JP6682532B2 - 神経毒ポリペプチドの生物学的活性を測定する方法 - Google Patents
神経毒ポリペプチドの生物学的活性を測定する方法 Download PDFInfo
- Publication number
- JP6682532B2 JP6682532B2 JP2017527367A JP2017527367A JP6682532B2 JP 6682532 B2 JP6682532 B2 JP 6682532B2 JP 2017527367 A JP2017527367 A JP 2017527367A JP 2017527367 A JP2017527367 A JP 2017527367A JP 6682532 B2 JP6682532 B2 JP 6682532B2
- Authority
- JP
- Japan
- Prior art keywords
- neurotoxin
- protein
- bont
- cell
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002581 neurotoxin Substances 0.000 title claims description 269
- 231100000618 neurotoxin Toxicity 0.000 title claims description 265
- 101710138657 Neurotoxin Proteins 0.000 title claims description 250
- 238000000034 method Methods 0.000 title claims description 95
- 230000004071 biological effect Effects 0.000 title claims description 60
- 229920001184 polypeptide Polymers 0.000 title description 85
- 102000004196 processed proteins & peptides Human genes 0.000 title description 85
- 108090000765 processed proteins & peptides Proteins 0.000 title description 85
- 210000004027 cell Anatomy 0.000 claims description 291
- 108090000623 proteins and genes Proteins 0.000 claims description 175
- 102000004169 proteins and genes Human genes 0.000 claims description 169
- 108030001720 Bontoxilysin Proteins 0.000 claims description 128
- 108020001507 fusion proteins Proteins 0.000 claims description 108
- 102000037865 fusion proteins Human genes 0.000 claims description 108
- 238000003776 cleavage reaction Methods 0.000 claims description 56
- 230000007017 scission Effects 0.000 claims description 56
- 239000005089 Luciferase Substances 0.000 claims description 37
- 230000000694 effects Effects 0.000 claims description 29
- 108060001084 Luciferase Proteins 0.000 claims description 20
- 230000008823 permeabilization Effects 0.000 claims description 16
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 claims description 13
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 claims description 13
- 210000004881 tumor cell Anatomy 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims description 12
- 108010006464 Hemolysin Proteins Proteins 0.000 claims description 11
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 11
- 229960001231 choline Drugs 0.000 claims description 11
- 239000003228 hemolysin Substances 0.000 claims description 11
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 10
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 230000004952 protein activity Effects 0.000 claims description 10
- 239000003053 toxin Substances 0.000 claims description 10
- 231100000765 toxin Toxicity 0.000 claims description 10
- 108700012359 toxins Proteins 0.000 claims description 10
- 102000003834 Histamine H1 Receptors Human genes 0.000 claims description 9
- 108090000110 Histamine H1 Receptors Proteins 0.000 claims description 9
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 8
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 8
- 108091006275 SLC5A7 Proteins 0.000 claims description 8
- 108091006047 fluorescent proteins Proteins 0.000 claims description 8
- 102000034287 fluorescent proteins Human genes 0.000 claims description 8
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 7
- 210000000130 stem cell Anatomy 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 102000035160 transmembrane proteins Human genes 0.000 claims description 6
- 108091005703 transmembrane proteins Proteins 0.000 claims description 6
- 108010052285 Membrane Proteins Proteins 0.000 claims description 5
- 241000239290 Araneae Species 0.000 claims description 4
- 102000018697 Membrane Proteins Human genes 0.000 claims description 4
- 101710183389 Pneumolysin Proteins 0.000 claims description 4
- BQSPOPBPQURHDF-QVYRLLROSA-N (4s)-2-amino-5-[(1r)-2-amino-1-hydroxyethyl]-4,5-dihydro-1h-imidazole-4-carboxylic acid Chemical compound NC[C@@H](O)C1NC(N)=N[C@@H]1C(O)=O BQSPOPBPQURHDF-QVYRLLROSA-N 0.000 claims description 3
- 241000270295 Serpentes Species 0.000 claims description 3
- BQSPOPBPQURHDF-UHFFFAOYSA-N Streptolidine Natural products NCC(O)C1NC(N)=NC1C(O)=O BQSPOPBPQURHDF-UHFFFAOYSA-N 0.000 claims description 3
- 101710179002 Hemolytic toxin Proteins 0.000 claims description 2
- 229940053031 botulinum toxin Drugs 0.000 description 108
- 230000014509 gene expression Effects 0.000 description 39
- 206010029260 Neuroblastoma Diseases 0.000 description 31
- 238000012360 testing method Methods 0.000 description 25
- 239000013598 vector Substances 0.000 description 24
- 108091033319 polynucleotide Proteins 0.000 description 22
- 102000040430 polynucleotide Human genes 0.000 description 22
- 239000002157 polynucleotide Substances 0.000 description 22
- 210000000170 cell membrane Anatomy 0.000 description 18
- 239000000758 substrate Substances 0.000 description 18
- 125000003275 alpha amino acid group Chemical group 0.000 description 17
- 238000003556 assay Methods 0.000 description 17
- 210000002569 neuron Anatomy 0.000 description 17
- 102000005962 receptors Human genes 0.000 description 16
- 108020003175 receptors Proteins 0.000 description 16
- 230000004069 differentiation Effects 0.000 description 13
- 230000001105 regulatory effect Effects 0.000 description 13
- 230000035945 sensitivity Effects 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- 230000001939 inductive effect Effects 0.000 description 11
- 230000001537 neural effect Effects 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 210000003061 neural cell Anatomy 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 9
- 108010055044 Tetanus Toxin Proteins 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 229940118376 tetanus toxin Drugs 0.000 description 9
- 230000005945 translocation Effects 0.000 description 9
- 102100030552 Synaptosomal-associated protein 25 Human genes 0.000 description 8
- 239000004098 Tetracycline Substances 0.000 description 8
- 230000000875 corresponding effect Effects 0.000 description 8
- 229960002180 tetracycline Drugs 0.000 description 8
- 229930101283 tetracycline Natural products 0.000 description 8
- 235000019364 tetracycline Nutrition 0.000 description 8
- 150000003522 tetracyclines Chemical class 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 108010057722 Synaptosomal-Associated Protein 25 Proteins 0.000 description 7
- 210000000172 cytosol Anatomy 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 101000584505 Homo sapiens Synaptic vesicle glycoprotein 2A Proteins 0.000 description 6
- 102100030701 Synaptic vesicle glycoprotein 2A Human genes 0.000 description 6
- 231100001103 botulinum neurotoxin Toxicity 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 231100000654 protein toxin Toxicity 0.000 description 6
- 201000009051 Embryonal Carcinoma Diseases 0.000 description 5
- 108090000331 Firefly luciferases Proteins 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- -1 perfringolidine O Proteins 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 241001112695 Clostridiales Species 0.000 description 4
- 108030001722 Tentoxilysin Proteins 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000012937 correction Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000010606 normalization Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 108010075210 streptolysin O Proteins 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 3
- 241000193155 Clostridium botulinum Species 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 102000000583 SNARE Proteins Human genes 0.000 description 3
- 108010041948 SNARE Proteins Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 230000007248 cellular mechanism Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 229960003722 doxycycline Drugs 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 230000035987 intoxication Effects 0.000 description 3
- 231100000566 intoxication Toxicity 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000034217 membrane fusion Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 210000002243 primary neuron Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 229930002330 retinoic acid Natural products 0.000 description 3
- 210000002504 synaptic vesicle Anatomy 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 108700020534 tetracycline resistance-encoding transposon repressor Proteins 0.000 description 3
- 108091006106 transcriptional activators Proteins 0.000 description 3
- 229960001727 tretinoin Drugs 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 241000193449 Clostridium tetani Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010068250 Herpes Simplex Virus Protein Vmw65 Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 208000007101 Muscle Cramp Diseases 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000005392 Spasm Diseases 0.000 description 2
- 208000004350 Strabismus Diseases 0.000 description 2
- 108010057266 Type A Botulinum Toxins Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000005462 in vivo assay Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000004897 n-terminal region Anatomy 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 239000002858 neurotransmitter agent Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- FGDZQCVHDSGLHJ-UHFFFAOYSA-M rubidium chloride Chemical compound [Cl-].[Rb+] FGDZQCVHDSGLHJ-UHFFFAOYSA-M 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 101710117542 Botulinum neurotoxin type A Proteins 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 102000002110 C2 domains Human genes 0.000 description 1
- 108050009459 C2 domains Proteins 0.000 description 1
- XMWRBQBLMFGWIX-UHFFFAOYSA-N C60 fullerene Chemical class C12=C3C(C4=C56)=C7C8=C5C5=C9C%10=C6C6=C4C1=C1C4=C6C6=C%10C%10=C9C9=C%11C5=C8C5=C8C7=C3C3=C7C2=C1C1=C2C4=C6C4=C%10C6=C9C9=C%11C5=C5C8=C3C3=C7C1=C1C2=C4C6=C2C9=C5C3=C12 XMWRBQBLMFGWIX-UHFFFAOYSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 208000014094 Dystonic disease Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 208000004095 Hemifacial Spasm Diseases 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 208000008454 Hyperhidrosis Diseases 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
- 241000254158 Lampyridae Species 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 101710164436 Listeriolysin O Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 108010063737 Myristoylated Alanine-Rich C Kinase Substrate Proteins 0.000 description 1
- 102000015695 Myristoylated Alanine-Rich C Kinase Substrate Human genes 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 241001483952 Peach chlorotic mottle virus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010056995 Perforin Proteins 0.000 description 1
- 102000004503 Perforin Human genes 0.000 description 1
- 101710182846 Polyhedrin Proteins 0.000 description 1
- 101000606032 Pomacea maculata Perivitellin-2 31 kDa subunit Proteins 0.000 description 1
- 101000606027 Pomacea maculata Perivitellin-2 67 kDa subunit Proteins 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 206010043376 Tetanus Diseases 0.000 description 1
- 206010044074 Torticollis Diseases 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 108010079650 abobotulinumtoxinA Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 1
- 230000001042 autoregulative effect Effects 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 230000009141 biological interaction Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 206010005159 blepharospasm Diseases 0.000 description 1
- 230000000744 blepharospasm Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940089093 botox Drugs 0.000 description 1
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001721 carbon Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 201000002866 cervical dystonia Diseases 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 229940098753 dysport Drugs 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003617 erythrocyte membrane Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 201000002904 focal dystonia Diseases 0.000 description 1
- 229910003472 fullerene Inorganic materials 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- CEAZRRDELHUEMR-UHFFFAOYSA-N gentamicin Chemical compound O1C(C(C)NC)CCC(N)C1OC1C(O)C(OC2C(C(NC)C(C)(O)CO2)O)C(N)CC1N CEAZRRDELHUEMR-UHFFFAOYSA-N 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 230000037315 hyperhidrosis Effects 0.000 description 1
- 229960004716 idoxuridine Drugs 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 108010024001 incobotulinumtoxinA Proteins 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000007422 luminescence assay Methods 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229960003248 mifepristone Drugs 0.000 description 1
- VKHAHZOOUSRJNA-GCNJZUOMSA-N mifepristone Chemical compound C1([C@@H]2C3=C4CCC(=O)C=C4CC[C@H]3[C@@H]3CC[C@@]([C@]3(C2)C)(O)C#CC)=CC=C(N(C)C)C=C1 VKHAHZOOUSRJNA-GCNJZUOMSA-N 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000023105 myelination Effects 0.000 description 1
- 230000007498 myristoylation Effects 0.000 description 1
- 210000005155 neural progenitor cell Anatomy 0.000 description 1
- 210000003757 neuroblast Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000026792 palmitoylation Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000004197 pelvis Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 108010094020 polyglycine Proteins 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 210000000063 presynaptic terminal Anatomy 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000003571 reporter gene assay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229940102127 rubidium chloride Drugs 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000001608 teratocarcinoma Diseases 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000007888 toxin activity Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 229940018272 xeomin Drugs 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6809—Determination of free amino acids involving fluorescent derivatizing reagents reacting non-specifically with all amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Neurology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
(a)(i)アンカー型タンパク質と、(ii)レポータータンパク質と、(iii)アンカー型タンパク質とレポータータンパク質との間に介在する神経毒切断部位とを含む融合タンパク質を神経毒感受性細胞に発現させ、
(b)(a)の神経毒感受性細胞を神経毒とともにインキュベートし、神経毒がその生物学的活性を示すことができる条件下で神経毒感受性細胞を培養し、
(c)(b)の神経毒感受性細胞への透過処理を、該透過処理した神経毒感受性細胞からレポータータンパク質は遊離するがアンカー型タンパク質は遊離しない条件下で行い、かつ
(d)(c)の透過処理した神経毒感受性細胞から遊離したレポータータンパク質の活性を定量化し、これにより神経毒の生物学的活性を測定するステップを含む方法に関する。
本発明はまた、以下に関する。
[項目1]
神経毒の生物学的活性を測定する方法であって、
(a)(i)アンカー型タンパク質と、(ii)レポータータンパク質と、(iii)前記アンカー型タンパク質と前記レポータータンパク質との間に介在する神経毒切断部位とを含む融合タンパク質を神経毒感受性細胞に発現させ、
(b)(a)の神経毒感受性細胞を神経毒とともにインキュベートし、前記神経毒がその生物学的活性を示すことができる条件下で前記神経毒感受性細胞を培養し、
(c)(b)の神経毒感受性細胞への透過処理を、前記透過処理した神経毒感受性細胞から前記レポータータンパク質は遊離するが、前記アンカー型タンパク質は遊離しない条件下で行い、且つ
(d)(c)の透過処理した神経毒感受性細胞から遊離した前記レポータータンパク質の活性を定量化し、
これにより前記神経毒の生物学的活性を測定するステップ、
を含む、前記方法。
[項目2]
前記神経毒感受性細胞が、腫瘍細胞株、初代細胞、幹細胞又は人工多能性幹細胞に由来する、項目1に記載の方法。
[項目3]
前記アンカー型タンパク質が、コリントランスポーター、H1−受容体、Gタンパク質共役型受容体(GPCR)及びSV2から成る群から選択される膜タンパク質である、項目1又は2に記載の方法。
[項目4]
前記神経毒切断部位が、BoNT/A、BoNT/B、BoNT/C1、BoNT/D、BoNT/E、BoNT/F、BoNT/G及びTeNTの切断部位から成る群から選択される、項目1〜3のいずれか1項に記載の方法。
[項目5]
前記レポータータンパク質が、ルシフェラーゼ、アルカリホスファターゼ、βガラクトシダーゼ、及びホースラディッシュペルオキシダーゼ(HRP)から成る群から選択される酵素であるか、又はGFP、YFP、BFP及びRFPから成る群から選択される蛍光タンパク質である、項目1〜4のいずれか1項に記載の方法。
[項目6]
前記神経毒感受性細胞の透過処理のために溶血毒を使用する、項目1〜5のいずれか1項に記載の方法。
[項目7]
前記溶血毒が、ストレプトリジンO、パーフリンゴリジンO、ニューモリシン、細菌性溶血素、及びヘビ又はクモ由来の膜孔形成毒素から成る群から選択される、項目6に記載の方法。
[項目8]
前記融合タンパク質が、コリントランスポーター−GFP−SNAP−25−ルシフェラーゼ、H1−受容体−SNAP−25−ルシフェラーゼ及びH1−受容体−SNAP−25−HRPから選択される融合タンパク質を含む、項目1〜7のいずれか1項に記載の方法。
[項目9]
前記レポータータンパク質の活性の定量化が、前記レポータータンパク質の活性を標準化することを含む、項目1〜8のいずれか1項に記載の方法。
[項目10]
前記レポータータンパク質の活性の標準化を、前記神経毒感受性細胞内若しくは前記神経毒感受性細胞上に残存する切断されていない融合タンパク質の残留レポータータンパク質活性又は前記融合タンパク質の総レポータータンパク質活性を測定することにより行う、項目9に記載の方法。
[項目11]
神経毒感受性細胞において神経毒の生物学的活性を測定するための、(i)アンカー型タンパク質と、(ii)レポータータンパク質と、(iii)前記アンカー型タンパク質と前記レポータータンパク質との間に介在する神経毒切断部位とから成る融合タンパク質。
[項目12]
前記アンカー型タンパク質が、コリントランスポーター、H1−受容体、Gタンパク質共役型受容体(GPCR)及びSV2から成る群から選択され、前記レポータータンパク質が、ルシフェラーゼ、アルカリホスファターゼ、βガラクトシダーゼ及びホースラディッシュペルオキシダーゼから成る群から選択される酵素であるか、又はGFP、YFP、BFP及びRFPから成る群から選択される蛍光タンパク質であり、且つ、前記神経毒切断部位が、BoNT/A、BoNT/B、BoNT/C1、BoNT/D、BoNT/E、BoNT/F、BoNT/G及びTeNTの切断部位から成る群から選択される、項目11に記載の融合タンパク質。
[項目13]
前記融合タンパク質が、コリントランスポーター−GFP−SNAP−25−ルシフェラーゼ、H1−受容体−SNAP−25−ルシフェラーゼ及びH1−受容体−SNAP−25−HRPから選択される融合タンパク質を含む、項目11又は12に記載の融合タンパク質。
[項目14]
項目11〜13のいずれか1項に記載の融合タンパク質を含むキット。
[項目15]
神経毒感受性細胞において、神経毒の生物学的活性を測定するための、項目11〜13のいずれか1項に記載の融合タンパク質の使用。
(a)(i)アンカー型タンパク質と、(ii)レポータータンパク質と、(iii)アンカー型タンパク質とレポータータンパク質との間に介在する神経毒切断部位とを含む融合タンパク質を、神経細胞に分化することのできる神経毒感受性細胞に発現させ、
(b)ステップ(a)の神経毒感受性細胞が神経分化細胞に分化する条件下および時間でステップ(a)の神経毒感受性細胞を培地中にて分化させ、
(c)ステップ(b)の神経分化細胞を神経毒とともにインキュベートし、該細胞を、神経毒がその生物学的活性を示すことができる条件下で培養し、
(d)ステップ(c)の神経分化細胞の透過処理を、該透過処理した神経分化細胞からレポータータンパク質は遊離させるがアンカー型タンパク質は遊離させない条件下で行い、
(e)ステップ(d)の細胞から遊離したレポータータンパク質の活性を定量化し、これにより神経毒の生物学的活性を測定する各ステップを含む。
Claims (15)
- 神経毒の生物学的活性を測定する方法であって、
(a)(i)アンカー型タンパク質と、(ii)レポータータンパク質と、(iii)前記アンカー型タンパク質と前記レポータータンパク質との間に介在する神経毒切断部位とを含む融合タンパク質を神経毒感受性細胞に発現させ、
(b)(a)の神経毒感受性細胞を神経毒とともにインキュベートし、前記神経毒がその生物学的活性を示すことができる条件下で前記神経毒感受性細胞を培養し、
(c)(b)の神経毒感受性細胞への透過処理を、前記透過処理した神経毒感受性細胞から前記レポータータンパク質は遊離するが、前記アンカー型タンパク質は遊離しない条件下で行い、且つ
(d)(c)の透過処理した神経毒感受性細胞から遊離した前記レポータータンパク質の活性を定量化し、
これにより前記神経毒の生物学的活性を測定するステップ、
を含む、前記方法。 - 前記神経毒感受性細胞が、腫瘍細胞株、初代細胞、幹細胞又は人工多能性幹細胞に由来する、請求項1に記載の方法。
- 前記アンカー型タンパク質が、コリントランスポーター、H1−受容体、Gタンパク質共役型受容体(GPCR)及びSV2から成る群から選択される膜タンパク質である、請求項1又は2に記載の方法。
- 前記神経毒切断部位が、BoNT/A、BoNT/B、BoNT/C1、BoNT/D、BoNT/E、BoNT/F、BoNT/G及びTeNTの切断部位から成る群から選択される、請求項1〜3のいずれか1項に記載の方法。
- 前記レポータータンパク質が、ルシフェラーゼ、アルカリホスファターゼ、βガラクトシダーゼ、及びホースラディッシュペルオキシダーゼ(HRP)から成る群から選択される酵素であるか、又はGFP、YFP、BFP及びRFPから成る群から選択される蛍光タンパク質である、請求項1〜4のいずれか1項に記載の方法。
- 前記神経毒感受性細胞の透過処理のために溶血毒を使用する、請求項1〜5のいずれか1項に記載の方法。
- 前記溶血毒が、ストレプトリジンO、パーフリンゴリジンO、ニューモリシン、細菌性溶血素、及びヘビ又はクモ由来の膜孔形成毒素から成る群から選択される、請求項6に記載の方法。
- 前記融合タンパク質が、コリントランスポーター−GFP−SNAP−25−ルシフェラーゼ、H1−受容体−SNAP−25−ルシフェラーゼ及びH1−受容体−SNAP−25−HRPから選択される融合タンパク質を含む、請求項1〜7のいずれか1項に記載の方法。
- 前記レポータータンパク質の活性の定量化が、前記レポータータンパク質の活性を標準化することを含む、請求項1〜8のいずれか1項に記載の方法。
- 前記レポータータンパク質の活性の標準化を、前記神経毒感受性細胞内若しくは前記神経毒感受性細胞上に残存する切断されていない融合タンパク質の残留レポータータンパク質活性又は前記融合タンパク質の総レポータータンパク質活性を測定することにより行う、請求項9に記載の方法。
- 神経毒感受性細胞において神経毒の生物学的活性を測定するための、(i)1つのアンカー型タンパク質と、(ii)1つのレポータータンパク質と、(iii)前記アンカー型タンパク質と前記レポータータンパク質との間に介在する神経毒切断部位とから成る融合タンパク質であって、ここで前記アンカー型タンパク質は膜貫通型タンパク質又は膜結合タンパク質である、前記融合タンパク質。
- 前記アンカー型タンパク質が、コリントランスポーター、H1−受容体、Gタンパク質共役型受容体(GPCR)及びSV2から成る群から選択され、前記レポータータンパク質が、ルシフェラーゼ、アルカリホスファターゼ、βガラクトシダーゼ及びホースラディッシュペルオキシダーゼから成る群から選択される酵素であるか、又はGFP、YFP、BFP及びRFPから成る群から選択される蛍光タンパク質であり、且つ、前記神経毒切断部位が、BoNT/A、BoNT/B、BoNT/C1、BoNT/D、BoNT/E、BoNT/F、BoNT/G及びTeNTの切断部位から成る群から選択される、請求項11に記載の融合タンパク質。
- 前記融合タンパク質が、コリントランスポーター−GFP−SNAP−25−ルシフェラーゼ、H1−受容体−SNAP−25−ルシフェラーゼ及びH1−受容体−SNAP−25−HRPから選択される融合タンパク質を含む、請求項11又は12に記載の融合タンパク質。
- 請求項11〜13のいずれか1項に記載の融合タンパク質を含むキット。
- 神経毒感受性細胞において、神経毒の生物学的活性を測定するための、請求項11〜13のいずれか1項に記載の融合タンパク質の使用。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP14194265 | 2014-11-21 | ||
EP14194265.6 | 2014-11-21 | ||
PCT/EP2015/077245 WO2016079310A1 (en) | 2014-11-21 | 2015-11-20 | Methods for the determination of the biological activities of neurotoxin polypeptides |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2017535274A JP2017535274A (ja) | 2017-11-30 |
JP6682532B2 true JP6682532B2 (ja) | 2020-04-15 |
Family
ID=51945763
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2017527367A Active JP6682532B2 (ja) | 2014-11-21 | 2015-11-20 | 神経毒ポリペプチドの生物学的活性を測定する方法 |
Country Status (8)
Country | Link |
---|---|
US (1) | US10634683B2 (ja) |
EP (1) | EP3221699B1 (ja) |
JP (1) | JP6682532B2 (ja) |
KR (1) | KR102409293B1 (ja) |
CN (1) | CN107003310B (ja) |
AU (1) | AU2015348254B2 (ja) |
CA (1) | CA2968284C (ja) |
WO (1) | WO2016079310A1 (ja) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MX360513B (es) | 2013-06-28 | 2018-11-07 | Merz Pharma Gmbh & Co Kgaa | Medios y metodos para la determinacion de la actividad biologica de polipeptidos de neurotoxina en celulas. |
WO2015088477A1 (en) * | 2013-12-09 | 2015-06-18 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Peptide substrates recognizable by type e botulinum neurotoxin |
TWI704156B (zh) * | 2015-03-04 | 2020-09-11 | 德商曼茲法瑪股份有限公司 | 用於增進肉毒桿菌神經毒素進入細胞之特定攝取的方法 |
JP6493316B2 (ja) * | 2016-06-21 | 2019-04-03 | 東亜ディーケーケー株式会社 | 変異型甲虫ルシフェラーゼ、遺伝子、組換えベクター、形質転換体、及び変異型甲虫ルシフェラーゼの製造方法 |
GB201702500D0 (en) | 2017-02-16 | 2017-04-05 | Univ Sheffield | Stable vamp reporter assay |
CN113015812A (zh) * | 2018-09-28 | 2021-06-22 | 益普生生物制药有限公司 | 基于细胞的梭菌神经毒素测定法 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7374896B2 (en) * | 2001-08-28 | 2008-05-20 | Allergan, Inc. | GFP-SNAP25 fluorescence release assay for botulinum neurotoxin protease activity |
EP2502930B1 (en) * | 2003-12-19 | 2016-06-29 | Wisconsin Alumni Research Foundation | Method and compositions for detecting botulinum neurotoxin |
GB0426397D0 (en) * | 2004-12-01 | 2005-01-05 | Health Prot Agency | Fusion proteins |
EP2208067B1 (en) * | 2007-09-14 | 2019-02-06 | BioMadison, Inc. | Resonance energy transfer assay with cleavage sequence and spacer |
CA2715033C (en) * | 2008-03-14 | 2014-11-25 | Allergan, Inc. | Immuno-based botulinum toxin serotype a activity assays |
SG174350A1 (en) | 2009-03-13 | 2011-10-28 | Allergan Inc | Cells useful for immuno-based botulinum toxin serotype a activity assays |
PT2425251T (pt) | 2009-04-27 | 2017-11-27 | Merz Pharma Gmbh & Co Kgaa | Meios e métodos para a determinação da quantidade de polipéptido de neurotoxina e das suas atividades catalítica e proteolítica |
US20150044709A1 (en) * | 2012-03-07 | 2015-02-12 | Merz Pharma Gmbh & Co Kgaa | Means and methods for determining neurotoxin activity based on a modified luciferase |
BR112015011255A2 (pt) * | 2012-11-21 | 2018-09-25 | Merz Pharma Gmbh & Co Kgaa | polinucleotídeo que codifica um polipeptídeo de fusão, vetor, polipeptídeo de fusão, célula hospedeira, método para determinar a atividade de neurotoxina, uso do polinucleotídeo e kit. |
-
2015
- 2015-11-20 EP EP15798407.1A patent/EP3221699B1/en active Active
- 2015-11-20 US US15/528,219 patent/US10634683B2/en active Active
- 2015-11-20 CA CA2968284A patent/CA2968284C/en active Active
- 2015-11-20 AU AU2015348254A patent/AU2015348254B2/en active Active
- 2015-11-20 CN CN201580062143.8A patent/CN107003310B/zh active Active
- 2015-11-20 KR KR1020177013483A patent/KR102409293B1/ko active IP Right Grant
- 2015-11-20 WO PCT/EP2015/077245 patent/WO2016079310A1/en active Application Filing
- 2015-11-20 JP JP2017527367A patent/JP6682532B2/ja active Active
Also Published As
Publication number | Publication date |
---|---|
US20180045733A1 (en) | 2018-02-15 |
EP3221699A1 (en) | 2017-09-27 |
CA2968284C (en) | 2023-11-14 |
KR102409293B1 (ko) | 2022-06-14 |
JP2017535274A (ja) | 2017-11-30 |
AU2015348254A1 (en) | 2017-05-25 |
CA2968284A1 (en) | 2016-05-26 |
CN107003310A (zh) | 2017-08-01 |
EP3221699B1 (en) | 2020-06-17 |
AU2015348254B2 (en) | 2021-03-25 |
KR20170084098A (ko) | 2017-07-19 |
WO2016079310A1 (en) | 2016-05-26 |
CN107003310B (zh) | 2019-07-19 |
US10634683B2 (en) | 2020-04-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6682532B2 (ja) | 神経毒ポリペプチドの生物学的活性を測定する方法 | |
US11788069B2 (en) | In vitro and cell based assays for measuring the activity of botulinum neurotoxins | |
AU2013349725B2 (en) | Means and methods for determination of botulinum neurotoxin biological activity | |
US8853360B2 (en) | Engineered botulinum neurotoxin C1 with selective substrate specificity | |
US10787696B2 (en) | System for the assessment of protease activity | |
JP2015509372A (ja) | 修飾ルシフェラーゼに基づいて神経毒活性を測定するための手段および方法 | |
RU2694191C2 (ru) | Ганглиозиды для стандартизации и повышения чувствительности клеток к нейротоксинам ботулизма в тест-системах in vitro |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
RD01 | Notification of change of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7426 Effective date: 20171205 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20171205 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20180802 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20190624 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20190709 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20191008 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20200317 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20200325 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6682532 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |