããäž ã§ã SMP 30ãæ¬ æããåç©ã¢ãã«ã®æäŸãå¿
é ã§ããã ãŸãã æ¬é¡çºæè
ã¯ã äžèšã®éãã ãã SMP 30ã§ã¥ãµã®ãå
ç«ããããšã« ãããæãã SMP 30ããªã¯ããŒãã«æäœãäœè£œããïŒç¹éå¹³ 7-97399å·å
¬å ±ïŒã ããããªããã ãã®æãã SMP 30ããªã¯ãäžãã«æäœã®ãã SMP 30ã«å¯Ÿ ããåå¿æ§ã¯ããŸãé«ããªããæºè¶³ã§ãããã®ã§ã¯ãªãã£ãããŸããç¹éå¹³ 7-97399 å·å
¬å ±èšèŒã®æ¹æ³ã«åŸã£ãŠãã SMP 30ã§å
ç«ããåç©ããåžžæ³ã«ããã¢ã㯠ããŒãã«æäœãäœè£œããããšããŠãã æãã SMP30ã¢ãã¯ããŒãã«æäœãåŸ ãããšã¯ã§ããªãã£ãã åŸã£ãŠã æ¬çºæã®ç®çã¯ã SMP 30ãæ¬ æããéããåç©ãæäŸããããšã§ ããã ãŸãã æ¬çºæã®ç®çã¯ã åŸæ¥æè¡ã§ã¯äœè£œããããšãã§ããªãã£ãåå¿æ§ ãé«ãæ SMP 30æäœãæäŸããããšã åã³ãã®äœè£œãå¯èœã«ããæ段ãæäŸ ããããšã§ããã
çºæã®é瀺
æ¬é¡çºæè
ãã¯ã äžèšèª²é¡ã解決ããããã«éææ€èšãéããçµæã åŸæ¥æè¡ ã«ãããŠã åå¿æ§ãé«ãæãã SMP 30æäœãæãã SMP 30ã¢ãã¯ãäžã ã«æäœãäœè£œããããšãã§ããªãã£ãåå ã¯ã SMP 30ã®ã¢ããé
žé
åã®ç°çš® éã§ã®çžåæ§ã®é«ãã«ããã®ã§ã¯ãªãããšããããšã«æ³å°ããã ããªãã¡ã å¥åžž ãªåç©åäœã§ã¯ã èªå·±ã®ã¿ã³ãã¯è³ªã«å¯Ÿããæäœã¯ç£çãããªã ïŒå
ç«å¯å®¹ïŒ ã ãã®ããã ãã®åç©ã«ãšã£ãŠã®èªå·±ã¿ã³ãã¯è³ªãšçžåæ§ã®é«ãç°çš®åç©ã®ã¿ã³ã ã¯è³ªãå
ç«ããŠãã å
Ÿå¯å®¹ã®ããã«ã èªå·±ã¿ã³ãã¯è³ªãšçžåãªéšåã«å¯Ÿããæ äœã¯çç£ãããã ç°ãªãé åã®ã¿ãå
ç«åãšããŠäœçšãã ãã®ããã åŸãããã ãªã¯ããŒãã«æäœã®åå¿æ§ãäœãã ãŸãã ã¢ãã¯ããŒãã«æäœãåŸããã確çã éåžžã«å°ãããªãããšãåå ã§ã¯ãªãããšããããšã«æ³å°ããã
ãããŠã ãããããªãã°ã åç©èªèº«ã® SMP 30éºäŒåãç Žå£ãã SMP 30 æ¬ æ ïŒããã¯ã¢ãŠãïŒ åç©ãäœè£œãã ããã«ãã SMP 30ãå
ç«ããã°ã ãã® æ¬ æåç©ã¯èª 3SMP 30ãæããªãã®ã§ã ç°çš® SMP 30ã®å
šäœãå
ç«åãšã ãŠäœçšãã é«ãåå¿æ§ãæããæ S MP 30æäœãã æ S MP 30ã¢ãã¯ãäžã ã«æäœãäœåºã§ããã®ã§ã¯ãªãããšããããšã«æ³å°ããã ãããŠã ããã«éæç 究ããçµæã SMP 30ã®æ©èœè§£æã®åŒ·åãªæ段ãšãªã SMP 30æ¬ æåç©ã¢ã ã«ã®ç¢ºç«ã«æåãã ãã®æ¬ æåç©ã«ç°çš® SMP 30ãå
ç«ããããšã«ããã åå¿ æ§ã®é«ãæ SMP30ããªã¯ãäžãã«æäœãã æ SMP30ã¢ãã¯ããŒãã«æäœ ãå®éã«äœåºããããšã«åããŠæåãã æ¬çºæãå®æããã å³ã¡ã æ¬çºæã® S MP 30æ¬ æéããåç©ã®ç¬¬ 1ã®æ
æ§ã¯ã SMP30ã®æ©èœ ãæ¬ æããéããåç©ã§ããã æ¬çºæã® S MP 30æ¬ æéããåç©ã®ç¬¬ 2ã®æ
æ§ã¯ã åèš S MP 30ã®æ©èœã® æ¬ æãã æè²äœ SMP 30éºäŒåã«ãããŠã å°ãªããšãäžéšã® DNAé
åãæ¬ å€± ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® DNAé
åãä»ã® é
åãžçœ®æããããåã¯ãããã®çµã¿åããã«ãããã®ã§ããããšãç¹åŸŽãšããã
SMP 30æ¬ æéããåç©ã§ããã æ¬çºæã® S MP 30æ¬ æéããåç©ã®ç¬¬ 3ã®æ
æ§ã¯ã SMP 30ã®æ©èœã®æ¬ æ ãã æè²äœ S M P 30éºäŒåã®çºçŸã®éçšãŸã㯠S M P 30ã®ããã»ãã·ã³ã°ã® éçšã«é¢ããéºäŒåç£ç©ãã³ãŒãããéºäŒåã«ãããŠã å°ãªããšãäžéšã® DNA é
åãæ¬ å€±ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® DNA é
åãä»ã®é
åã«çœ®æãããã åã¯ãããã®çµã¿åããã«ãããã®ã§ããããšã ç¹åŸŽãšããã SMP30æ¬ æéããåç©ã§ããã æ¬çºæã® S MP 30æ¬ æéããåç©ã®ç¬¬ 4ã®æ
æ§ã¯ã åèšä»ã®é
åãè¬å€èæ§ éºäŒåã§ããããšãç¹åŸŽãšããã SMP 30æ¬ æéããåç©ã§ããã æ¬çºæã® SMP 30æ¬ æéããåç©ã®ç¬¬ 5ã®æ
æ§ã¯ã åèšè¬å€èæ§éºäŒåãã æè²äœ S MP 30éºäŒåã®ã§ããœã³ã«æ¿å
¥ãããããªãã€ã·ã³èæ§éºäŒåã§ã ãããšãç¹åŸŽãšããã SMP 30æ¬ æéããåç©ã§ããã æ¬çºæã® SMP 30æ¬ æéããåç©ã®ç¬¬ 6ã®æ
æ§ã¯ã SMP 30æ¬ æéããå ç©ãã åºä¹³é¡ã§ããããšãç¹åŸŽãšããã SMP 30æ¬ æéããåç©ã§ããã æ¬çºæã® S MP 30æ¬ æéããåç©ã®ç¬¬ 7ã®æ
æ§ã¯ã SMP 30æ¬ æéããå ç©ããã€æ¯é¡ã§ããããšãç¹åŸŽãšããã SMP 30æ¬ æéããåç©ã§ããã æ¬çºæã® SMP 30æ¬ æéããåç©ã®ç¬¬ 8ã®æ
æ§ã¯ã SMP 30æ¬ æéããå ç©ãããŠã¹1ã§ããããšãç¹åŸŽãšããã SMP 30æ¬ æéããåç©ã§ããã æ¬çºæã«ãããŠã¯ããã« SMP 30æ¬ æéããåç©ãå
ç«ããŠåŸãããæäœäŸ¡ ã®é«ãæ SMP30æäœãæäŸããã å³ã¡æ¬çºæã®æäœã®ç¬¬ 1ã®æ
æ§ã¯ã SMP 30æ¬ æéããåç©ã SMP 30ã§å
ç«ããŠåŸããã SMP 30åã¯ãã®äžéšãº ããããæåãšããæäœã§ããã
æ¬çºæã®æäœã®ç¬¬ 2ã®æ
æ§ã¯ã äžèšèšèŒã® SMP 30æ¬ æéããåç©ã®ããã ããå
ç«ããŠåŸãããã SMP 30åã¯ãã®äžéšãããããæåãšããæäœã§ã ãã æ¬çºæã®æäœã®ç¬¬ 3ã®æ
æ§ã¯ã èæ€åç©ã® SMP 30è¥ããã¯ãã®äžéšãºãã ãåã¯èæ€åç©ã®äžéšã® S MP 30è¥ããã¯ãã®äžéšãããããå
±éæåãšãã ããšãç¹åŸŽãšããæäœã§ããã æ¬çºæã®æäœã®ç¬¬ 4ã®æ
æ§ã¯ã äžèšèæ€åç©ãåºä¹³åç©ã§ããããšãç¹åŸŽãšã ãæäœã§ããã æ¬çºæã®æäœã®ç¬¬ 5ã®æ
æ§ã¯ã ããŠã¹ã ã©ã
ãã ããã ã¥ãµã®ã ã¥ã·ã ã£ãã ãå€ã åã³åãšã«ã®ãã¡ãããã 1皮㮠SMP 30è¥ããã¯ãã®äžéšãºããã㧠å
ç«ããŠåŸãããæäœã§ã ããŠã¹ã ã©ããã ããã ã¥ãµã®ã ã¥ã·ã ã£ãã ãå€ã åã³åãšã«ãããŠã®ã è¥ããã¯ãããã®ãã¡ã® 2çš®é¡ä»¥äžã®ã SMP30è¥ãã ã¯ãã®äžéšã¹ããããå
±éæåãšããããšãç¹åŸŽãšããæäœã§ããã æ¬çºæã®æäœã®ç¬¬ 6ã®æ
æ§ã¯ã ã©ãã SMP 30ã SMP 30æ¬ æããŠã¹ã«å
ç«ããŠåŸãããã ããŠã¹ã ã©ã ãã ããã ã¥ãµã®ã ã¥ã·ã ã£ãã ãå€ã åã³ã«ã§ ã«ãã¹ãŠã® SMP 30è¥ããã¯ãã®äžéšãºããããå
±éæåãšããããšãç¹åŸŽãš ããæäœã§ããã æ¬çºæã®æäœã®ç¬¬ 7ã®æ
æ§ã¯ã æ S MP 30æäœãã ã¢ããé
žã®ä»å ã 眮æã æ¬ å€±ã æ¿å
¥ã«ãã修食ããã S MP 30ã§å
ç«ããŠåŸãããããšãç¹åŸŽãšããæ äœã§ããã æ¬çºæã®æäœã®ç¬¬ 8ã®æ
æ§ã¯ã äžèšã®æäœãã ããªã¯ããŒãã«æäœã§ããããš ãç¹åŸŽãšããæäœã§ããã
ããã«æ¬çºæã®æäœã®ç¬¬ 9ã®æ
æ§ã¯ã äžèšæäœãã ã¢ãã¯ããŒãã«æäœåã¯ã ã®æåçµåæ§ãã©ã°ã¡ã³ãã§ããããšãç¹åŸŽãšããã æäœã§ããã ãŸãã ãã㧠ãã ããã©ã°ã¡ã³ãã ãšã¯ã æåçµåæ§ãã©ã°ã¡ã³ããããã æåã«éçµåæ§ã® ãã©ã°ã¡ã³ã ïŒäŸãã° Fcãã©ã°ã¡ã³ãïŒ ã¯å«ãŸãªãã äžæ¹æ¬çºæã«ãããŠã¯ã S M P 3 0æ¬ æéããåç©äœè£œæ¹æ³ãæäŸããã å³ã¡ æ¬çºæã® S MP 3 0æ¬ æéããåç©äœè£œæ¹æ³ã®ç¬¬ 1ã®æ
æ§ã¯ã æè²äœ S MP 3 0 éºäŒåã®å°ãªããšãäžéšã® D N Aé
åãæ¬ å€±ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® D N Aé
åãä»ã®é
åã§çœ®æãããã åã¯ãããã®çµ ã¿åããã«ãã€ãŠæ©èœãæ¬ æããå€ç°å S M P 3 0éºäŒåãäœè£œããããšãç¹åŸŽ ãšããã S M P 3 0æ¬ æåç©ã®äœè£œæ¹æ³ã§ããã æ¬çºæã® S M P 3 0æ¬ æéããåç©äœè£œæ¹æ³ã®ç¬¬ 2ã®æ
æ§ã¯ã æè²äœ S M P 3 0éºäŒåã®çºçŸã®éçšãŸã㯠S MP 3 0ã®ããã»ãã·ã³ã°ã®éçšã«é¢ããéºäŒå ç£ç©ãã³ãŒãããéºäŒåã«ãããŠãå°ãªããšãäžéšã® D N Aé
åãæ¬ å€±ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® D N Aé
åãä»ã®é
åã§çœ®æ ããããåã¯ãããã®çµã¿åããã«ããã S MP 3 0æ¬ æåç©ã®äœè£œæ¹æ³ã§ããã ããã«æ¬çºæã¯ãçš®éã®çžåæ§ã ãæäœå ã§ããªãããããã¯äœè£œããŠãæäœäŸ¡ ã®é«ãæäœå W富ãããªã ã®ãæäœ æ³ãç°Ÿãããå³ã¡æ¬çºæã«ãããæäœäœé» æ³ã®ç¬¬ 1ã®éã¯ã SMP 3 0ã®æ¬ æéããåãå
ç«ããããšãéãšããã SMP 3 0 ãŸãã¯ãã®äžéšãæåãšããæ SMP 3 0æäœã®äœ Ÿæ³ã§ããã æ¬çºæã®æäœäœ æ³ã®ç¬¬ 2ã®æ
æ§ã¯ãäžèš3Ÿäœããäžèšã® SMP 3 0æ¬ æéããåç©ã® ãã¡ã®ãããããçšããŠäžèš ŸÏäœã®ãããããé«ãããæ SMP 3 0æäœã®äœ Ÿæ³ã§ã ãã æ¬çºæã®æäœäœ æ³ã®ç¬¬ 3ã®!^ã¯ãäžèšèªæ³ã«ãã£ãŠäœè£œãããæäœããããªã¯ãäž
ãã«æäœåã¯ã¢ãã¯ãäžãã«æ è¥ããã¯ãã®ãå^!çãã©ã°ã¡ã³ãã§ããããšã^ ãšããæ SMP 3 0æäœã®äœé»æ³ã§ããã æ¬çºæã®æ ί*0äœ Â»æ³ã®ã ©Iä¹ã®, Jitã¯ãäžèš Οæ³ã«ãã£ãŠ ãããæäœãã ãã S MP 3 0ãŸãã¯ãã® ã« SJiTãã瀺ããã¢ãã¯ããŒãã«æäœåã¯ã㮠ίλ ^æ§ãã©ã°ã¡ã³ ãã§ããããšã ãšããäœ æ³ã§ããã æ¬çºæã®ãã€ããªãäžãäœ ÂŸæ³ã®ç¬¬ 1㮠ίçžã¯ã SMP 3 0æ¬ æéããåç©ãå
ç«ã㊠SMP 3 0ãŸãã¯ãã®" Ÿãæªãšããæ SMP 3 0æäœèãã€ããªããŒããçºããã ãã€ããªããŒãäœè£œæ¹æ³ã§ããã æ¬çºæã®ãã€ããªããŒãäœ SŸæ³ã®ç¬¬ 2ã®ç¹ã¯ãäžèš ãœã£ããªããŒãã®æ²ããæ SM P 3 0æäœãã äžèšã«èš B®ã®æäœã®ããããã§ããã ããã€ããªããŒã ©{ä¹å€æ³ã§ããã ãŸããæ¬çºæã¯ã SMP 3 0ã«éãããçš®éã§é«ãçžåæ§ã瀺ãã¿ã³ãã¯è³ªãæŒãšãã æäœã®æ²ã® HISçæ¹æ³ã瀺ããå³ã¡ãæ¬çºæã®æäœã®äœ!^æ³ Â©Î¯ä¹ã®çºã¯ãèæ£ç©é ã®ã¡ããé
žã®çžåæ§ã 4 0 %以äžã§ããå€ã³ãã¯è³ªã ãšããæäœåã¯ãã®ç¹^ ^'ç ãã©ã°ã¡ã³ã ©{ä¹äžæ³ã§ãã£ãŠãåœ |ŸÎåã®æ¬ æåç©ãå
ç«ããŠåœ ã«å¯Ÿããæäœå ã¯ãã®æå^æ§ãã©ã°ã¡ã³ããäœè£œãããæäœ Â®Î¯ä¹ ^ãæ³ã§ãããæ¬çºæã¯è βç©éã® ã¢ããé
žã®çžåæ§ã 5 0 %以äžã®:^ã«ç¹ã«æå¹ã§ããã ããã«ãæ¬çºæã®æäœã®äœ Ÿæ³ã®ä»ã® JŸã¯ãåºä¹³åç©éã®ã¢ããé
žã®çžåæ§ã 5 0% 以äžã§ããå€ã³ã ã¯è³ªã芳ãšããæäœåã¯ãã® çµå I 'çãã©ã°ã¡ã³ãã®äœé»æ³ã§ã ã€ãŠãåœ |ŸŸ ^ã®ã¿ 3å¡åç©ãå
ç«ããŠåœ^^ã«å¯Ÿããæäœåã¯ãã®ãæº 1^1'çãã©ã°ã¡ã³ ããäž«ä¹è£œãããæäœã® ^æ³ã§ãããæ¬çºæ㯠(flLåç©éã®ã¢ããé
žã®çžåæ§ã 6 0 %以 äžã® ã«ç¹ã« åã§ããã l Ÿåç©éã®çžåæ§ã 6 0 % ã® ã¯ã ã«çšããã ããã£éºµã ( Xä¹ã§ã¯çžåæ§ãããã«é«ãã»ãšãã©ã®é©ãæäœäœè£œç§ãšãªããããæ¬ çºæã¯çš®éã§ä¿åãããŠããå€ã³ãã¯è³ªã®æäœãäœè£œããæ¹æ³ãšããŠæ¥µããŠæå¹ãªæ段ã ãããã®ã§ããã
å³é¢ã®ç°¡åãªèª¬æ
å³ 1ã¯ã 129ZSvããŠã¹ããã¯ããŒãã³ã°ãã SMP30éºäŒåã®æŠç¥å³ ã§ããã
å³ 2ã¯ã å€äžã²ã
ãã£ã³ã°ãã¯ã¿äžäœè£œã«äœ¿çšããã ããã¢ãŒã¿ãŒãå«ã ne
0èæ§éºäŒåã®æŠç¥å³ã§ããã
å³ 3ã¯ã å€ãŒã²ããã£ã³ã°ãã¯ã¿äžäœè£œã®æŠå¿µå³ã§ããã
å³ 4ã¯ã çžåçµæããæ€åºãã P CRã·ã¹ãã ã®æŠç¥å³ã§ããã
å³ 5ã¯ã SMP 30éºäŒåæ¬ æããŠã¹ã®ç¬¬äžäžä»£ãã第äžäžä»£ãŸã§ã®éºäŒåå åã³è¡šçŸåã§ããã çºæãå®æœããããã®æè¯ã®åœ¢æ
SMP 30éºäŒåã«ã€ããŠã¯ãåè¿°ã®ããã«ã ãããããŠã¹ã ã©ã
ãã ã¥ãµã®ã ã¥ã·ã ãã®ããªã åãšã«çãããã® cDNAãã¯ãäžãã³ã°ããã cDNAé
å åã³ã³ãŒãèçœè³ª ïŒSMP30) ã®ã¢ããé
žé
åãå ±åãããŠãã ïŒåè¿° Fuj it aãã ãŸã㯠Mi s awaãã®è«æïŒ ã ãŸãããŠã¹ã§ã¯æè²äœ SMP 30éº äŒåã®æ§é ãå ±åãããŠãã ïŒFujitaãã Biochim Biophys第 1308å·»ã 49 ã57ã 1996å¹ŽïŒ ã ãããã® S MP 30ã¯ãã¹ãŠ 299ã¢ããé
žãããªãã çŽ 70%ã92%ã®é«ãçžåæ§ã瀺ãã ç¹ã«ããŠã¹ãšããã§ã¯ 89%ã ããŠã¹ãš ã©ã
ãã§ã¯ 94%ãšé«å€ã§ãã£ã ïŒFujitaãã Biochim Biophys 第 1308å·ã 49ã57ã 1996å¹ŽïŒ ã æ¬çºæã«ãããã SMP 30ãŸãã¯ãããã³ãŒããã SMP 30éºäŒåã¯ã æ¢ å ±ã®ããã ããŠã¹ã ã©ã
ãã ã¥ãµã®ã ã¥ã·ã ãã®ããªçã® SMP 30ã®ã¢ããé
ž é
åã S M P 30éºäŒåã® c D N Aé
åãŸãã¯ãžãšããã㯠D N Aé
åãšçžåæ§ ãæãããã®ã§ããã°ã æ¢å ±ã®é
å以å€ã®ãã®ãã ãããã®åç©ç±æ¥ä»¥å€ã®ãã® ãå«ãŸããã ãã®ãã㪠SMP 30ãŸãã¯ãããã³ãŒããã SMP 30éºäŒåãš ããŠã¯ã äŸãã° ïŒ1)æ¢å ±ã®ã NCBIã¢ã¯ã»ã¹çªå· Q 64374 (Eur op e a n Mo le cular Bio lo y Laborat ory (EMB
L) ã«èšèŒãããããŠã¹ã®ã¢ããé
žé
å ïŒSEQ ID No ïŒ 1) ãæããè çœè³ªãŸãã¯ãããã³ãŒãããéºäŒåã ïŒ2)æ¢å ±ã®ã NCBIã¢ã¯ã»ã¹çªå· Q6 4374 ( E MB L)ã«èšèŒãããããŠã¹ã®ã¢ããé
žé
åïŒSEQ ID NoïŒ 1) ã«ãããŠã 1ãããã¯æ°åã®ã¢ããé
žæ®åºãä»å ã æ¬ å€±ãããã¯çœ®æããã ã¢ããé
žãæããèçœè³ªãŸãã¯ãããã³ãŒãããéºäŒåã ãæããããã ããã§ã¢ããé
žã®ä»å ã æ¬ å€±ã ãããã¯çœ®æã¯ã éåžž 1ãçŽ 90åã 奜ãŸãã 㯠1ãçŽ 60åããã奜ãŸãã㯠1ãçŽ 30åãããã«å¥œãŸãã㯠1ãçŽ 15åã ãŸãããã«å¥œãŸãã㯠1ãæ°åã§ããã ãŸãæ¬çºæã«ããã SMP 30ã¯ã æ¢å ±ã®ã NCB Iã¢ã¯ã»ã¹çªå· Q 6437 4 (EMBL) (SEQ ID NoïŒ 1) ã«èšèŒãããããŠã¹ã®ã¢ããé
žé
å ãšéåžž 70%以äžã 奜ãŸãã㯠80%以äžã ãã奜ãŸãã㯠90%以äžã ãã㫠奜ãŸãã㯠95%以äžã®ã¢ããé
žé
åã®çžåæ§ãæããã ãªãã ã¢ããé
žé
åã® çžåæ§ã¯ã FAS TAã®ãããªåšç¥ã®ã³ã³ãã¥ãŒã¿ãŒãœãããçšããŠå®¹æã«ç®åº ããããšãã§ãã ãã®ãããªãœããã¯ã£ã³å€äžãããã«ãã£ãŠãå©çšã«äŸãã㊠ããã ãŸãæ¬çºæã«ããã SMP 30éºäŒå cDNAã¯ã NCB Iã¢ã¯ã»ã¹çªå· U2 8937 (DDB J) (SEQ ID N oïŒ 2 ) ã«èšèŒããã c D N Aé
åãš éåžž 60%以äžã 奜ãŸãã㯠70%以äžã ãã奜ãŸãã㯠80%以äžã ããã«å¥œ ãŸãã㯠90 %以äžã®å¡©åºé
åäžã®çžåæ§ãæããã
I æ¬çºæã«ã ('ã㊠ãSMP 30ã®æ©èœãæ¬ æããéããåç©ã ïŒä»¥äžã SMP3
0æ¬ æåç©ãšçŠŸ |Îãããã ïŒ ãšã¯ã æè²äœäžã«å°å
¥ãããå€ç°ã«ããã SMP 30ã ç£çãããªããã ãããã¯æ©èœãæ¬ æããå€ç°å SMP30ãç£çããéããå ç©ãæå³ããã SMP30ã®æ¬ æã¯ã æ©èœãæ¬ æããå€ç°å SMP 30éºäŒåã æããããšã«èµ·å ããã®ãæãäžè¬çã§ããã éºäŒåå·¥åŠçã«ãææ³ã確ç«ã㊠ããã æ¬çºæã«ãããŠãã ä»ã®é
å ïŒéºäŒåæ¬æ¥ã®é
åãšç°ãªãé
åïŒ ã®æ¿å
¥ã«
ããå€ç°å SMP 30éºäŒåãæãã SMP 30æ¬ æåç©ãã ãã®å®æœã®äžäŸãš ããŠãããŠããã ãã ã SMP 30ã®æ¬ æã¯å¿
ãããæè²äœ SMP 30éºäŒåã«ãããå€ç°ã«é å®ããããã®ã§ã¯ãªãã ã¿ã³ãã¯è³ªãæ£åžžã«æ©èœããªãåå ã¯å¿
ããããã®ã¿ã³ ãã¯è³ªãã³äžãããéºäŒåã«éããã 転åãšç¿»èš³ãå«ããäžé£ã®åæéçšã«æ¬ é¥ ãããå Žåãæ³å®ã§ããããã§ããã ç¹ã«ç°çš®é
åå°å
¥åŸã®ã¹ã¯ãªãŒãã³ã°ãæ SMP30æäœçãçšããŠã SMP 30ã®ç£çãããªãã§è¡ãã°ã ç®çãšããå€ ã³ãã¯è³ªã®éºäŒåãšã¯ãŸã£ããç°ãªãéºäŒåãæ¹å€ããããšã«ãã£ãŠ SMP 30 æ¬ æã®è¡šçŸåãåŸãããå¯èœæ§ãããã æ©èœãæ¬ æããå€ç°å SMP 30éºäŒåãšããŠã¯ã å€ç°ã®ãªãæ£åžžéºäŒåãšæ¯ èŒããŠã éºäŒåçºçŸãæå¶ããããã ãŸãã¯éºäŒåç£ç©ã®æŽ»æ§ãå°ãªããšãäžéš 倱ãããéºäŒåãæããããã å
žåäŸãšããŠã¯ã éºäŒåçºçŸãå®å
šã«æå¶ããã ãã ãŸãã¯éºäŒåç£ç©ã®æŽ»æ§ãå®å
šã«æ¶å€±ããéºäŒåãæããããã æ¬çºæã®åç©ãšããŠã¯ã ãã®æ§æ现èã®ãã¹ãŠã«ãããŠã å€ç°å SMP30éº äŒåãæè²äœäžã§ã ãã¢ã®ç¶æ
ã§ååšããŠãããã® ïŒãã¢æ¬ æåç©ïŒ ã ãŸãã¯ãž ããã®ç¶æ
ã§ååšããŠãããã® ïŒãããæ¬ æåç©ïŒ ãæããããã ãŸãã ãã®æ§ æ现èã®äžéšã®ã¿ã«ãããŠå€ç°å SMP 30éºäŒåããã¢ãŸãã¯ãžããã®ç¶æ
㧠ååšããã ãã¡ã©åç©ãæããããã éããåç©ãšããŠã¯ãäŸãã°ã ããŠã¹ã ã©ããã ã¥ãµã®ã ãå€ãªã©ã®åºä¹³åç©ã ãã®ããªãªã©ã®é³¥é¡ã åãšã«ãªã©ã®äž¡çé¡ã ãªã©ãæããããã ãããã®ãã¡ã ãŠã¹ãã©ãããªã©ã®ãã€æ¯é¡åç©ã¯ã ã©ã€ããµã€ã¯ã«ãçãç¹æ®ã容æã§ããã éºäŒçæ¬ æåç©ã®äœè£œæè¡ã確ç«ããŠããããšãªã©ã®ç¹ã§æå©ã§ããããã 奜㟠ããã æ¬çºæã® S MP 30æ¬ æåç©ã¯ã æãäžè¬çãªæ¹æ³ãšããŠã æè²äœã® SMP 3
0éºäŒåã«äººçºçã«å€ç°ãå°å
¥ãããã®æ©èœãæ¬ æãããããšã«ããäœè£œã§ããã å
·äœçã«ã¯ã æè²äœäžã® S MP 30éºäŒåã®å°ãªããšãäžéšã® DN Aé
åãæ¬ å€± ããããã ä»ã®é
åãæ¿å
¥ãããã ãããã¯ä»ã®é
åã«çœ®æããããšã«ããã S MP 30éºäŒåã®æ©èœãæ¬ æãããããšãã§ããã æ¬ å€±ãŸãã¯ä»ã®é
åãæ·å
¥ '眮æãããéšäœ ïŒã¿ãŒã²ããéšäœïŒ ã¯ã SMP3 0éºäŒåå
ã®ããã¢ãŒã¿ãŒé åã é翻蚳é åã 翻蚳é åãªã©ã®ãããã§ãã£ãŠã ããã ãããã®ã©ã®éšåãã¿ãŒã²ãããšãããã«ãã€ãŠã SMP30éºäŒåã®è»¢ åç£ç© ïŒmRNA) ãç£çãããªããªã£ããã 転åç£ç©ãäžå®å®åãããããã ãšãå¯èœã§ããã ãŸã掻æ§ã®å€±ã£ã SMP 30 (èçœè³ªïŒ ããã®æçãç£çãã ãããšãã§ããã éºäŒåã®æ©èœãå®å
šã«æ¬ æãããã«ã¯ã ããã¢ãŒã¿ãŒé åãŸã ã¯ç¿»èš³é åãå€ãŒã²ãããšããããšã奜ãŸããã åèšã®ãããªæè²äœäžã® S MP 30éºäŒåãžã®å€ç°å°å
¥ã¯ã éºäŒåçžåçµæã ã«ããå®æœã§ããã å
·äœçã«ã¯ã äŸãã°ä»¥äžã®ããã«å®æœã§ããã
ãŸãçžåçµæãã«äœ¿çšããå€äžã²ããã£ã³ã°ãã¯ã¿ãŒãäœè£œããã å€äžã²ãã ã£ã³ã°ãã¯ã¿ãŒã«ã¯çžåçµæãã«ãã£ãŠçœ®ãæããã æè²äœç±æ¥ã® SMP 30éº äŒåã®å
šéšãŸãã¯äžéšãšã ããŒã«ãŒéºäŒåãå«ãã ããŒã«ãŒéºäŒåã®äžæµãšäžæµ ã¯ã SMP30éºäŒåã®ã æ¿å
¥ããããšããéšåãšçžåãªå¡©åºé
åéšäœã«ãã£ãŠ å²ãŸããã åèš SMP 30éºäŒåã®å
šéšãŸãã¯äžéšã¯ã SMP 30éºäŒåãæ¬ æãããã ãšããåç©çš®ã®æè²äœ DNAãã SMP30éºäŒåé åãåé¢ããŠåŸãããã äŸ ãã°å®æ³ã«ãããã®åç©çš®ã®æè²äœ DN A (ãžãšããã㯠DNA) ã©ã€ãã©ãªãŒ ããã PCRæ³ ïŒPolymerase Chain Reaction)ã ã³ããäžãã€ããªãã£ãŒäžã·ã§ ã³æ³ããã©ãŒã¯ãã€ããªãã£ãŒãŒã·ãšã³æ³ããããã¯ãããã®çµã¿åããã«ããã SMP 30éºäŒåã®ã¯ããŒã³ãåŸãããšãã§ããã å€ãŒã²ããã£ã³ã°ãã¯ã¿âã«ã¯ã ããŒã«äžéºäŒåãæ·å
¥ããã ããã¯ã ãã¯ã¿
âã现èå
ã«å°å
¥ããéã«ã å°å
¥ãã现èã®ã¿ãéžæããããã®ãã®ã§ããã ã ã®ãããªããŒã«äžéºäŒåãšããŠã¯ã äŸãã°ããªãã€ã·ã³èæ§éºäŒå ïŒneo)ã ãžãããªã¢ããã·ã³ Aãã©ã°ã¡ã³ãéºäŒå (DT-A) ãã£ã°ããã€ã·ã³èæ§éºäŒåã ããã©ãµã€ã¯ãªã³èæ§éºäŒåã ã¹ãã¬ãããã€ã·ã³èæ§éºäŒåã ãã«ãºã¹ãŠã£ã« ã¹ãããžã³ãããŒãŒéºäŒå ïŒHSV-tk)çã éåžžã®è¬å€èæ§éžæã«çšããéºäŒå ãçšããããšãã§ããã äŸãã°ããªãã€ã·ã³èæ§éºäŒåãå°å
¥ããã现èã¯ã G 418ãªã©ã®ããªãã€ã·ã³ã«èæ§ã瀺ãã®ã§ã 现èã®å¹é€ã®éã«ããªãã€ã·ã³ã å ããäºã«ãã£ãŠã ãã¯ã¿äžãå«ã现èã®ã¿ãéžæã§ããã ãŸãã SMP 30éºäŒåã«ã¬ãäžå€äžéºäŒåãæ¿å
¥ããŠãããã ããã¯å€äžã² ã
ãã£ã³ã°ã¹ã¯å€äžãå°å
¥ãã现èå
ãã ç®çãšãã SMP30æ¬ æåç©ã®çŽ°è å
ã§ã® SMP 30éºäŒåã®è»¢å掻æ§ãã¢ãã¿äžããããã®ãã®ã§ããããã®å Žåã æ¿å
¥ããéçžåçé
åéšåã« S MP 30éºäŒåãšã¬ãäžå€äžéºäŒåã®ç¿»èš³ãã¬ãŒ ã ãäžèŽããããã«èšèšãããã¬ãäžå€äžéºäŒåã¯äžè¬ã«çšãããããã®ã§ããã äŸãã° lacZ (倧è
žèäžã¬ã©ã¯ãã·ãäžãŒïŒ éºäŒåã CAT (ã¯ãã©ã ããšäº ã³ãŒã«ã¡ã»ãã«ãã©ã³ã¹ãã§ã©ãŒãŒïŒ éºäŒåã GUS ( ?âã°ã«ã³ãããŒãŒïŒ éº äŒåã ã«ã·ãã±ã©ãŒãŒéºäŒåã ã§ã¯ãªãªã³éºäŒåã ã¿ãŠããªã³éºäŒåçãçšãã ããšãã§ããã ããã«ãã¯ã¿ãŒã现èå
ã§éçžåççµæããèµ·ãããå Žåã«çŽ°èãæ»æ»
ããã ã㪠DTâ Aã«ã»ãããå ããããšãå¯èœã§ããã ããã«ãã€ãŠå€ãŒã²ã
ãã£ã³ ã°ãã¯ã¿âãç®çã®æè²äœé åã«çµã¿èŸŒãŸãã现èãã éåžžããé«ãå²åã§éžæ ããããšãã§ããã
çžåçµæãçšå€ãŒã²ããã£ã³ã°ãã¯ã¿äžã¯ä»¥äžã®ããã«æ§ç¯ããã å³ã¡ã åè¿° ã®ããã«ããŠåé¢ããæè²äœç±æ¥ S M P 30ãé©åœãªå¶éé
µçŽ ã§åæããŠåŸãã ãæçãã ãäžåäžéºäŒåã ã¬ããŒã¿ãŒéºäŒåã DTâ Aã«ã»ããçãšé©åœãªé åºã§é£çµãããã ãã®æã«å¿
èŠãªãã°åæãããªã³ã«ãŒïŒ DNAãçšããŠãããã ãŸãã åé¢ããæè²äœç±æ¥ S MP 30éºäŒåã¯ã å¿
èŠã«å¿ã㊠PCI å»çã«ãã æçã®äžéšãå¢å¹
ããããšãå¯èœã§ããã çžåçµæãçšã¿ãŒã²ããã£ã³ã°ãã¯ã¿
âã¯ã éåžžã® D N Açµæãæè¡ã«ãã容æã«äœè£œããããšãã§ããã 次ã«ã çžåçµæãçšå€äžã²ããã£ã³ã°ãã¯ã¿äžãã çŽç·åããŠã é©åœãªçŽ°èãž å°å
¥ããã 现èã¯ã æœåšçã«å
šãŠã®çŽ°èã«ååãåŸããã®ãçšããã äŸãã°ã㊠ã¹ã®å Žåã å粟åµãåæèã ããã㯠E S现è ïŒèæ§å¹¹çŽ°èïŒ ã®ãããªå¹é€çŽ°è. ã§ãããã ãã®ãã¡ E S现èã¯æè¡çã«ã確ç«ããŠããããšããæã奜ãŸããã å°å
¥ããæ¹æ³ã¯ã éåžžã®æ¹æ³ã äŸãã°ãšã¬ã¯ãããã¬äžã·ãšã³æ³ã ãã€ã¯ãã€ã³ ãžã§ã¯ã·ãšã³æ³çãçšããããã ïŒ
å€ãŒã²ããã£ã³ã°ãã¯ã¿äžãåã蟌ãŸãã现èã¯ã ãã¯ã¿äžå
ã®ãäžåäžéºäŒ åã®çºçŸã«ãã£ãŠéžæã§ããã äŸãã°ããŒã«ãŒãè¬å€èæ§éºäŒåã§ããã°ã é©åœ ãªæéã è¬å€ååšäžã§å¹é€ããããšã«ããéžæã§ããã éžæããã现èå
ã®çžå ççµæã㯠P C Ræ³ããµã¶ã³ãã€ããªãã€ãŒäžã·ãšã³ã«ãã£ãŠç¢ºèªã§ããã D T äž Aã«ã»ãããæ¿å
¥ããå Žåã¯ã éåžžãããçžåççµæãã®å²åãäžæãããã ãããã«ããŠãæ¿å
¥ç¢ºèªã®éã«çµæãã®éšäœã確èªããå¿
èŠãããã çµæãã«ãã£ãŠç®ç D N Aãæ¿å
¥ãã现è ïŒäŸãã° E S现èïŒ ã 8现èæã®å®¿ äž»èã«åéæ³ã«ãã泚å
¥ãã èç€èæãŸã§çºçãããåŸã ãããåœåŠåš åç©ã®å å®®ã«ç§»æ€ããŠãã¡ã©ã®ç£ä»ãåŸãã
ãããã®ãã¡ã©åç©ã®ç£ä»ã§çæ®çŽ°èã E S现èã«ç±æ¥ãããã®ãéçåãšé 亀é
ã㊠S M P 3 0éºäŒåãžããæ¬ æåç©ãåŸãããšãã§ããã ãŸãåŸããã S M P 3 0ãããæ¬ æåç©å士ã亀é
ããŠãã¢æ¬ æãšããããšãå¯èœã§ããã ãã ãã®åç©ã®éºäŒååã¯ã ãã®åç©ã®äœã®äžéš ïŒäŸãã°å°Ÿéšå
ç«¯ïŒ ããåŸãæè²äœ D NAã«ã€ããŠã P C Ræ³ããµã¶ã³ãã€ããªãã£ãŒäžã·ãšã³æ³çã«ããã 解æã 確èªã§ããã æ¬çºæã®åç©ã¯ã æ£åžžãªåç©ãšåæ§ãªæ¹æ³ã«ãã飌è²ããããšãã§ãã
S MP 3 0æ¬ æéããåç©ã¯ã S MP 3 0æ¬ æã«ããçŸç
ãšãã®æ²»çæ³ã®è§£æ ã«çšããããšãã§ããã
ãŸãã æ¬çºæã® S M P 3 0æ¬ æéããåç©ãçšããããšã«ããã åŸæ¥ã¯äžå¯èœ
ã§ãã€ãæåæäŸåç©ãšæäœç£çåç©ãçµã¿åãããããšãã ããããçš®ã«æå¹ ãªã åå¿æ§ã®é«ãæ SMP 30æäœãŸãã¯ãã®ãã©ã°ã¡ã³ããäœè£œããããšãå¯ èœãšãªã ïŒããã§ãã ããã©ã°ã¡ã³ãã ãšã¯ã æåçµåæ§ãã©ã°ã¡ã³ããããã æåã«éçµåæ§ã®ãã©ã°ã¡ã³ã ïŒäŸãã° Fcãã©ã°ã¡ã³ãïŒ ã¯å«ãŸãªãïŒ ã äŸãã°ã æäœã®äœè£œã«ã¯ããŠã¹ãã©ããçã®ãã€æ¯é¡ãçšããã®ãäžè¬çã§ã ããã åè¿°ã®ããã«ã SMP 30ã®ã¢ããé
žé
åã¯ããã ããŠã¹ã ã©ã
ãã ã¥ãµ ã®ã«ãããŠãéåžžã«é«ãçžåæ§ãç¶æããŠããïŒFuj i t a Ÿ,Mech Ageing Dev. 第 107å·ã 271â 280ã 1999å¹ŽïŒ ïŒM i s a w aãã Int J Mol MedN 第 6å·»ã 191ã196ã 2000å¹ŽïŒ ã ãããã®ãã€æ¯é¡ã«ç°çš® SMP 30ã 泚å
¥ããŠãã ç°çš®ã¿ã³ãã¯è³ªãšã¯èªèããªãã®ã§ã åå¿æ§ã®é«ãæ SMP 30æ äœã ç¹ã«ã¢ãã¯ãäžãã«æäœãäœè£œããã®ã¯å°é£ã§ãã£ãã ããããã®åé¡ã¯æ¬çºæã§ãã SMP30æ¬ æåç©ãçšããããšã§è§£æ±ºã§ãã c äŸãã° S MP 30æ¬ æããŠã¹ã«ä»»æã®åç©çš®ã® SMP 30ãå
ç«ããããšã«ã〠ãŠã ã¢ãã¯ããŒãã«æäœã ããªã¯ããŒãã«æäœã®ããããäœæå¯èœãšãªãã ãã ã«åŸæ¥ã§ã¯ã»ãšãã©äžå¯èœã§ãã£ããããªçžåæ§ã®é«ãæåæäŸåç©ãšæäœç£ç åç©ã®çµã¿åããã äŸãã°ããŠã¹ã«æã©ãã SMP30ãç£çãããããšãå¯èœ ãšãªãã çžåæ§ã®å²åã«ãããããèªç±ã«çµã¿åãããŠæäœãäœæããããšã㧠ããã æäœãè€æ°ã®æåãå
±éæåãšããŠèªèããŠãããã©ããã¯ã çš®ã®ç°ãªã
2çš®é¡ä»¥äžã® SMP 30ãšåå¿ãããã©ããã確èªããããšã§ç°¡åã«å€æã§ãã c æåãšããŠè§ãã SMP 30ã¯ãæ¢å ±ã®ããã ããŠã¹ã ã©ã
ãã ã¥ãµã®ã ã¥ã·ã ãã®ããªçã® SMP30ã®ã¢ããé
žé
åãšçžåæ§ãæãããã®ã§ããã°ã æ¢å ±ã® é
å以å€ã®ãã®ãããããã®åç©ç±æ¥ä»¥å€ã®ãã®ãããã«ã¡ããé
žãä»å ã眮æã æ¬ å€±ã æ¿å
¥ã«ãã修食ããããã®ãå«ãŸããã ãã®ãã㪠SMP30ã¯ã äŸãã°
(1)æ¢å ±ã®ã NCB Iã¢ã¯ã»ã¹çªå· D 31662ã«èšèŒãããã©ããã®ã¡ãã é
žé
åã ïŒ2)æ¢å ±ã®ã NCB Iã¢ã¯ã»ã¹çªå· D 31662ã«èšèŒãããã©ãã ã®ã¢ããé
žé
åã«ãããŠã 1ãããã¯æ°åã®ã¢ããé
žæ®åºãä»å ã 眮æã æ¬ å€±ã ããã¯æ¿å
¥ãããã¢ããé
žãæããèçœè³ªãæããããã
ããã§ã¢ããé
žã®ä»å ã 眮æã æ¬ å€±ã ãããã¯æ¿å
¥ã¯ã éåžž 1ãçŽ 90åã 奜 ãŸãã㯠1ãçŽ 60åã ãã奜ãŸãã㯠1ãçŽ 30åã ããã«å¥œãŸãã㯠1ãçŽ 15åã ãŸãããã«å¥œãŸãã㯠1ãæ°åã§ããã ãŸãæ¬çºæã«ãããŠæåãšããŠçšãã SMP 30ã¯ã æ¢å ±ã®é
åã äŸãã°äžèš ã®ã©ã
ãã®é
åãã åãšã« ïŒNCB Iã¢ã¯ã»ã¹çªå· AB 033368) ã ããŠã¹ (Q 64374å㯠D 86217) ã ã¥ã· ïŒAB 035446) ã ãã ïŒD31 815)çã®ã¢ããé
žé
åãšéåžž 70%以äžã 奜ãŸãã㯠80%以äžã ãã奜㟠ãã㯠90%以äžã ããã«å¥œãŸãã㯠95%以äžã®ã¢ããé
žé
åã®çžåæ§ãæã ãã ãããã ãçžåæ§ãé«ãã ãšã¯ã©ã®ãããã®å²åãæãã®ãã¯å€åã«ç 究è
㮠䞻芳ã«å§ããããŠãããã äžè¬ã«ã¯ã¢ããé
žã¬ãã«ã§ 70%以äžçžåãªã¿ã³ã㯠質å士㯠ãé²åçã«éåžžã«ããä¿åãããŠããã ã å³ã¡çžåæ§ãé«ããšè¡šçŸãã ãïŒShcherban ABãã Genet icaã第 110å·ã 43â 53ã 2000幎ã Swidz inski JAãã Genome, 第 44å·ã 394 â 400ã 2001å¹ŽïŒ ã ãŸãã å®éã®æäœäœæã«ãããŠçŸåšæãé »ç¹ã«äœ¿çšã ããŠãããã€æ¯é¡ã¯ã äŸãã° SMP 30ã«ã€ããŠã¯ã åãšã«ãšã©ã
ãã®çžåæ§ã¯ 60. 1%ã§ãããã ã©ã
ããšããŠã¹ã®éã§ã¯çžåæ§ 93. 8%ã§ããããã«ïŒN CBIãäžå€ïŒ ïŒ ã äºãã«é²åçã«è¿ãã®ã§ã äžè¬ã«ããã«é«ãçžåæ§ã瀺ãã ãšãäºæ³ãããã ãããã£ãŠã çŸå®ã®å®éšå·¥çšã«ãããŠã¯ã åºä¹³åç©éã§ã¯ 50%以äžã èæ€å ç©ãšããŠã¯ 40%以äžã®çžåæ§ã瀺ãã¿ã³ãã¯è³ªã«ãããŠã æäœäœè£œãå°é£ã«ãª ããšèããããã çžåæ§ãåºä¹³åç©é㧠60%以äžã èæ€åç©é㧠50%以äžãš ãªããšã 確ççã«æäœã®äœè£œã¯ããªãé£ããã å®éšã§ã¯ãã£ã±ãããªã¯ãäžãã« æäœã§ã ãããæ床ã®äœããã®ã䜿ããããåŸãªãã
ãã㧠SMP 3 0ã«éãããçžåæ§ã®é«ãã¿ã³ãã¯è³ªã®æäœäœè£œ ã«ãããŠãæ¬ æèœ ãäœæããæäœäž«æã«çšããããš asããŠæ²ã«ã§ãããæ¬ æèœã«ãšã€ãŠåœææã¿ã³ã㯠質ã¯å®å
šã«é£ã¿ã³ãã¯è³ªã§ããã®ã§ãã¿ã³ãã¯é
綱çžåæ§ã®ããéšåãã§ããäžããšãã ããšãã§ããããã§ããã æ¬çºæã¯é銳éã®ã¢ããé
žã®çžåæ§ã 4 0 %以äžã® i絊㫠ã«ã§ããããŸããæ¬çºæ ã¯èæ€åéã®ã¢ããé
žã®çž æ§ã 5 0 %W_hã® ã¯ããæ©ã§ãããã«çžå' Iãçã 6 0 % 以äžã® i ã¯ç¹ã«è¢«åã§ããããŸããæ¬çºæã¯åŒç¡éã®ã¢ããé
žã®çžåæ§ã 5 0 %&± ã® ã«æ§åã§ãã¢ããé
žã®çžåæ§ã 6 0 %^±㮠ã¯ããæ§åã§ãããããã«åºä¹³åç© éã®ã¡ããé
žã®çžåæ§ã 7 0 %以äžã® ί ã¯ç¹ã«æ žåã§ãããå³ã¡ ç©éã®çžåæ§ã 4 0 %以äžã® ί ãããã¯åºä¹³åç©éã®çžåæ§ã 5 0 %以äžã®^ã¯ãå® ã«çšããããã ã€æ¯é¡ã ä¹ã§ã¯çžåæ§ãããã«é« ã»ãšãã©ã®éœ¢ãæäœäœè£œå°é£ãšãªãã æ¬çºæ ã¯çš®éã§ä¿åãããŠããã¿ã³ãã¯è³ªã®æäœãäœè£œããæ¹æ³ãšããŠæ¥µããŠæå¹ãªæ段ã ãããã®ã§ããã ãŸããã®æäœã¯åŸæ¥ã®ãã®ããé«ãæ±çšæ§ãæã€ããšãäºæ³ãããã å³ã¡äœæ ããæäœãã ç°çš®éã§ä¿åãããŠããã¢ããé
žé
åéšåãã§ããäžããšããæäœ ã§ããã°ããã®æäœ 1çš®é¡ã®ã¿ã§ãããããçš®ã®åçš®ã¿ã³ãã¯è³ªã«åå¿ããããšã« ãªãããã®ããšã¯ãè€æ°ã®ã§ãããŒãã«åå¿ããããªã¯ãäžãã«æäœã®å Žåã«ãã ãã¡ãã該åœãããã ã¢ãã¯ããŒãã«æäœã®äœæã«ãããŠãã ãã®ãããªåºãå å¿æ§ãæã€æäœç£ç现èãéžæããããšãå¯èœã§ããã ãŸãå
ç«ãããåç©ã«ãšã£ãŠã ãã®æåããŸã£ããã®ç°ç©ã§ããããšããã é åžžã«æ床ã®é«ãããªã¯ããŒãã«æäœãç£çãããããšãäºæ³ãããã å³ã¡æåãš ããŠäœå
ã«å°å
¥ãããã¿ã³ãã¯è³ªã®ããããéšåãã§ãããŒããšããŠæå¹ã§ããã ãããã«åå¿ããç¡æ°ã®æäœã®çµåãããã匷ãåå¿ã瀺ãããã§ããã ããã« ãã®ããšã¯ã¢ãã¯ããŒãã«æäœã®äœè£œã«ãããŠãã æå©ã«äœçšããã ããªã¯ã㌠ãã«æäœãååã«åå¿æ§ã瀺ããªãå Žåã¯ã æå¹ãªãã€ããªãäžããäœè£œããã ãšãå°é£ã§ããããšã¯ç 究è
ã«ã¯åºãç¥ãããŠããããéã«åå¿æ§ã®é«ãå Žåã¯ã
æ§ã
ãªã§ãããŒãã«åºã¥ããå€æ§ãªãã€ããªããŒããå¹çããäœè£œãããããšã æåŸ
ã§ããã äžè¬ã«éºäŒåæ¬ æåç©ã§ã¯ã åå¿æ§ã®é«ãæäœã¯ã éºäŒåãæ¬ æããã E S现 èããåŸããã第 1äžä»£ã®æ¬¡ã
äžä»£ ïŒç¬¬ 3äžä»£ïŒ ã§äœè£œãå¯èœãšãªã ïŒå³ 5 ) ã äŸãã°ããŠã¹ã§ã¯ S M P 3 0éºäŒå㯠Xæè²äœäžã«äœçœ®ããã®ã§ã 第 1äžä»£ã® S M P 3 0æ¬ æããŠã¹ã®éã¯éºäŒåå ïŒY/âïŒ ã éã®éºäŒååã¯ã»ãšãã©ã®å Žå ããã ïŒ+/â ) ãšãªãã ãã®ç¬¬ 1äžä»£ã® ïŒY/âïŒ ã®é㯠S MP 3 0æ¬ æè¡šçŸ åã瀺ããã æ¯èŠªã®èå
ã§å
ç«å¯å®¹æ§ãç²åŸããŠããã®ã§ã å€éšããã® S MP 3 0ãç°çš®ã¿ã³ãã¯è³ªãšèªèããªãã ãŸã次äžä»£ã®ããŠã¹ã¯é ïŒY/âïŒ ã ãã¢æ¬ æã®é ïŒäž/â) ã®ãããããã æ¯èŠªãããã ïŒ+Zâ ) ã§ããããšããã ã㯠ãæ¯èŠªã®èå
ã§å
ç«å¯å®¹æ§ãç²åŸããŠããŸãã å®éã«æå¹æ§ã®é«ãæäœãã€ãã ã®ã¯ã S MP 3 0ã«ã€ããŠèªå·±çç£èœã倱ã£ãã ããã第 2äžä»£ããŠã¹ã芪ãšã ãŠçãŸãã第 3äžä»£ã§ããããšãäºæ³ãããã ãã®ããã«ããŠåŸãããæäœã¯ã äŸãã°äœæ¶² ·çµç¹äžã® S M P 3 0éã®æž¬å®ã« çšããããã è¡æ¶²ã å°¿ã è液ã è液ã èæ±ã é«æ¶²çã ã®ããããäœæ¶²ãçµç¹ã«ã¯ S M P 3 0ãååšããŠããã åçš®ã®èåšé害ã«ããéãå¢æžããã ãããã¯æ S M P 3 0æäœãçšããŠæž¬å®ããããšãã§ããã 枬å®æ¹æ³ãšããŠã¯ã E L I S Aã ãµã³ãã€ããæ³çã äžè¬ç枬å®æ¹æ³ã®ããããçšããŠãããã éçºããã枬å®æ¹ æ³ã¯ã åè¿°ã®ãšããçš®ãè¶
ããŠæå¹ã§ããã äŸãã°ããã®ã¿ãªããã 家çã ãºã ãã å®éšåç©çã«çšããããšãå¯èœã§ããã ãŸãåè¿°ã®æäœã¯ã å®æ³ã«ããã ç
çåŠççµç¹èšºæã«ãããŠå
ç«çµç¹åŠçã«å© çšããããšãå¯èœã§ããã
S MP 3 0ã¯ã ã»ãŒå
šèº«ã®èåšã«çºçŸããŠãããã ç¹ã«è¬å€é害ã®äž»èŠãªæšç èåšã§ããè现èåã³è
尿现管äžç®çŽ°èã«ã¯åŒ·ãçºçŸãããã S M Pæ¬ æåç©ã« ãããŠã¯ãããã®æšç现èãè¬ç©é害ã«å¯ŸããŠè匱ãšãªãã®ã§ã å»è¬åã®ããã
ã®èåšã«å¯Ÿããå¯äœçšãé«æ床ã§æ€åºããããšãå¯èœã§ããã SMP30ãæ¬ æ ããåç©ã¯ã å»è¬åéçºã«ãããå¯äœçšã®é«æ床æ€åºã¢ãã«ãšããŠæå¹ã§ããã åç©å®éšã«ãããŠã é
žåã¹ãã¬ã¹ã«ãã£ãŠ S M P 30ã®çºçŸãäœäžããããšã ç¥ãããŠããã ãŸã S MP 30ã®çºçŸã¯å 霢ã«äŒŽãæžå°ãã ãã®çµæè¡äž S MP 30èçœè³ªéãäœäžããããšãã ïŒFuj i t aãã Biochim Biophys Acta, 第 1308å·ã 49ã57ã 1996幎ïŒã é
žåã¹ãã¬ã¹ã«ãã£ãŠçŽ°èã®èåãäº¢é² ããŠããããšãæšå®ãããã ãã®ããšããã äŸãã°è¡äž SMP 30èçœè³ªéã枬 å®ããããšã«ããã SMP 30ã®çºçŸã®äœäžãã èå床ã®ãã©ã¡äžå€äžãšããŠçš ããããšãã§ããã
SMP 30æ¬ æåç©ã®è¡šç®çŽ°èã§ã¯ãã€ã©ã°ãªã³ã®å解ãäœäžãã ä¿æ°Žæ§ã倧 ããéé¢ã¡ããé
žéãæžå°ããã S M P 30枬å®æ³ã S M P 30æ¬ æåç©çãçµ åããŠã ç®èã®ä¿æ°Žæ§ã亢é²ãããå»è¬åãå粧åã®éçºãå¯èœã§ããã èç现èã« S MP 30 cDNAã移å
¥ããããšã«ãã£ãŠç现èã®ååãçã ãã S MP 30æ¬ æåç©åã³æ S MP 30æäœãçšããŠã SMP30æå¶ã«ãã ç现èã®åå ·èªå°ã®å¶åŸ¡ã å³ã¡ã ç现èã®ä¿åççæ³ã«å¿çšãå¯èœãšãªãã 以äžã å®æœäŸãæããŠæ¬çºæãå
·äœçã«èª¬æãããã æ¬çºæã¯ãããã«éå®ã ãããã®ã§ã¯ãªãã å®æœäŸ å®æœäŸ 1 SMP 30éºäŒåæ¬ æåç©äœè£œçš DN Aæçã®èª¿è£œ
ã©ã
ãã® SMP30ã® cDNAã§ããå
¥ SMP 8 (NCB Iã¢ã¯ã»ã¹çªå·ïŒ X
69021 (DDBJ) ) ( F u j i t aãã Biochim Biophys Actaã 第 11
32å·ã 297ã305ã 1992å¹ŽïŒ ã E c o R I /P s t Iã«ãã£ãŠå¶éé
µ çŽ åŠçããŠåŸãããã ã©ã
ã SMP 30 cDNAã®å
šã³äžãã£ã³ã°é åãå«ãã
çŽ 1 Kbpã® DNAæçãããäžã ããŠã 129/SV系統ããŠã¹ç±æ¥ã®ã²ãã ã©ã€ãã©ãªäž ïŒStratagene瀟補ã ããã¡ãŒãžïŒ ã«ã€ããŠãã©ãŒã¯ãã€ããªãã£ãŒ âã·ãšã³ã«ããã ããŠã¹ SMP 30éºäŒå ïŒçŽ 14Kbp) ãã¯ããŒãã³ã°ãã (å³ 1) ã ãããå¶éé
µçŽ C la Iã§æ¶åãã 第 1ã§ã¯ãœã³ãšç¬¬ 2ã§ã¯ãœã³ãå« ãçŽ 6 Kb pã®æçãå«ãçŽ 14 Kbpã® C la Iæç ïŒé·è
ïŒ ãåŸãã ããã« C 57 B L/ 6ããŠã¹ç±æ¥ãžãšããã
㯠DNAã® pGM6 SMP 30 (NCBIã¢ã¯ã»ã¹çªå·ïŒ U 32170 (DDB J)) ( F u j i t aãã Biochim Biophys第 1308å·ã 49ã57ã 1996å¹ŽïŒ ãé¡åãšããŠã ã»ã³ã¹ãã©ã€ ã㌠ïŒSEQ ID NoïŒ 3) ã第 3ã§ã¯ãœã³ã® C 1 a Iãã 3ïŒæ¹åã«ã ãã® äžæµçŽ 2 Kbpéšäœã«ã¢ã³ãã»ã³ã¹ãã©ã€ã㌠ïŒSEQ ID No : 4)ã 5ïŒ æ¹åã«èšå®ãã çŽ 2Kbpæç ïŒçè
ïŒ ã PCI å»ã«ãã調補ãã ïŒå³ 1) ã 㪠ãäžèšé¡åã«çšãã D N Aæçã®é
åã¯æ±ºå®ãããŠããªãã®ã§ã ãã®å¯Ÿå¿ããéš åãå«ãé åã®ã¿ DNAé
åã決å®ããããã«ãã® 5ïŒç«¯ã S a 1 Iæ端ãšãªãã ãã«é
åãå ããŠã ã¢ã³ãã»ã³ã¹ãã©ã€ããŒãèšèšããã 次㫠3804bpã®pMC 1 ne oãã©ã¹ã ã ïŒStratagene瀟補ïŒãã Xhol ãš S a 1 Iã§æ¶åãã N e oèæ§éºäŒåãšããã¢ãŒå€äžãå«ãã 1083 b pã® æçãåŸã ïŒå³ 2)ã ãã®æçã® 5ïŒç«¯ãã¢ããã¿äžã«ãã Sal Iæ端ã«æ¹å€ã ã ο ä»¥äž 3ã€ã® DN Aæçã å³ã¡ã S MP 30ã®é·è
ãšçè
ã Neoèæ§éºäŒåã ãã©ã¹ã ã pBluescript S K +å
ã®ãã«ãã¯ããŒãã³ã°ãµã€ åã® C 1 a Iãš S a 1 Iã®éã«é·è
ã n e oã çè
ã®é ã«çµã¿èŸŒãã ïŒå³ 3 Aã B) ã 次ã«ã ãã®ãã©ã¹ã ãããå¶éé
µçŽ No t Iãš Xho Iã«ããåŸãããçŽ 8.
5Kbpã® DNAæçãã ãã©ã¹ã ã pMCDTâ A (A + T/p au) (ã©ã€ ãããã¯ãªãªãšã³ã瀟補ã Yag iãã AnalBiochemã第 214å·ã 77ã86ã
1993å¹ŽïŒ ã® No t I/Xho Iã®éã«æ¿å
¥ãã ïŒå³ 3 C) ã ãã®çµæå³ 3D
ã«ç€ºãå€ãŒã²ããã£ã³ã°ãã¯ã¿ãŒãåŸãããã ãããéåžžã®æ¹æ³ã«ãã倧éã«èª¿ 補ãã No t Iã§åæããŠã SMP 30ã®çžåçµæãã«çšããã çŽç·åãã DN AãåŸã ïŒå³ 3E) ã å®æœäŸ 2 E S现èãçšãã SMP 30éºäŒåã®çžåçµæã
ããŠã¹èæ§å¹¹çŽ°è ïŒE S现èïŒ ïŒ1[äž 2现èæ ªã Â¥æè²äœïŒ ïŒYag iãã Anal Biochemã 第 2 14å·ã Î 0ã76, 19 93å¹ŽïŒ ãå®æ³ã«ããå¹é€ãã 㧠ã¬ã¯ãããã¬ãŒã·ãšã³æ³ã«ããäžèšã® No t IåŠçã«ããçŽç·åããå€äžã²ãã ã€ã³ã°ã¹ã¯ã¿äžã E S现èã«å°å
¥ããã ãšã¬ã¯ãããã¬ãŒã·ãšã³å®æœåŸ G 418 ãå«æããå¹å°äžã§éžå¥çã«å¹é€ãã 502åã®ããªãã€ã·ã³èæ§ã¯ãäžã³ ïŒå€ âã²ã
ãã£ã³ã°ãã¯ã¿ãŒãå°å
¥ããã E S现èïŒ ãååŸããã ããã 502åã®ã¯ããŒã³ã«ã€ããŠããµã¶ã³ãã€ããªãã€ãŒãŒã·ãšã³æ³ã«ããã çžåçµæãã®æç¡ã確èªããã å³ã¡ã æœåºãã现è DNAãã E c oR Iã«ãã åæãã é»æ°æ³³åã®åŸã¡ã³ãã¬ã³ã«ç§»ãã D N Aæçã«ã€ããŠ32 Pæšè Fããäž ãã§ãã€ããªãã€ãŒãŒã·ãšã³ãè¡ã£ããéçåïŒWT)ã§ã¯ 7.5 Kb pã®ãã³ã ãæ€åºãããã®ã«å¯ŸããŠçžåçµæããçãã现è ïŒKãïŒ ã§ã¯ 7Kbpã§ãã£ã (å³ 4)ãããã¯ç¬¬ 3ã§ã¯ãœã³å
ã«æ¿å
¥ããã N e 0èæ§éºäŒåå
ã« E c oRã§å æéšäœãååšããããã§ããã ãŸããã®ãšãã«äœ¿çšãã Fããäžãã¯ã ããŠã¹ S MP 3âŠéºäŒåã®ç¬¬ 3ã§ã¯ãœã³ãšç¬¬ 4ã§ã¯ãœã³éããæœåºãã 0.52 Kb pã®æ çã§ããïŒå³ 1)ã 502åã®ããªãã€ã·ã³èæ§ã¯ããŒã³ã解æããçµæã 3åïŒ# 41ã # 60ã # 16 9) ã®çžåçµæããçãã现èãæ€åºããã å®æœäŸ 3 S MP 30æ¬ æããŠã¹ã®æš¹ç«
åŠåš ããŠã¹ãã 8现èæèãæ¡åããããã«ééã® I CRããŠã¹ã亀é
ããã ãã©ã°ã確èªãã åŠåš ãããšæãããéããŠã¹ã®åå®®ããæ¯ç€èãæåºãã 8现 èæèãåŸãŠã SMP 30éºäŒåã®çžåçµæããçãã E S现è ïŒ#4 1) ãå éæ³ã«ããèè¡šé¢ã«åžçãããã ãã®åŸããã« 1æ¥å¹é€ããŠæ¯ç€èã«çºçããã ã®ã仮芪ã®åå®®ã«ç§»æ€ããã åºçãããã¡ã©ããŠã¹ã®éºäŒååããµã¶ã³ãã€ããª
ãã€ãŒãŒã·ãšã³ã PCRæ³ã«ãã確èªããã å
·äœçã«ã¯åè¿°ã®ã¯ããŒã³ã®ç¢ºèªãš åæ§ã ããŠã¹èèããæœåºãã DNAãå¶éé
µçŽ Ec oRIã§åæãã 7Kbp ã®æçã確èªããã ãŸã PCRæ³ã«ãã解æã§ã¯ã å³ 4ã«ç€ºãããã«ã éçåã ãŠã¹ã§ã¯ TS3 (SEQ ID NoïŒ 5) ãš T A4 (SEQ ID NoïŒ 6) ãäžçµã®ãã©ã€ãäžãšã㊠280 bpã®ãã³ããã æ¬ æããŠã¹ã§ã¯ NE 01 (S E Q ID NoïŒ 7) ãš TA4ã®çµã¿åããã«ãã 323bpã®ãã³ãåã³ T S 3ãš TA4ã®çµã¿åããã«ãã 1363 bpã®ãã³ããèªããããã
SMP 30éºäŒåã«ãããŠçžåçµæããçããããŠã¹ãšçããŠããªãããŠã¹ã ãããã®èèã«ããã S M P 30éºäŒåã®çºçŸããŠãšã¹ã¿ã³ããããæ³ã«ãã解 æããã ã©ã
ã SMP 30ã家å
ã«å
ç«ããŠäœè£œããããªã¯ãäžãã«æäœãçšã ãŠãŠãšã¹ã¿ã³ãããããè¡ããšã éçåããŠã¹ã§ã¯ SMP30ã®ååéã«è©²åœã ãçŽ 30 KD aã®éšäœã«ãã³ããèªãããããã æ¬ æããŠã¹ã§ã¯èªããããªã〠ãã ãã®çµæããã SMP 30éºäŒåã®çžåçµæãã«ããã SMP30æ¬ æã㊠ã¹ãæš¹ç«ããããšã確èªããã å®æœäŸ 4 SMP 30æ¬ æããŠã¹ã«ãããæ SMP 30æäœç£çã®æå¹æ§ã®ç¢º åŸããã SMP 30æ¬ æããŠã¹ãçšããŠã åå¿æ§ã®é«ãæ S MP 30ããªã¯ã ãŒãã«æäœãåŸããããã©ããã®ç¢ºèªãè¡ã£ãã ããªã¯ãäžãã«æäœäœè£œæ³ã¯ç ç©åŠã®åéã§ã¯åºãäžè¬çã«ç¥ãããæè¡ã§ããã çšããããŠã¹ã¯ã 第 1äžä»£ã® Mlã ïŒ F 1ãšãããã䞡芪ããŠçãŸãã M2ãš F 2ã ããã«ãããã䞡芪ãšãã M 3ã® 5çš®é¡ã®ããŠã¹ã§ã éºäŒååã¯ã Ml (Y/â )ã F 1 (+/-)ã M2 (Y/âïŒ ã F 2 (äž/äžïŒ ã M3 (Y/âïŒ ã§ãã ïŒSMP 30㯠Xæè²äœäž ã«äœçœ®ããã®ã§ã ã©ã¹ã®æ¬ æããŠã¹ã®éºäŒåå㯠Y/âãšãªãïŒ ã ãããã®è¡šçŸ åã¯ãžããã® F 1ã®ã¿ã SMP 30ãçºçŸãããã®ä»ã¯ãã¹ãŠ SMP 30æ¬ æã§ã éºäŒååãšäžèŽããã ãããã®ããŠã¹ã«ç²Ÿè£œã©ã
ã S M P 30ã§äœè£œããå
ç«çšæåã§ãã«ãžã§ã³
ãç®äžæ³šå°ãã ãã®åŸ 2é±éããã« 3åè¿œå å
ç«ããã ãã®åŸåŸãããè¡æž
ãã ãŠã¹ S MP 30ãçšããŠãŠãšã¹ã¿ã³ãããããè¡ã£ããšãã M 3ã«åŒ·ããã³ã ãæ€åºãããã ãããã®çžå¯Ÿç匷床ã¯ä»¥äžã®è¡šã®ãšããã§ããã åœåããäºæ³ã ããçè«å€ãšå®å
šã«äžèŽããã
å®æœäŸ 5 ã¢ãã¯å£äžãã«æäœã®äœè£œãšã»ã«ã©ã€ã³ã®ç¢ºç«
(1) ããšå£äžã现èã®èª¿æŽ
ããšããŒã现è PX 63 Ag 8. 653ã American Type Cu 1 t u r e Co l lect ion (Rockvi l leã MDã ç±³åœ) ããå
¥ æãã åœæ¥ 5â1 Ox 107åã®çŽ°èãåŸãããããã«èåã® 1é±éåã«å¹é€ã éå§ããã åœæ¥çŽ°èã 4°C 1200 rpm㧠5åéé å¿ãã æ²æ®¿ã«ã€ãŒã°ã« ME Mãå ãå床æžæ¿ãšé å¿ãè¡ãã ã€ãŒã°ã« MEM 20mlãå ãæžæ¿ãã现èæ° ãæ°ããã
( 2 ) èŸçŽ°èã®èª¿æŽ
å
ç«ããããŠã¹ããåãåºããèŸèãã¹ãã³ã¬ã¹ã¡ãã·ã¥äžã«ãããŠçŽ°èããã åºãã ã€ãŒã°ã« MEM (Nissui) ã§ã¹ãã³ã¬ã¹ã¡ãã·ã¥ãæŽã£ãŠçŽ°èãå¹ é€ç¿ã«èœãšããã ã°ã©ã¹ãŠãŒã«ã§çŽ°èæžæ¿æ¶²ãæ¿Ÿéãã 濟液ã 4°C 1000 rp m㧠5åéé å¿ããŠçŽ°èãååãã ããã« 40 mlã®ã€ãŒã°ã« MEMãå ã现è
æ°ãæ°ããã
(3) 现èèå
ããšå£äžã现èãšèŸçŽ°èã 1察 2ã«ãªãããã«æ··ãã 4°C 1000 rpm㧠5å éé å¿ããã äžæž
ãé€ã P E Gâã€ãŒã°ã« M E M 1 m 1ãåŸã
ã«å ã现èãã»ã ãã ãã®åŸ 37 °Cã®ã€ãŒã°ã« MEM 3 OmlãåŸã
ã«æ·»å ããã é å¿ ïŒ800 r pmã 宀枩ã 5åïŒ åŸäžæž
ãé€ãã 37°Cã® 10%F CS+RPMIâ 1640 ã 1 Omlå ãã 37 °C㧠60åæŸçœ®åŸèããããŠå€©å°ã«ãã€ãããšæ¯ã£ãŠæ²æ®¿ ãã»ãããã 10%FCS+RPMIâ 1640ã 4 Omlå ãã 25mlã¥ã£ å泚ãã 10%FCS+RPMIâ 1640ãå ã 5 Omlãšããã
å
端ãã«ããããã¯ãªã¹ã¿ã«ãã
ã㧠96ç©Žãã¬ãŒãã« 10 O lãã€ãŸãã 3 7 °Cã§äžæ©å¹é€ããã
(4) HATéžæ
èåã®ç¿æ¥ã 10%FCS+RPMIâ 164(H;:50xHAT (SI GMAç€ŸïŒ ãå ã 2xHATãäœè£œãã åã¥ãšã«ã« 10 O lãã€å ããã ãã®åŸ 2ã 3æ¥ ããã« 100ã1ãã€åå°ã亀æãã èå 10æ¥åŸãã㯠2xHT/l 0%FC S+RPMI - 1640ã§å¹é€ããã
(5) ã¹ã¯ãªãŒãã³ã°åã³ã¯ããŒãã³ã°
èå 10æ¥åŸããããŠã¹èè S MP 30ãæåãšãã EL I S Aæ³ã«ããã¹ã¯ãª âãã³ã°ãéå§ããã åŸãããéœæ§ã³ãããŒãéå€åžéæ³ã«ããã¯ããŒãã³ã°ã ãã å³ã¡ã ã¥ãšã«åœãã 0. 3现èã«ãªãããã«ãã€ã㪠ããŒããåžéã㊠10 æ¥éå¹é€ãã å床 ELI SAæ³ã§éœæ§ã³ãããŒãã¹ã¯ãªãŒãã³ã°ããã ãã®æäœ ãå床è¡ãã 20åã®æ SMP 30æäœç£çã¯ããŒã³ãåŸãã å®æœäŸ 6 ã¢ãã¯ãäžãã«æäœã®åå¿æ§ã®ç¢ºèª
çŽ 2 X 107现è/ m 1ã®åè¿°ã®ã¢ãã¯å£äžãã«æäœç£ç现èã S C I DããŠã¹
(é床å
ç«äžå
šããŠã¹ïŒ ã®è
¹è
å
ã«æäžããã 2é±éåŸè
¹æ°Žãæ¡åãã 30%ç¡«
å®æ²æ®¿åã³ PBSã«ããéæãè¡ãã ããŠã¹ã ã©ããã ããã ã¥ãµã®ã åãšã«ã® èè现èå
S MP 30ãšã®åå¿æ§ããŠãšã¹ã¿ã³ããããã£ã³ã°ã§ç¢ºèªãããšããã ããããããŠã® SMP 30ã«åå¿ããããšã瀺ããã çš®éã§é«åºŠã«ä¿åãããŠã ãéšäœã«å¯ŸããŠãåå¿ããŠããããšã瀺åãããã éåžžã®ããŠã¹ãçšããå Žåã« åŸãããæäœã¯ã äžè¬ã«èªå·±ã® SMP30ã¡ããé
žé
åãšç°ãªã€ãéšäœãã§ãã âããšãããã®ã§ããããšããã äžèšã¢ãã¯ããŒãã«æäœã¯ S MP 30æ¬ æã㊠ã¹ãçšãããæ
ã«åŸãããæäœã§ããããšãæšå®ãããã æ¬çºæã® S MP 30ã®æ©èœãæ¬ æããåç©ã¯ã SMP 30ã®çç©åŠçæ©èœã®è§£ æã çŸç
ãšã®é¢é£ã æ²»çæ¹æ³ã®æ¢çŽ¢ã«æçšã§ããã SMP 30ã¯å 霢ã«äŒŽãå
šèº« ã®èåšã§æžå°ããããšããã å 霢ã«äŒŽãçŸæ£ã®çºçæ©åºã®è§£æã«ãæçšã§ããã ãŸãè现èçãè匱ãªããã æ°è¬ã®å¯äœçšã¹ã¯ãªãŒãã³ã°ãšããŠäœ¿çšã§ããã ã ãã« SMP 30æ¬ æåç©ãçšããŠè£œäœããæ SMP 30æäœã¯ã éçåãçšãã å Žåããåå¿æ§ãé«ãã çš®ãè¶
ããŠæå¹ã§ããã S MP 3âŠã®æž¬å®æ³ã®éçºãã® ä»ã«åºãå¿çšã§ããã
Claims
è«æ±ã®ç¯å²
I . SMP 30ã®æ©èœãæ¬ æãã SMP 30æ¬ æéããåç©ã
2. åèš SMP 30ã®æ©èœã®æ¬ æãã æè²äœ SMP 30éºäŒåã«ãããŠã å°ãª ããšãäžéšã® DNAé
åãæ¬ å€±ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãª ããšãäžéšã® DN Aé
åãä»ã®é
åãžçœ®æãããã åã¯ãããã®çµã¿åããã«ã ããã®ã§ããããšãç¹åŸŽãšããã è«æ±é
1ã«èšèŒã® S M P 30æ¬ æéããåç©ã 3. åèš SMP 30ã®æ©èœã®æ¬ æãã æè²äœ SMP 30éºäŒåã®çºçŸã®éçšãŸ ã㯠S M P 30ã®ããã»ãã·ã³ã°ã®éçšã«é¢ããéºäŒåç£ç©ãã³ãŒãããéºäŒå ã«ãããŠãå°ãªããšãäžéšã® D N Aé
åãæ¬ å€±ãããããä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® DNAé
åãä»ã®é
åã«çœ®æãããã åã¯ãããã®çµ ã¿åããã«ãããã®ã§ããããšãç¹åŸŽãšããã è«æ±é
1ã«èšèŒã® SMP 30æ¬ æ éããåç©ã
4. åèšä»ã®é
åãè¬å€èæ§éºäŒåã§ããããšãç¹åŸŽãšããã è«æ±é
2ãŸã㯠3ã«èšèŒã® S MP 30æ¬ æéããåç©ã
5. åèšè¬å€èæ§éºäŒåãã ããªãã€ã·ã³èæ§éºäŒåã§ã ãã€æ¿å
¥éšäœãæè² äœ SMP 30éºäŒåã®ã§ããœã³ã§ããããšãç¹åŸŽãšããã è«æ±é
4ã«èšèŒã® SM P 30æ¬ æéããåç©ã
6. åèš S M P 30æ¬ æéããåç©ãåºä¹³é¡ã§ããããšãç¹åŸŽãšããè«æ±é
1 ãã 5ã®ãããã 1é
ã«èšèŒã® SMP 30æ¬ æéããåç©ã
7. åèš SMP 30æ¬ æéããåç©ããã€æ¯é¡ã§ããããšãç¹åŸŽãšããã è«æ± é
1ãã 6ã®ãããã 1é
ã«èšèŒã® S MP 30æ¬ æéããåç©ã
8. åèš SMP 30æ¬ æéããåç©ãããŠã¹ã§ããããšãç¹åŸŽãšããã è«æ±é
1ãã 7ã®ãããã 1é
ã«èšèŒã® SMP30æ¬ æéããåç©ã
9. SMP 30æ¬ æéããåç©ã SMP 30ã§å
ç«ããŠåŸãããã SMP30 åã¯ãã®äžéšãããããæåãšããæäœã
10. è«æ±é
1ãã 8ã®ãããã 1é
ã«èšèŒã® S MP 30æ¬ æéããåç©ãå
ç« ããŠåŸãããã SMP 30åã¯ãã®äžéšãºããããæåãšããæäœã
I I. èæ€åç©ã® SMP 30è¥ããã¯ãã®äžéšããããåã¯èæ€åç©ã®äžéšã®
SMP30è¥ããã¯ãã®äžéšã¹ããããå
±éæåãšããæäœã
12. åèšèæ€åç©ãã åºä¹³åç©ã§ããããšãç¹åŸŽãšããã è«æ±é
11ã«èšèŒ ã®æäœã
13. ããŠã¹ã ã©ããã ããã ã¥ãµã®ã ã¥ã·ã ã£ãã ãå€ã åã³åãšã«ã®ãã¡ ãããã 1皮㮠SMP 30è¥ããã¯ãã®äžéšããããã§å
ç«ããŠåŸãããæäœã§ã ããŠã¹ã ã©ããã ããã ã¥ãµã®ã ã¥ã·ã ã£ãã ãå€ã åã³åãšã«ãããŠã®ã è¥ã ãã¯ãããã®ãã¡ã® 2çš®é¡ä»¥äžã®ã SMP30è¥ããã¯ãã®äžéšãããããå
±é æåãšããããšãç¹åŸŽãšããæäœã
14. ã©ã
ã SMP 30ã SMP 30^æããŠã¹ã«å
ç«ããŠåŸããããããŠã¹ã ã©ã
ãã ããã ã¥ãµã®ã ã¥ã·ã ã£ãã ãå€ã åã³åãšã«ãããŠã® SMP 30è¥ã ãã¯ãã®äžéšãããããå
±éæåãšããããšãç¹åŸŽãšããæäœã
15. åèšæäœãã ã¢ããé
žã®ä»å ã 眮æã æ¬ å€±ã æ¿å
¥ã«ãã修食ããã SMP 30ã§å
ç«ããŠåŸãããããšãç¹åŸŽãšããã è«æ±é
9ãã 14ã®ãããã 1é
ã« èšèŒã®æäœã
16. åèšæäœãã ããªã¯ããŒãã«æäœã§ããããšãç¹åŸŽãšããã è«æ±é
9ã ã 15ã®ãããã 1é
ã«èšèŒã®æäœã
17. åèšæäœããã¢ãã¯ããŒãã«æäœåã¯ãã®æåçµåæ§ãã©ã°ã¡ã³ãã§ãã ããšãç¹åŸŽãšããã è«æ±é
9ãã 15ã®ãããã 1é
ã«èšèŒã®æäœã
18. æè²äœ S MP 30éºäŒåã«ãããŠã å°ãªããšãäžéšã® DNAé
åãæ¬ å€± ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® DNAé
åãä»ã® é
åã§çœ®æãããã åã¯ãããã®çµã¿åããã«ãã€ãŠæ©èœãæ¬ æããå€ç°å S M P 30éºäŒåãäœè£œããããšãç¹åŸŽãšããã SMP30æ¬ æéããåç©ã®äœè£œæ¹ æ³ã
19. æè²äœ SMP 30éºäŒåã®çºçŸã®éçšãŸã㯠S MP 30ã®ããã»ãã·ã³ ã°ã®éçšã«é¢ããéºäŒåç£ç©ãã³äžãããéºäŒåã«ãããŠã å°ãªããšãäžéšã® D NAé
åãæ¬ å€±ããããã ä»ã®é
åãæ¿å
¥ãããã è¥ããã¯å°ãªããšãäžéšã® D NAé
åãä»ã®é
åã§çœ®æãããã åã¯ãããã®çµã¿åããã«ããã SMP30 æ¬ æéããåç©ã®äœè£œæ¹æ³ã
20. SMP 30æ¬ æéããåç©ãå
ç«ããŠã SMP 30ãŸãã¯ãã® ãæåãšããæ
SMP 30æäœãäœè£œããã æäœã® æ³ã
2 1. è«é 2 0ã«èšé©ã®æ¹æ³ã«ãããŠäœè£œããæ SMP 3 0æäœããè« é 1 0ãã 1 5ã®ãããã 1é
ã«èšè€ã®æ ãããæäœã® i i^æ³ã
2 2. èšéæ±é
2 0ã«èš Bttã®æ¹æ³ã«ãããŠä¹è£œããæ S MP 3 0æäœããèšéæ±é
1 6ãŸã㯠è«ãé 1 7ã«èš 3«ã®æäœ:ã§ããããšã»ãšããã æäœ ä¹^æ³ã
2 3. SMP 3 0æ¬ æéããåç©ãå
ç«ããŠã SMP 3 0ãŸãã¯ãã® ~Ÿãæºãšããæ SMP 3 0æäœã; βãããã€ããªããŒããäœè£œããã ãã€ããªããŒãäœ SŸŸã
2 4. t ŸŸSMPæäœããè«æ£ 9ãã 1 5ã®ãããã 1é
ã«è¬¹ã®æäœã§ãããè«æ± é
2 3ã« Î ã®ãã€ããªããŒãäœ ÂŸæ³ã
2 5. èæ£ç©éã®ã¢ããé
žã®çžåæ§ã 4 0%miã§ããã¿ã³ãã¯è³ªãé¡ãšããæäœå ã¯ãã®ãæº^æ§ãã©ã°ã¡ã³ãã®äœäžæ³ã§ãã£ãŠãåœ^^ã®æ¬ æåç©ãå
ç«ããŠåœ! åã«å¯Ÿããæäœåã¯ãã®Âœ ^^æ§ãã©ã°ã¡ã³ããäœè£œããã æäœã® æ³ã
2 6. åºå£ç§éã®ã¢ããé
žã®çžåæ§ã 5 0 % ã§ããã¿ã³ãã¯è³ªã芳ãšããæäœå ã¯ãã®ãæº 1^1ãçãã©ã°ã¡ã³ãã®äœ^æ³ã§ãã£ãŠãåœ |ŸŸ ^ã®æ¬ æåç©ãå
ç«ããŠåœ |ŸŸ åã«å¯Ÿããæäœåã¯ã㮠ŸŸ æ§ãã©ã°ã¡ã³ããäœè£œããã æäœã® im^æ³ã
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000-322234 | 2000-10-23 | ||
| JP2000322234A JP2004267002A (ja) | 2000-10-23 | 2000-10-23 | ã»ããã»ã³ã¹ããŒã«ãŒãããã€ã³ïŒïŒæ¬ æåç©ãæäœããã³ãã®äœè£œæ¹æ³ |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2002034041A1 true WO2002034041A1 (fr) | 2002-05-02 |
Family
ID=18800142
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2001/009243 WO2002034041A1 (fr) | 2000-10-23 | 2001-10-22 | Animal non humain a proteine-marqueur de la senescence 30 defectueuse, anticorps et son procede de production |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2004267002A (ja) |
| WO (1) | WO2002034041A1 (ja) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1437043A1 (en) * | 2001-09-20 | 2004-07-14 | Japan Science and Technology Agency | Model animal with overexpression of regucalcin |
| WO2006044336A1 (en) | 2004-10-12 | 2006-04-27 | Translational Medicines, Inc. | Methods for increasing the diversity of monoclonal antibodies produced against an antigen |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007054042A (ja) * | 2005-07-26 | 2007-03-08 | Japan Science & Technology Agency | ã€ã³ã¿ãŒãã§ãã³èªå°ååïœïœïœâïŒ |
-
2000
- 2000-10-23 JP JP2000322234A patent/JP2004267002A/ja active Pending
-
2001
- 2001-10-22 WO PCT/JP2001/009243 patent/WO2002034041A1/ja active Application Filing
Non-Patent Citations (6)
| Title |
|---|
| CASTROP J. ET AL., IMMUNOBIOL., vol. 193, 1995, pages 281 - 287, XP001013453 * |
| DECLERCK P.J. ET AL., J. BIOL. CHEM., vol. 270, 1995, pages 8397 - 8400, XP002174630 * |
| ENOMOTO N. ET AL., HEPATOLOGY, vol. 32, no. 4, PT. 2, 2000, pages 323A, XP002907860 * |
| ENOMOTO N. ET AL., HEPATOLOGY, vol. 69, no. 4, PT. 2, 1999, pages 510A, XP002907861 * |
| FUJITA T. ET AL., BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1308, 1996, pages 49 - 57, XP002907863 * |
| FUJITA T. ET AL., MECHANISMS OF AGEING AND DEVELOPMENT, vol. 107, 1999, pages 271 - 280, XP002907862 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1437043A1 (en) * | 2001-09-20 | 2004-07-14 | Japan Science and Technology Agency | Model animal with overexpression of regucalcin |
| EP1437043A4 (en) * | 2001-09-20 | 2007-01-17 | Japan Science & Tech Agency | ANIMAL MODEL HAVING OVEREXPRESSION OF REGUCALCIN |
| US7355093B2 (en) | 2001-09-20 | 2008-04-08 | Japan Science And Technology Agency | Model animal with overexpression of regucalcin |
| WO2006044336A1 (en) | 2004-10-12 | 2006-04-27 | Translational Medicines, Inc. | Methods for increasing the diversity of monoclonal antibodies produced against an antigen |
| US8389793B2 (en) | 2004-10-12 | 2013-03-05 | Acton Biotech Consulting | Methods for increasing the diversity of monoclonal antibodies produced against an antigen |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2004267002A (ja) | 2004-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6187992B1 (en) | Transgenic mouse having a disrupted amyloid precursor protein gene | |
| KR20040015711A (ko) | ìœë¬Œíì ë° ë ì±íì ì°êµ¬ì© ížëì€ì ë ë¹ìžê° ë묌 | |
| EP1871163A2 (en) | Transgenic models for different genes and their use for gene characterization | |
| US20200359610A1 (en) | Humanized transgenic animal | |
| US6452065B2 (en) | Transgenic mouse expressing non-native wild-type and familial Alzheimer's Disease mutant presenilin 1 protein on native presenilin 1 null background | |
| JP2001211782A (ja) | ïœïœïœéºäŒåæ¬ æããã¯ã¢ãŠãéããåºä¹³åç© | |
| KR101954726B1 (ko) | íë늬ë ìŠíêµ° ë묌 ëªšëž ë° ìŽì ì ì¡° ë°©ë² | |
| WO2002034041A1 (fr) | Animal non humain a proteine-marqueur de la senescence 30 defectueuse, anticorps et son procede de production | |
| JP5888693B2 (ja) | é質æ§èºçã¢ãã«åã³ãã®çšé | |
| WO2006016692A1 (ja) | ããã¯ã¢ãŠãéããåç© | |
| US20220217956A1 (en) | Rodent Model Of Increased Bone Mineral Density | |
| JP6267986B2 (ja) | ããã®ç¹å®ååãšçµåããååæšçç©è³ªã®ïœïœïœïœïœïœè©äŸ¡æ³ | |
| JP4374438B2 (ja) | ïœïœïœïŒïœéºäŒåæ¬ æããŠã¹ | |
| JP2007129938A (ja) | ããã¯ã¢ãŠãããŠã¹ | |
| JP4217782B2 (ja) | ãã¹ã¢ã³ãã»ãã ãœã³çå矀ã®ç¹åŸŽã瀺ãããŠã¹åã³ãã®äœè£œæ¹æ³ | |
| JP4940477B2 (ja) | ïœïœïœïœéºäŒåæ¬ æããŠã¹ | |
| US7541511B2 (en) | Mouse exhibiting characteristics of Rothmund-Thomson syndrome and preparation method thereof | |
| JP2009225697A (ja) | ïœïœïœïŒïŒéºäŒåæ©èœæ¬ æããŠã¹ | |
| JP2006325452A (ja) | ïœïœïŒïœïœïœâïœéºäŒåããã¯ã¢ãŠãéããåºä¹³åç©ããã®äœè£œæ¹æ³ãããã³ãã®å©çšæ¹æ³ | |
| JP2006025647A (ja) | ïœïœïŒïŒããã¯ã¢ãŠãåç© | |
| JP2006042683A (ja) | ãã¥ãŒãã°ãªã«ã³ïœéºäŒåã®çºçŸãæå¶ãããéããåç© | |
| JP2003169679A (ja) | ããβïŒå容äœçºçŸåç© | |
| WO2007080710A1 (ja) | å°èæ§ïœïœïœïœãã©ã³ã¹ããŒã¿ãŒéºäŒåæ¬ æããŠã¹ | |
| JP2004298180A (ja) | ã²ãã ãæ¹å€ãããããŠã¹ | |
| JP2008061557A (ja) | ïœïœ ïœïœ ïœïœïœïœïœïœïœïœïœ ãç¥çµçŽ°èç¹ç°çã«çºçŸããããŠã¹ |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 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 | ||
| 122 | Ep: pct application non-entry in european phase |