JPWO2002081515A1 - Insulin-like growth factor binding protein - Google Patents
Insulin-like growth factor binding protein Download PDFInfo
- Publication number
- JPWO2002081515A1 JPWO2002081515A1 JP2002579900A JP2002579900A JPWO2002081515A1 JP WO2002081515 A1 JPWO2002081515 A1 JP WO2002081515A1 JP 2002579900 A JP2002579900 A JP 2002579900A JP 2002579900 A JP2002579900 A JP 2002579900A JP WO2002081515 A1 JPWO2002081515 A1 JP WO2002081515A1
- Authority
- JP
- Japan
- Prior art keywords
- protein
- disease
- abnormal
- dna
- diseases
- 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.)
- Withdrawn
Links
- 108091022911 insulin-like growth factor binding Proteins 0.000 title description 80
- 102000028416 insulin-like growth factor binding Human genes 0.000 title description 76
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 500
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 409
- 238000000034 method Methods 0.000 claims abstract description 243
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 231
- 201000010099 disease Diseases 0.000 claims abstract description 203
- 239000003814 drug Substances 0.000 claims abstract description 53
- 230000000069 prophylactic effect Effects 0.000 claims abstract description 17
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 16
- 108020004414 DNA Proteins 0.000 claims description 166
- 210000004027 cell Anatomy 0.000 claims description 161
- 230000014509 gene expression Effects 0.000 claims description 125
- 230000002159 abnormal effect Effects 0.000 claims description 108
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 90
- 239000002773 nucleotide Substances 0.000 claims description 76
- 125000003729 nucleotide group Chemical group 0.000 claims description 76
- 108091034117 Oligonucleotide Proteins 0.000 claims description 72
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 51
- 241000282414 Homo sapiens Species 0.000 claims description 51
- 239000013598 vector Substances 0.000 claims description 45
- 230000000694 effects Effects 0.000 claims description 44
- 230000004663 cell proliferation Effects 0.000 claims description 43
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 38
- 150000001875 compounds Chemical class 0.000 claims description 35
- 238000003752 polymerase chain reaction Methods 0.000 claims description 34
- 201000011510 cancer Diseases 0.000 claims description 32
- 230000002829 reductive effect Effects 0.000 claims description 31
- 230000009471 action Effects 0.000 claims description 29
- 150000001413 amino acids Chemical class 0.000 claims description 29
- 239000000523 sample Substances 0.000 claims description 28
- 238000012360 testing method Methods 0.000 claims description 28
- 102000013275 Somatomedins Human genes 0.000 claims description 27
- 206010012601 diabetes mellitus Diseases 0.000 claims description 27
- 230000004069 differentiation Effects 0.000 claims description 26
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 25
- 206010009944 Colon cancer Diseases 0.000 claims description 25
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 25
- 238000004519 manufacturing process Methods 0.000 claims description 25
- 230000004097 bone metabolism Effects 0.000 claims description 24
- 230000024245 cell differentiation Effects 0.000 claims description 23
- 208000035475 disorder Diseases 0.000 claims description 23
- 210000000329 smooth muscle myocyte Anatomy 0.000 claims description 23
- 238000012258 culturing Methods 0.000 claims description 21
- 239000000122 growth hormone Substances 0.000 claims description 21
- 210000004408 hybridoma Anatomy 0.000 claims description 21
- 238000012216 screening Methods 0.000 claims description 21
- 108010051696 Growth Hormone Proteins 0.000 claims description 20
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 20
- 206010060862 Prostate cancer Diseases 0.000 claims description 20
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 20
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 20
- 210000002363 skeletal muscle cell Anatomy 0.000 claims description 20
- 238000009396 hybridization Methods 0.000 claims description 19
- 210000004698 lymphocyte Anatomy 0.000 claims description 19
- 230000035772 mutation Effects 0.000 claims description 19
- 239000013612 plasmid Substances 0.000 claims description 19
- 230000027119 gastric acid secretion Effects 0.000 claims description 18
- 238000013518 transcription Methods 0.000 claims description 18
- 230000035897 transcription Effects 0.000 claims description 18
- 208000019553 vascular disease Diseases 0.000 claims description 18
- 230000008595 infiltration Effects 0.000 claims description 17
- 238000001764 infiltration Methods 0.000 claims description 17
- 208000027866 inflammatory disease Diseases 0.000 claims description 17
- 230000035755 proliferation Effects 0.000 claims description 17
- 206010006187 Breast cancer Diseases 0.000 claims description 15
- 208000026310 Breast neoplasm Diseases 0.000 claims description 15
- 208000001132 Osteoporosis Diseases 0.000 claims description 15
- 206010046798 Uterine leiomyoma Diseases 0.000 claims description 15
- 208000020832 chronic kidney disease Diseases 0.000 claims description 15
- 201000010260 leiomyoma Diseases 0.000 claims description 15
- 201000007270 liver cancer Diseases 0.000 claims description 15
- 208000014018 liver neoplasm Diseases 0.000 claims description 15
- 201000007954 uterine fibroid Diseases 0.000 claims description 15
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 claims description 14
- 208000007536 Thrombosis Diseases 0.000 claims description 14
- 208000029742 colonic neoplasm Diseases 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 14
- 230000036961 partial effect Effects 0.000 claims description 14
- 230000002792 vascular Effects 0.000 claims description 14
- 206010020751 Hypersensitivity Diseases 0.000 claims description 13
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 claims description 13
- 210000004102 animal cell Anatomy 0.000 claims description 13
- 208000022831 chronic renal failure syndrome Diseases 0.000 claims description 13
- 208000035285 Allergic Seasonal Rhinitis Diseases 0.000 claims description 12
- 206010003645 Atopy Diseases 0.000 claims description 12
- 208000023275 Autoimmune disease Diseases 0.000 claims description 12
- 208000014181 Bronchial disease Diseases 0.000 claims description 12
- 206010012689 Diabetic retinopathy Diseases 0.000 claims description 12
- 206010018364 Glomerulonephritis Diseases 0.000 claims description 12
- 208000009329 Graft vs Host Disease Diseases 0.000 claims description 12
- 206010019280 Heart failures Diseases 0.000 claims description 12
- 241000238631 Hexapoda Species 0.000 claims description 12
- 208000031226 Hyperlipidaemia Diseases 0.000 claims description 12
- 206010020772 Hypertension Diseases 0.000 claims description 12
- 206010040047 Sepsis Diseases 0.000 claims description 12
- 208000007107 Stomach Ulcer Diseases 0.000 claims description 12
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 12
- 208000026935 allergic disease Diseases 0.000 claims description 12
- 230000007815 allergy Effects 0.000 claims description 12
- 208000006673 asthma Diseases 0.000 claims description 12
- 206010008118 cerebral infarction Diseases 0.000 claims description 12
- 208000026106 cerebrovascular disease Diseases 0.000 claims description 12
- 238000001415 gene therapy Methods 0.000 claims description 12
- 208000024908 graft versus host disease Diseases 0.000 claims description 12
- 208000015181 infectious disease Diseases 0.000 claims description 12
- 208000014674 injury Diseases 0.000 claims description 12
- 230000000813 microbial effect Effects 0.000 claims description 12
- 208000010125 myocardial infarction Diseases 0.000 claims description 12
- 208000031225 myocardial ischemia Diseases 0.000 claims description 12
- 201000008383 nephritis Diseases 0.000 claims description 12
- 230000002093 peripheral effect Effects 0.000 claims description 12
- 208000037803 restenosis Diseases 0.000 claims description 12
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 12
- 230000008736 traumatic injury Effects 0.000 claims description 12
- 206010000599 Acromegaly Diseases 0.000 claims description 11
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims description 11
- 208000014644 Brain disease Diseases 0.000 claims description 11
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 11
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 11
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 11
- 230000000302 ischemic effect Effects 0.000 claims description 11
- 206010002383 Angina Pectoris Diseases 0.000 claims description 10
- 208000000419 Chronic Hepatitis B Diseases 0.000 claims description 10
- 206010013883 Dwarfism Diseases 0.000 claims description 10
- 238000010367 cloning Methods 0.000 claims description 10
- 208000027744 congestion Diseases 0.000 claims description 10
- 201000005917 gastric ulcer Diseases 0.000 claims description 10
- 208000002672 hepatitis B Diseases 0.000 claims description 10
- 230000001771 impaired effect Effects 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 230000002265 prevention Effects 0.000 claims description 9
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 8
- 206010028417 myasthenia gravis Diseases 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 206010061876 Obstruction Diseases 0.000 claims description 7
- 230000005856 abnormality Effects 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 7
- 230000036428 airway hyperreactivity Effects 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 108020004511 Recombinant DNA Proteins 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 6
- 108091026890 Coding region Proteins 0.000 claims description 5
- 206010020880 Hypertrophy Diseases 0.000 claims description 5
- 108700008625 Reporter Genes Proteins 0.000 claims description 5
- 230000037431 insertion Effects 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 4
- 238000012217 deletion Methods 0.000 claims description 4
- 230000037430 deletion Effects 0.000 claims description 4
- 230000003248 secreting effect Effects 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 238000011002 quantification Methods 0.000 claims description 3
- 210000002784 stomach Anatomy 0.000 claims description 3
- 102000018997 Growth Hormone Human genes 0.000 claims 6
- 230000028327 secretion Effects 0.000 claims 2
- 208000018556 stomach disease Diseases 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 abstract description 13
- 235000018102 proteins Nutrition 0.000 description 350
- 239000002299 complementary DNA Substances 0.000 description 79
- 102100029228 Insulin-like growth factor-binding protein 7 Human genes 0.000 description 59
- 108010008598 insulin-like growth factor binding protein-related protein 1 Proteins 0.000 description 57
- 239000002609 medium Substances 0.000 description 56
- 239000000243 solution Substances 0.000 description 39
- 230000006870 function Effects 0.000 description 38
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 28
- 238000002360 preparation method Methods 0.000 description 28
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 27
- 108020004999 messenger RNA Proteins 0.000 description 27
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 26
- 229940024606 amino acid Drugs 0.000 description 26
- 235000001014 amino acid Nutrition 0.000 description 26
- 239000012634 fragment Substances 0.000 description 26
- 241000588724 Escherichia coli Species 0.000 description 25
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 25
- 101001055320 Myxine glutinosa Insulin-like growth factor Proteins 0.000 description 25
- 238000006243 chemical reaction Methods 0.000 description 25
- 108090000765 processed proteins & peptides Proteins 0.000 description 25
- 210000001519 tissue Anatomy 0.000 description 25
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 24
- 239000013615 primer Substances 0.000 description 24
- 230000001965 increasing effect Effects 0.000 description 23
- 241001465754 Metazoa Species 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 241000196324 Embryophyta Species 0.000 description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 19
- 230000027455 binding Effects 0.000 description 19
- 239000012228 culture supernatant Substances 0.000 description 18
- 239000000203 mixture Substances 0.000 description 18
- 238000010369 molecular cloning Methods 0.000 description 17
- 229920005989 resin Polymers 0.000 description 16
- 239000011347 resin Substances 0.000 description 16
- 238000013519 translation Methods 0.000 description 16
- 239000000427 antigen Substances 0.000 description 15
- 108091007433 antigens Proteins 0.000 description 15
- 102000036639 antigens Human genes 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 15
- 239000007758 minimum essential medium Substances 0.000 description 15
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- KAQKFAOMNZTLHT-OZUDYXHBSA-N prostaglandin I2 Chemical compound O1\C(=C/CCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 KAQKFAOMNZTLHT-OZUDYXHBSA-N 0.000 description 15
- 235000002639 sodium chloride Nutrition 0.000 description 15
- 102100038803 Somatotropin Human genes 0.000 description 14
- 239000000126 substance Substances 0.000 description 14
- 101000862089 Clarkia lewisii Glucose-6-phosphate isomerase, cytosolic 1A Proteins 0.000 description 13
- 210000004556 brain Anatomy 0.000 description 13
- 239000013604 expression vector Substances 0.000 description 13
- 102000004877 Insulin Human genes 0.000 description 12
- 108090001061 Insulin Proteins 0.000 description 12
- 102000004374 Insulin-like growth factor binding protein 3 Human genes 0.000 description 12
- 108090000965 Insulin-like growth factor binding protein 3 Proteins 0.000 description 12
- 229940125396 insulin Drugs 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000007423 decrease Effects 0.000 description 11
- 244000005700 microbiome Species 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 102000004375 Insulin-like growth factor-binding protein 1 Human genes 0.000 description 9
- 108090000957 Insulin-like growth factor-binding protein 1 Proteins 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 210000005013 brain tissue Anatomy 0.000 description 8
- 229940125904 compound 1 Drugs 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- -1 secretory production Proteins 0.000 description 8
- 210000002027 skeletal muscle Anatomy 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 206010003445 Ascites Diseases 0.000 description 7
- 101000840577 Homo sapiens Insulin-like growth factor-binding protein 7 Proteins 0.000 description 7
- 108090000964 Insulin-like growth factor binding protein 2 Proteins 0.000 description 7
- 102000004372 Insulin-like growth factor binding protein 2 Human genes 0.000 description 7
- 108090000969 Insulin-like growth factor-binding protein 4 Proteins 0.000 description 7
- 102000004369 Insulin-like growth factor-binding protein 4 Human genes 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000003018 immunoassay Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 108090000961 Insulin-like growth factor binding protein 5 Proteins 0.000 description 6
- 102000004371 Insulin-like growth factor binding protein 5 Human genes 0.000 description 6
- 102000003982 Parathyroid hormone Human genes 0.000 description 6
- 108090000445 Parathyroid hormone Proteins 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- 210000000628 antibody-producing cell Anatomy 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 230000001419 dependent effect Effects 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 210000004379 membrane Anatomy 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 210000000963 osteoblast Anatomy 0.000 description 6
- 239000000199 parathyroid hormone Substances 0.000 description 6
- 229960001319 parathyroid hormone Drugs 0.000 description 6
- 230000001766 physiological effect Effects 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 6
- 241000701161 unidentified adenovirus Species 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 108090001014 Insulin-like growth factor-binding protein 6 Proteins 0.000 description 5
- 102000004883 Insulin-like growth factor-binding protein 6 Human genes 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 108091081024 Start codon Proteins 0.000 description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 238000012136 culture method Methods 0.000 description 5
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 210000000265 leukocyte Anatomy 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 241000701447 unidentified baculovirus Species 0.000 description 5
- QTWZCODKTSUZJN-LJAQVGFWSA-N (2s)-5-[[amino-[(2,2,5,7,8-pentamethyl-3,4-dihydrochromen-6-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C(C(C)=C1C)=C(C)C2=C1OC(C)(C)CC2 QTWZCODKTSUZJN-LJAQVGFWSA-N 0.000 description 4
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 4
- 229960005508 8-azaguanine Drugs 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 208000013600 Diabetic vascular disease Diseases 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 241000588722 Escherichia Species 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 4
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 4
- 206010022998 Irritability Diseases 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 4
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 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 4
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 201000009101 diabetic angiopathy Diseases 0.000 description 4
- 201000002249 diabetic peripheral angiopathy Diseases 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 210000001671 embryonic stem cell Anatomy 0.000 description 4
- 230000002708 enhancing effect Effects 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000003862 glucocorticoid Substances 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 208000006454 hepatitis Diseases 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 208000032839 leukemia Diseases 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- 229920002523 polyethylene Glycol 1000 Polymers 0.000 description 4
- 229940113116 polyethylene glycol 1000 Drugs 0.000 description 4
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229930002330 retinoic acid Natural products 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 229940037128 systemic glucocorticoids Drugs 0.000 description 4
- 229960001727 tretinoin Drugs 0.000 description 4
- 241001430294 unidentified retrovirus Species 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 210000003556 vascular endothelial cell Anatomy 0.000 description 4
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 3
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 241000186146 Brevibacterium Species 0.000 description 3
- 102000011632 Caseins Human genes 0.000 description 3
- 108010076119 Caseins Proteins 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 101001081567 Homo sapiens Insulin-like growth factor-binding protein 1 Proteins 0.000 description 3
- 101001044927 Homo sapiens Insulin-like growth factor-binding protein 3 Proteins 0.000 description 3
- 101000840572 Homo sapiens Insulin-like growth factor-binding protein 4 Proteins 0.000 description 3
- 101000840566 Homo sapiens Insulin-like growth factor-binding protein 5 Proteins 0.000 description 3
- 101000840582 Homo sapiens Insulin-like growth factor-binding protein 6 Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 206010000891 acute myocardial infarction Diseases 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 210000001367 artery Anatomy 0.000 description 3
- 206010003549 asthenia Diseases 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 238000013357 binding ELISA Methods 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000000032 diagnostic agent Substances 0.000 description 3
- 229940039227 diagnostic agent Drugs 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 230000001605 fetal effect Effects 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 102000047065 human IGFBP1 Human genes 0.000 description 3
- 102000057148 human IGFBP3 Human genes 0.000 description 3
- 210000003917 human chromosome Anatomy 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000010841 mRNA extraction Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 210000005155 neural progenitor cell Anatomy 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- 239000000825 pharmaceutical preparation Substances 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 208000007056 sickle cell anemia Diseases 0.000 description 3
- 210000000813 small intestine Anatomy 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 239000006188 syrup Substances 0.000 description 3
- 235000020357 syrup Nutrition 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 210000001550 testis Anatomy 0.000 description 3
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 3
- DSNBHJFQCNUKMA-SCKDECHMSA-N thromboxane A2 Chemical compound OC(=O)CCC\C=C/C[C@@H]1[C@@H](/C=C/[C@@H](O)CCCCC)O[C@@H]2O[C@H]1C2 DSNBHJFQCNUKMA-SCKDECHMSA-N 0.000 description 3
- 230000005030 transcription termination Effects 0.000 description 3
- 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 3
- 229940035893 uracil Drugs 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 2
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 2
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 2
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 108020004491 Antisense DNA Proteins 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 102100031170 CCN family member 3 Human genes 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 2
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 101000777577 Homo sapiens CCN family member 1 Proteins 0.000 description 2
- 101000777550 Homo sapiens CCN family member 2 Proteins 0.000 description 2
- 101000777555 Homo sapiens CCN family member 3 Proteins 0.000 description 2
- 101001044940 Homo sapiens Insulin-like growth factor-binding protein 2 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241001467578 Microbacterium Species 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 238000009004 PCR Kit Methods 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 239000012979 RPMI medium Substances 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 206010043561 Thrombocytopenic purpura Diseases 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000007488 abnormal function Effects 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000003916 acid precipitation Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 229960003896 aminopterin Drugs 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000003816 antisense DNA Substances 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229960005261 aspartic acid Drugs 0.000 description 2
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000036765 blood level Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- NFLRWRCXIJABMY-UHFFFAOYSA-M cesium diaminomethylideneazanium 2,2,2-trifluoroacetate thiocyanate Chemical compound [Cs+].[S-]C#N.NC(N)=N.OC(=O)C(F)(F)F NFLRWRCXIJABMY-UHFFFAOYSA-M 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000001447 compensatory effect Effects 0.000 description 2
- 210000004351 coronary vessel Anatomy 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 229960001123 epoprostenol Drugs 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 239000006481 glucose medium Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 210000000224 granular leucocyte Anatomy 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000023597 hemostasis Effects 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 102000049732 human CCN1 Human genes 0.000 description 2
- 102000047612 human CCN2 Human genes 0.000 description 2
- 102000050770 human IGFBP2 Human genes 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 230000034217 membrane fusion Effects 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000013059 nephrectomy Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 229960002446 octanoic acid Drugs 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 229940043274 prophylactic drug Drugs 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 235000002020 sage Nutrition 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 239000008159 sesame oil Substances 0.000 description 2
- 235000011803 sesame oil Nutrition 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 2
- 229960001052 streptozocin Drugs 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 238000002636 symptomatic treatment Methods 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 229960002898 threonine Drugs 0.000 description 2
- 230000001732 thrombotic effect Effects 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- HHLJUSLZGFYWKW-UHFFFAOYSA-N triethanolamine hydrochloride Chemical compound Cl.OCCN(CCO)CCO HHLJUSLZGFYWKW-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 1
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 1
- SXOUIMVOMIGLHO-AATRIKPKSA-N (E)-3-(indol-2-yl)acrylic acid Chemical compound C1=CC=C2NC(/C=C/C(=O)O)=CC2=C1 SXOUIMVOMIGLHO-AATRIKPKSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- DURPTKYDGMDSBL-UHFFFAOYSA-N 1-butoxybutane Chemical compound CCCCOCCCC DURPTKYDGMDSBL-UHFFFAOYSA-N 0.000 description 1
- PJUPKRYGDFTMTM-UHFFFAOYSA-N 1-hydroxybenzotriazole;hydrate Chemical compound O.C1=CC=C2N(O)N=NC2=C1 PJUPKRYGDFTMTM-UHFFFAOYSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- OQUFOZNPBIIJTN-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;sodium Chemical compound [Na].OC(=O)CC(O)(C(O)=O)CC(O)=O OQUFOZNPBIIJTN-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-M 4-hydroxybenzoate Chemical compound OC1=CC=C(C([O-])=O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-M 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 101710197633 Actin-1 Proteins 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241001203868 Autographa californica Species 0.000 description 1
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 229920001342 Bakelite® Polymers 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 239000012619 Butyl Sepharose® Substances 0.000 description 1
- QLNSPJMIYWEWNF-UHFFFAOYSA-N CC(C)CCCC(C)CCCC(C)CCCC(C)C.CC(C)CCCC(C)CCCC(C)CCCC(C)C Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C.CC(C)CCCC(C)CCCC(C)CCCC(C)C QLNSPJMIYWEWNF-UHFFFAOYSA-N 0.000 description 1
- 101100327917 Caenorhabditis elegans chup-1 gene Proteins 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 102100033620 Calponin-1 Human genes 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241001517047 Corynebacterium acetoacidophilum Species 0.000 description 1
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 1
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 108020001019 DNA Primers Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241001452028 Escherichia coli DH1 Species 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 241001302160 Escherichia coli str. K-12 substr. DH10B Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 108010001498 Galectin 1 Proteins 0.000 description 1
- 102100021736 Galectin-1 Human genes 0.000 description 1
- 102100024637 Galectin-10 Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101001011019 Gallus gallus Gallinacin-10 Proteins 0.000 description 1
- 101001011021 Gallus gallus Gallinacin-12 Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101001055314 Homo sapiens Immunoglobulin heavy constant alpha 2 Proteins 0.000 description 1
- 101001076292 Homo sapiens Insulin-like growth factor II Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 206010070511 Hypoxic-ischaemic encephalopathy Diseases 0.000 description 1
- 101150102264 IE gene Proteins 0.000 description 1
- 102100026216 Immunoglobulin heavy constant alpha 2 Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- 229930182821 L-proline Natural products 0.000 description 1
- 102000008192 Lactoglobulins Human genes 0.000 description 1
- 108010060630 Lactoglobulins Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 206010028372 Muscular weakness Diseases 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 101710141795 Ribonuclease inhibitor Proteins 0.000 description 1
- 229940122208 Ribonuclease inhibitor Drugs 0.000 description 1
- 102100037968 Ribonuclease inhibitor Human genes 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 241000311088 Schwanniomyces Species 0.000 description 1
- 208000034189 Sclerosis Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000881765 Serratia ficaria Species 0.000 description 1
- 241000218654 Serratia fonticola Species 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 1
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 1
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241001634922 Tausonia pullulans Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 241000723873 Tobacco mosaic virus Species 0.000 description 1
- 241000255985 Trichoplusia Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 208000024248 Vascular System injury Diseases 0.000 description 1
- 208000012339 Vascular injury Diseases 0.000 description 1
- 101100068489 Vicia faba AGPC gene Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 230000031016 anaphase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- YJFXSWKKBWMMCM-UHFFFAOYSA-N azanium;3-ethyl-2h-1,3-benzothiazole-6-sulfonate Chemical compound [NH4+].[O-]S(=O)(=O)C1=CC=C2N(CC)CSC2=C1 YJFXSWKKBWMMCM-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000004637 bakelite Substances 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000000692 cap cell Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- YDQXYRCYDMRJGD-UHFFFAOYSA-N chloroform;phenol;thiocyanic acid Chemical compound SC#N.ClC(Cl)Cl.OC1=CC=CC=C1 YDQXYRCYDMRJGD-UHFFFAOYSA-N 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 239000006451 grace's insect medium Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 208000037824 growth disorder Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 102000044162 human IGF1 Human genes 0.000 description 1
- 102000057877 human IGF2 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 208000013403 hyperactivity Diseases 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- PLVPPLCLBIEYEA-UHFFFAOYSA-N indoleacrylic acid Natural products C1=CC=C2C(C=CC(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-UHFFFAOYSA-N 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012577 media supplement Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- IOPLHGOSNCJOOO-UHFFFAOYSA-N methyl 3,4-diaminobenzoate Chemical compound COC(=O)C1=CC=C(N)C(N)=C1 IOPLHGOSNCJOOO-UHFFFAOYSA-N 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000036473 myasthenia Effects 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229940066827 pertussis vaccine Drugs 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 230000036417 physical growth Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 229960002429 proline Drugs 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 230000004648 relaxation of smooth muscle Effects 0.000 description 1
- 230000033904 relaxation of vascular smooth muscle Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 235000011088 sodium lactate Nutrition 0.000 description 1
- 229940005581 sodium lactate Drugs 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 235000011067 sorbitan monolaureate Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- ZERULLAPCVRMCO-UHFFFAOYSA-N sulfure de di n-propyle Natural products CCCSCCC ZERULLAPCVRMCO-UHFFFAOYSA-N 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 208000001608 teratocarcinoma Diseases 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
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 210000004325 uterine smooth muscle cell Anatomy 0.000 description 1
- 231100000216 vascular lesion Toxicity 0.000 description 1
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 1
- 230000002883 vasorelaxation effect Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 235000021249 α-casein Nutrition 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- 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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4743—Insulin-like growth factor binding protein
-
- 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
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Diabetes (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physical Education & Sports Medicine (AREA)
- Biomedical Technology (AREA)
- Cardiology (AREA)
- Endocrinology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Pulmonology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Heart & Thoracic Surgery (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Zoology (AREA)
- Obesity (AREA)
- Virology (AREA)
- Communicable Diseases (AREA)
- Cell Biology (AREA)
Abstract
本発明の蛋白質、該蛋白質をコードするDNA、該蛋白質を認識する抗体を用いることにより、本発明の蛋白質に関与する疾患の判定法、および本発明の蛋白質に関与する疾患の診断薬、予防薬および治療薬を提供できる。A method for determining a disease associated with the protein of the present invention by using the protein of the present invention, a DNA encoding the protein, and an antibody recognizing the protein, and a diagnostic or prophylactic agent for a disease associated with the protein of the present invention And therapeutic agents.
Description
技術分野
本発明は、新規なインスリン様増殖因子結合蛋白質、該蛋白質をコードするDNAおよび該蛋白質を認識する抗体、および該蛋白質が関与する疾患の判定法、診断薬、予防薬または治療薬に関する。
背景技術
インスリン様増殖因子結合蛋白質(insulin−like growth factor binding protein、以下、「IGFBP」という)は、体液中のインスリン様増殖因子(insulin−like growth factor、以下、「IGF」という)が高分子複合体として存在することから発見された分子群で、現在までに、IGFBP−1〜10までの10種類の分子の存在が報告されており、スーパーファミリーを形成していることが知られている〔Endocr.Rev.,18,801(1997)、Prog.Growth Factor Res.,3,243(1991)、Mol.Reprod.Dev.,35,368(1993)、Proc.Natl.Acad.Sci.USA,94,12981(1997)〕。
IGFBPの機能としては、インテグリンへの結合などによる直接作用の存在も示唆されているが、主な作用は、IGFやインスリンに結合してその活性や分布、代謝などを調節することによって発揮されると考えられ〔Endocr.Rev.,18,801(1997)、Bio Science用語ライブラリー サイトカイン・増殖因子 改訂版p14−17(1988)〕、具体的にはIGFBPは、血中および血管外へのIGFの運搬と分解の抑制、受容体への結合調節などによりその機能を発揮していると考えられている。
IGFBPスーパーファミリーのうち、IGFBP−1〜6までの6種類の分子は構造的にも類似しており、インスリンよりもIGFに高い親和性をもって結合することから、IGF高親和性IGFBPとしてサブファミリーに分類されている〔Mol.Endocrinol.,2,404(1988)、EMBO J.,8,2497(1989)、Mol.Endocrinol.,2,1176(1989)、Mol.Endocrinol.,4,1806(1990)、Biochem.Biophys.Res.Commun.,176,219(1991)、J.Biol.Chem.,266,9043(1991)、J.Biol.Chem.,266,10646(1991)〕。
IGF高親和性IGFBPのサブファミリー分子間のアミノ酸配列の相同性はヒトで49%〜60%である。また、IGFBP−6を除く5種類のIGFBPでは18個のシステイン残基が保存されており、このうちN末端の3個がGly−Cys−Gly−Cys−Cys−X−X−Cysで代表される相同配列〔Xは任意のアミノ酸を表しており、該相同配列はインスリン様増殖因子結合モチーフと呼ばれ(以下、「IGFBPモチーフ」ともいう)〕を形成し、IGFの結合に関与することが示されている〔Prog.Growth Factor Res.,3,243(1991)〕。
IGFBP−1と3は、IGFの作用を抑制する場合と促進する場合の両方があることが知られている。また、IGFBP−2、4、6は抑制性の、IGFBP−5は促進性のIGF結合蛋白質である。IGFには、成長ホルモン依存的に肝、骨組織などで産生され、身体的成長を促進させる増殖因子として機能しているIGF−Iと中枢神経系、骨組織などに多量に発現し、主として胎生期の成長に重要な役割を果たしていると推定されるIGF−IIとが存在するが、IGFBP−1、3、4はIGF−I及びIGF−IIに同等の結合活性を示し、IGFBP−2、5、6は主としてIGF−IIに強い結合活性を示すことが知られている。
また、IGF高親和性IGFBPのサブファミリー分子の組織分布は、各分子によって異なっており、IGFBP−1は主に羊水や胎児血清中に、IGFBP−2は主に胎児肝臓や成人脳に、IGFBP−3は主に肝臓や血清中に、IGFBP−4は主に腎糸球体、皮膚および腸上皮に、IGFBP−5は主に腸上皮や骨に、IGFBP−6は主に皮膚や心臓に存在していることが知られている。
IGFBPと病態との関わりに関しては、以下にあげるような知見が報告されている。
小人症や末端肥大症患者ではIGFやIGFBPの発現変動が病態生理に直接関与していることが知られている。また、小児慢性腎不全においてもしばしば成長障害が起こるが、成長ホルモンやIGFの発現レベルは正常であり、多くはIGFBP−2や3の増加によるIGFの機能障害が原因であることが知られている〔Miner.Electrolyte Mrtab.,18,320(1992)〕。IGFBP−4および5は骨代謝に重要な機能を有しており、骨粗鬆症ではIGFBP−5の発現量が減少し、副甲状腺ホルモンの上昇を伴う老人女性の骨折患者ではIGFBP−4の発現が上昇することが報告されている。また、腎摘出後や小腸切除後の代償性肥大においてIGF−Iの傍分泌作用の亢進が観察されているが、IGF−IのmRNA量は変化せず、IGFBP−3の発現低下によって遊離のIGF−Iが増加することが報告されている〔J.Fuller;Baillieres Clin.Endocrinol.Metab.,8,165(1994)〕。さらに、子宮内膜の悪性腫瘍では、良性腫瘍に比べてIGFBP−1の発現が低下していることが報告されている〔Growth Regul.,3,74,(1993)〕。
一方、GFBPスーパーファミリーのうち、IGFBP−7〜10までの4種類の分子は構造的に類似し、かつ、IGFに低い親和性を有するという共通の性質を持つと考えられることから、IGF低親和性IGFBPとして別のサブファミリーに分類されている〔Proc.Natl.Acad.Sci.USA,94,12981(1997)、Cancer Res.,59,2787(1999)〕。
IGFBP−7については様々な生理活性についての報告がなされ、特に癌およびその病態との関係について多くの報告がなされている。
IGFBP−7は、老化したヒト上皮細胞で発現が上昇している〔J.Clin.Endocrinol.Metab.,4,715(1993)〕一方で、カルシノーマ細胞株などでは発現が低下していることから、癌抑制活性遺伝子としての機能を有するのではないかと考えられている〔Proc.Natl.Acad.Sci.USA,92,4472(1995)〕。
IGFBP−7のヒト染色体上の遺伝子座は4q12で、ヒト上皮細胞をレチノイン酸で処理することでその発現が上昇することも知られている〔Proc.Natl.Acad.Sci.USA,92,4472(1995)〕。
乳癌組織では、染色体4q12〜13の位置にLOH(loss of heterozygosity)が約50%の頻度で観察され、かつ、IGFBP−7の発現が減少していることが確認されている〔Oncogene,16,2459(1998)〕。前立腺癌組織でもIGFBP−7の発現がmRNAレベルで減少しており、特に悪性の前立腺癌由来の細胞株では検出されないことが報告されている〔J.Clin.Endocrinol.Metab.,83,4355(1998)〕。
さらに、IGFBP−7の発現が減少した前立腺癌細胞株にIGFBP−7を強制発現させると、細胞***時間の延長、軟寒天培地中でのコロニー形成能の低下、ヌードマウス移植時の腫瘍形成能の低下、薬剤処理によるアポトーシス誘導率の上昇が観察されており、IGFBP−7の発現と前立腺癌の悪性度との関係が示唆されている〔Cancer Res.,59,2370(1999)〕。
白血病患者では、脳脊髄液中のIGFBP−7およびIGFBP−3の濃度が上昇することが報告されており、白血病の病態との関係が注目されている〔J.Clin.Endocrinol.Metab.,84,1283(1999)〕。
大腸癌では、大腸癌組織および大腸癌細胞株においてIGFBP−7の発現が上昇していることから、病態との関係が注目されている〔J.Gastroenterology,33,213(1998)〕。
大型の子宮平滑筋腫部位では、IGFBP−7の発現が低下しており、ゴナドトロピン分泌促進ホルモン(Gonadotropin−releasing hormone)治療をうけた患者ではIGFBP−7の発現が上昇することが報告されている〔J.Reprod.Immunol.,43,53(2000)〕。
SV40T抗原で誘導されたマウス肝臓癌細胞では、IGFBP−7遺伝子の5’上流域がメチル化され発現量が減少していることから、癌化にともなうIGFBP−7の発現調節に遺伝子のメチル化による機構が提唱されている〔Biochem.Biophys.Res.Commun.,267,109(2000)〕。
IGFBP−7と糖尿病との関連についても報告されている。
血管内皮細胞に作用しプロスタサイクリンPGI2の産生を促進する因子(PGI2−stimulating factor、以下「PSF」と略す)が、IGFBP−7と同一であることが示され〔Biochem.J.,303,591,(1994)〕、血管内皮細胞及び平滑筋細胞において発現している該因子は〔Thromb Haemost.,74,1407(1995)〕、ストレプトゾトシン投与によるI型糖尿病モデルでは腎臓および血管障害部位で発現が低下していることが報告されている〔Diabetes,45,S111(1996)、J.Diabetes & its Complications,12,252(1998)〕。またII型糖尿病患者の冠状動脈平滑筋細胞においてもIGFBP−7の発現低下が蛋白質レベルで観察されている〔Diabetes,46,1627(1997)〕。さらに、牛の動脈由来平滑筋細胞を高グルコース培地で培養するとIGFBP−7の発現量が低下することがmRNAおよび蛋白レベルで確認されている〔Diabetes,46,1627(1997)、Diabetologia,41,134(1998)〕。
TGF−β(Transforming growth factor−β)、副甲状腺ホルモン(PTH)およびプロスタグランジンE2(PGE2)によってIGFBP−7の発現が骨芽細胞において上昇することから、IGFBP−7が骨芽細胞に生理的作用を及ぼすことが示唆されている〔Endocrinology,140,1998(1999)〕。また、骨芽細胞をグルココルチコイドで処理するとIGF−Iの発現を抑制する一方でIGFBP−7の発現を上昇させることも報告されている〔Endocrinology,140,228(1999)〕。
また、IGFBP−7は骨格筋への分化にもIGFの分化促進作用を抑制することで影響していることが示唆されている〔Exp.Cell Res.,237,192(1997)、Endocrinology,141,100(2000)〕。
IGFBP−7がプロスタサイクリンPGI2の産生を促進する因子PSFと同一分子であったことから、IGFBP−7の生理機能の一部はPGI2をエフェクター分子として発揮されていると考えられている。
プロスタグランジンの一種であるPGI2は強力な血小板凝集抑制作用と血管弛緩作用を有し、逆の作用を有するTXA2と拮抗的に作用し生体内の恒常性の維持に関与していることが知られており〔Br.J.Pharmac.,76,3(1982)〕、血栓症や動脈硬化症などではTXA2およびPGI2の産生の不均衡、とりわけPGI2の産生が低下し血管障害が発症することが知られている〔Br.J.Pharmac.,76,3(1982)〕。糖尿病性血管障害の発症や進展においても、血小板由来のTXA2の産生亢進に加え〔Thromb.Res.,19,211(1980)、J.Lab.Clin.Med.,97,87(1981)〕、血管由来のPGI2の産生低下が血小板凝集の亢進を引き起こすことが糖尿病患者や実験糖尿病動物で確認されている〔Lancet,1,325(1979)、Lancet,2,1365(1979)、N.Engl.J.Med.,300,366(1979)、Life Sci.,23,351(1978)〕。
また、PSFは血流中に存在する因子であり、血管壁でのPGI2産生を刺激すること〔Nature,271,549(1978)〕、および該因子は、溶血性***症候群〔Lancet,2,871(1978)〕、血栓性血小板減少性紫斑病〔Lancet,2,748(1979)〕、鎌形赤血球性貧血症〔Br.J.Haematol.,48,545(1981)〕、急性心筋梗塞〔Coronary,2,49(1985)〕、糖尿病性血管障害〔Metabolism,38,837(1989)、Haemostasis,16,447(1986)、Diab.Res.Clin.Pract.,3,243(1987)〕、動脈硬化性疾患において血中レベルが低下していることが報告されている。
すなわち、IGFBP−7は血管内皮細胞のPGI2産生を促し血中のPGI2濃度を高めることにより血小板凝集抑制作用、平滑筋弛緩作用、胃酸分泌抑制作用を発揮することが明らかになっている。
以上のように、IGFBPスーパーファミリーに属する因子は、IGFやインスリンの機能の調節、妊娠、消耗性疾患における代償作用、骨代謝、骨格筋細胞の分化、血管内皮細胞に対するPGI2産生促進、PGI2を介した血小板擬集抑制、血管平滑筋弛緩、気管支平滑筋弛緩、胃酸分泌抑制など様々な生理現象に関与していることが示されている。また、小人症、末端肥大症、小児慢性腎不全、骨粗鬆症、乳癌、前立腺癌、急性白血病、大腸癌、子宮平滑筋腫、肝臓癌、I型糖尿病、II型糖尿病、血栓症、動脈硬化症、溶血性***症候群、血栓性血小板減少性紫斑病、鎌形赤血球性貧血症、急性心筋梗塞、糖尿病性血管障害等の疾患に関与していることも示されている。
したがって、IGFBPスーパーファミリーに属するIGFBPとしての活性を有する蛋白質、該蛋白質をコードする遺伝子、アンチセンスDNA、蛋白質を認識する抗体は、異常な細胞増殖を伴う疾患、血管障害を伴う疾患、骨代謝の異常を伴う疾患、IGFや成長ホルモン作用の障害を伴う疾患、異常な平滑筋細胞の分化増殖を伴う疾患、異常な骨格筋細胞の分化増殖を伴う疾患、胃酸分泌の異常を伴う疾患または異常なリンパ球浸潤を伴う炎症性の疾患の判定、治療または予防のための医薬になりうると考えられており、IGFBPスーパーファミリーに属する因子は有用な新薬開発のターゲットとして非常に注目されている。
また、IGFBPスーパーファミリーに属する新規な因子が存在する可能性も想起され、新規IGFBP遺伝子を取得できれば、該IGFBPのアミノ酸配列と既知IGFBPのアミノ酸配列とを比較したり、該IGFBP遺伝子の転写物の発現分布を調べることにより、該IGFBPの機能を推定し、医薬品開発に有用な情報を得ることができる。また、新規IGFBP遺伝子が取得できれば、該IGFBPの発現または機能を抑制する物質をスクリーニングすることが可能になる。該スクリーニングにより得られる化合物は有用な医薬品として期待される。
発明の開示
本発明は、新規インスリン様増殖因子結合蛋白質、該蛋白質をコードするDNA、該蛋白質を認識する抗体、該蛋白質が関与する疾患の判定方法、診断薬、予防薬および治療薬を提供する。
本発明者らは、上記課題を解決すべく鋭意検討を行った結果、IGFBPファミリーに属する新規インスリン様増殖因子結合蛋白質および該蛋白質をコードするDNAを取得することに成功し、本発明を完成させるに至った。
すなわち、本発明は、以下の(1)〜(43)を提供するものである。
(1) 配列番号1で表されるアミノ酸配列からなる蛋白質。
(2) 配列番号1で表されるアミノ酸配列において、1以上のアミノ酸が欠失、置換、挿入および/または付加されたアミノ酸配列からなり、かつ(1)に記載の蛋白質と実質的に同一の活性を有する蛋白質。
(3) 配列番号1で表されるアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつ(1)に記載の蛋白質と実質的に同一の活性を有する蛋白質。
(4) 配列番号1で表されるアミノ酸配列において、アミノ酸番号62〜69番のアミノ酸配列を含む部分アミノ酸配列からなり、かつ(1)に記載の蛋白質と実質的に同一の活性を有する蛋白質。
(5) (4)に記載の蛋白質のアミノ酸配列において、1以上のアミノ酸が欠失、置換、挿入および/または付加されたアミノ酸配列からなり、かつ(1)に記載の蛋白質と実質的に同一の活性を有する蛋白質。
(6) (4)に記載の蛋白質のアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつ(1)に記載の蛋白質と実質的に同一の活性を有する蛋白質。
(7) (1)〜(6)のいずれか1項に記載の蛋白質をコードするDNA。
(8) 配列番号2で表される塩基配列のコード領域を含むDNA。
(9) (1)〜(6)のいずれか1項に記載の蛋白質をコードするDNA、または配列番号2で表される塩基配列のコード領域を含むDNAとストリンジェントな条件下でハイブリダイズし、かつ(1)に記載の蛋白質と実質的に同一の活性を有する蛋白質をコードするDNA。
(10) (7)〜(9)のいずれか1項に記載のDNAをベクターに組込んで得られる組換え体DNA。
(11) (10)に記載の組換え体DNAを宿主細胞に導入して得られる形質転換体。
(12) 宿主細胞が、細菌、酵母、昆虫細胞、植物細胞または動物細胞からなる群から選ばれる細胞であることを特徴とする、(11)に記載の形質転換体。
(13) (11)または(12)に記載の形質転換体を培地に培養し、培養物中に(1)〜(6)のいずれか1項に記載の蛋白質を生成蓄積させ、該培養物から該蛋白質を採取することを特徴とする、(1)〜(6)のいずれか1項に記載の蛋白質の製造方法。
(14) (1)〜(6)のいずれか1項に記載の蛋白質を認識する抗体。
(15) 寄託番号がFERM BP−7977であるハイブリドーマが産生するモノクローナル抗体。
(16) ハイブリドーマFERM BP−7977。
(17) (14)または(15)に記載の抗体を用いる、(1)〜(6)のいずれか1項に記載の蛋白質を免疫学的に検出または定量する方法。
(18) (7)〜(9)のいずれか1項に記載のDNAの塩基配列中の連続した5〜60塩基からなる配列を有するオリゴヌクレオチド、該オリゴヌクレオチドと相補的な配列を有するオリゴヌクレオチド、またはそれらオリゴヌクレオチドの誘導体。
(19) (7)〜(9)のいずれか1項に記載のDNAまたは(18)に記載のオリゴヌクレオチドまたはオリゴヌクレオチド誘導体をプローブとして用いてハイブリダイゼーションを行うことを含む、(1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の発現を検出または定量する方法。
(20) (18)に記載のオリゴヌクレオチドまたはオリゴヌクレオチド誘導体をプライマーとして用いてポリメラーゼ・チェイン・リアクションを行うことを含む、(1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の発現の検出方法、または発現量の定量方法。
(21) (7)〜(9)のいずれか1項に記載のDNAまたは(18)に記載のオリゴヌクレオチドまたはオリゴヌクレオチド誘導体を用いてハイブリダイゼーションを行うことを含む、(1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の変異を検出する方法。
(22) (18)に記載のオリゴヌクレオチドまたはオリゴヌクレオチド誘導体を用いてポリメラーゼ・チェイン・リアクションを行うことを含む、(1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の変異を検出する方法。
(23) 以下の(A)または(B)に記載の疾患の判定方法。
(A)(1)〜(6)のいずれか1項に記載の蛋白質をコードするDNAの変異の検出または発現量の測定を行ない、健常人と比較することを特徴とする、疾患の判定方法。
(B)(14)または(15)に記載の抗体を用いて、(1)〜(6)のいずれか1項に記載の蛋白質の変異の検出または発現量の測定を行ない、健常人と比較することを特徴とする、疾患の判定方法。
(24) 疾患が、異常な細胞増殖を伴う疾患、血管障害を伴う疾患、骨代謝の異常を伴う疾患、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、異常な平滑筋細胞の分化増殖を伴う疾患、異常な骨格筋細胞の分化増殖を伴う疾患、胃酸分泌の異常を伴う疾患または異常なリンパ球浸潤を伴う炎症性の疾患である、(23)に記載の判定方法。
(25) 異常な細胞増殖を伴う疾患が、急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、脳腫瘍、悪性腫瘍または固形腫瘍であり、血管障害を伴う疾患が心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血または脈絡膜循環障害であり、骨代謝の異常を伴う疾患が骨粗鬆症であり、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患が小人症、末端肥大症または小児慢性腎不全であり、異常な平滑筋細胞の分化増殖を伴う疾患が動脈硬化、気管支疾患または再狭窄であり、異常な骨格筋細胞の分化増殖を伴う疾患が重症筋無力症であり、胃酸分泌の異常を伴う疾患が胃潰瘍であり、異常なリンパ球浸潤を伴う炎症性の疾患が微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏または自己免疫疾患である、(24)に記載の判定方法。
(26) 判定方法が、以下の(A)または(B)に記載の方法によるものである、(23)〜(25)のいずれか1項に記載の判定方法。
(A) (21)または(22)に記載の検出方法。
(B) (17)、(19)または(20)に記載の検出方法、または定量方法。
(27) (1)〜(6)のいずれか1項に記載の蛋白質を含有する医薬。
(28) (7)〜(9)のいずれか1項に記載のDNA、または(18)に記載のオリゴヌクレオチド若しくはオリゴヌクレオチド誘導体を含有する医薬。
(29) 医薬が、遺伝子予防用ベクターまたは遺伝子治療用ベクターである、(28)に記載の医薬。
(30) (14)または(15)に記載の抗体を含有する医薬。
(31) 医薬が、異常な細胞増殖を伴う疾患、血管障害を伴う疾患、骨代謝の異常を伴う疾患、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、異常な平滑筋細胞の分化増殖を伴う疾患、異常な骨格筋細胞の分化増殖を伴う疾患、胃酸分泌の異常を伴う疾患または異常なリンパ球浸潤を伴う炎症性の疾患に対する診断薬、予防薬または治療薬である、(27)〜(30)に記載の医薬。
(32) 異常な細胞増殖を伴う疾患が急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、脳腫瘍、悪性腫瘍または固形腫瘍であり、血管障害を伴う疾患が心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血または脈絡膜循環障害であり、骨代謝の異常を伴う疾患が骨粗鬆症であり、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患が小人症、末端肥大症または小児慢性腎不全であり、異常な平滑筋細胞の分化増殖を伴う疾患が動脈硬化、気管支疾患または再狭窄であり、異常な骨格筋細胞の分化増殖を伴う疾患が重症筋無力症であり、胃酸分泌の異常を伴う疾患が胃潰瘍であり、異常なリンパ球浸潤を伴う炎症性の疾患が微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏または自己免疫疾患である、(31)に記載の医薬。
(33) (i)(1)〜(6)のいずれか1項に記載の蛋白質を発現する細胞での該蛋白質の発現量と、(ii)該蛋白質を発現する細胞と被験試料を接触させた場合の該蛋白質の発現量とを比較し、被験試料より(1)〜(6)のいずれか1項に記載の蛋白質の発現量を制御する化合物を選択することを特徴とする、(1)〜(6)のいずれか1項に記載の蛋白質の発現量を制御する化合物のスクリーニング方法。
(34) (i)(1)〜(6)のいずれか1項に記載の蛋白質を発現する細胞の機能と(ii)該蛋白質を発現する細胞と被験試料を接触させた場合の細胞の機能とを比較し、被験試料より(1)〜(6)のいずれか1項に記載の蛋白質の機能を制御する化合物を選択することを特徴とする、(1)〜(6)のいずれか1項に記載の蛋白質の機能を制御する化合物のスクリーニング方法。
(35) (1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の転写を制御する領域の下流にレポーター遺伝子の連結されたDNAを含むプラスミドで形質転換された形質転換体と被験試料とを接触させ、被験試料より(1)〜(6)に記載の蛋白質をコードする遺伝子の発現を制御する化合物を選択することを特徴とする、(1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の発現を制御する化合物のスクリーニング方法。
(36) (33)〜(35)のいずれか1項に記載のスクリーニング法によって得られる化合物またはその薬理学的に許容される塩。
(37) (36)に記載の化合物またはその薬理学的に許容される塩を含有する医薬。
(38) 異常な細胞増殖を伴う疾患、血管障害を伴う疾患、骨代謝の異常を伴う疾患、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、異常な平滑筋細胞の分化増殖を伴う疾患、異常な骨格筋細胞の分化増殖を伴う疾患、胃酸分泌の異常を伴う疾患または異常なリンパ球浸潤を伴う炎症性の疾患に対する予防薬または治療薬である、(37)に記載の医薬。
(39) 異常な細胞増殖を伴う疾患が急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、脳腫瘍、悪性腫瘍または固形腫瘍であり、血管障害を伴う疾患が心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血または脈絡膜循環障害であり、骨代謝の異常を伴う疾患が骨粗鬆症であり、インスリン様増殖因子や成長ホルモン作用の障害を伴う疾患が小人症、末端肥大症または小児慢性腎不全であり、異常な平滑筋細胞の分化増殖を伴う疾患が動脈硬化、気管支疾患または再狭窄であり、異常な骨格筋細胞の分化増殖を伴う疾患が重症筋無力症であり、胃酸分泌の異常を伴う疾患が胃潰瘍であり、異常なリンパ球浸潤を伴う炎症性の疾患が微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏または自己免疫疾患である、(38)に記載の医薬。
(40) (1)〜(6)のいずれか1項に記載の蛋白質と被験試料を接触させ、被験試料より該蛋白質と特異的に結合する蛋白質を選択することを特徴とする、(1)〜(6)のいずれか1項に記載の蛋白質と特異的に結合する蛋白質のスクリーニング法。
(41) (40)に記載のスクリーニング法により取得される、(1)〜(6)のいずれか1項に記載の蛋白質と特異的に結合する蛋白質。
(42) (1)〜(6)のいずれか1項に記載の蛋白質をコードする遺伝子の発現が低下または完全に抑制されたノックアウト非ヒト動物。
(43) (1)〜(6)のいずれか1項に記載の蛋白質の有する機能が低下または完全に抑制されたノックアウト非ヒト動物。
本発明の蛋白質としては、
(a) 配列番号1で表されるアミノ酸配列からなる蛋白質
(b) 配列番号1で表されるアミノ酸配列において、1以上のアミノ酸が欠失、置換、挿入および/または付加されたアミノ酸配列からなり、かつ(a)に記載の蛋白質と実質的に同一の活性を有する蛋白質
(c) 配列番号1で表されるアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつ(a)に記載の蛋白質と実質的に同一の活性を有する蛋白質
(d) 配列番号1で表されるアミノ酸配列において、アミノ酸番号62〜69番のアミノ酸配列を含む部分アミノ酸配列からなり、かつ上記(a)に記載の蛋白質と実質的に同一の活性を有する蛋白質
(e) 上記(d)に記載の蛋白質を表すアミノ酸配列において、1以上のアミノ酸が欠失、置換、挿入および/または付加されたアミノ酸配列からなり、かつ上記(a)に記載の蛋白質と実質的に同一の活性を有する蛋白質
(f) 上記(d)に記載の蛋白質を表すアミノ酸配列と80%以上の相同性を有するアミノ酸配列からなり、かつ上記(a)に記載の蛋白質と実質的に同一の活性を有する蛋白質をあげることができる。
インスリン様増殖因子結合蛋白質スーパーファミリーに属する因子は、IGFあるいはインスリンと親和性を有する蛋白性の因子であり、インスリン様増殖因子結合モチーフ(IGFBPモチーフ)を有している。該モチーフはインスリン様増殖因子結合蛋白質スーパーファミリーに属する蛋白質のN末端部分で保存されており、Gly−Cys−Gly−Cys−Cys−X−X−Cysで代表されるアミノ酸配列からなる蛋白質の部分(Xは任意のアミノ酸を表す)をさす。インスリン様増殖因子結合蛋白質スーパーファミリーに属する蛋白質では、IGFBPモチーフを中心に少なくとも10個のシステイン残基が保存されており、IGFBPモチーフ部分を介してIGFあるいはインスリンに親和性を示す。配列番号1で表されるアミノ酸配列においては、アミノ酸番号62〜69番で表されるアミノ酸配列がIGFBPモチーフである。
上述した配列番号1で表されるアミノ酸配列からなる蛋白質と実質的に同一な活性を有する蛋白質としては、配列番号1で表されるアミノ酸配列からなる蛋白質と、結合するインテグリン、IGF、インスリンまたは受容体等の蛋白質が同一である蛋白質があげられる。実質的に同一とは、活性が性質的に同一であることを示す。したがって、結合活性の程度、蛋白質の分子量などの量的要素は異なっていてもよい。
本発明の蛋白質である配列番号1で表されるアミノ酸配列において1以上のアミノ酸が欠失、置換、挿入および/または付加されたアミノ酸配列からなり、かつ配列番号1で表されるアミノ酸配列からなる蛋白質と実質的に同一の活性を有する蛋白質は、Molecular Cloning,A Laboratory Manual,Second Edition,Cold Spring Harbor Laboratory Press(1989)(以下、モレキュラー・クローニング第2版と略す)、Current Protocols in Molecular Biology,John Wiley & Sons(1987−1997)(以下、カレント・プロトコールズ・イン・モレキュラー・バイオロジーと略す)、Nucleic Acids Research,10,6487(1982)、Proc.Natl.Acad.Sci.,USA,79,6409(1982)、Gene,34,315(1985)、Nucleic Acids Research,13,4431(1985)、Proc.Natl.Acad.Sci USA,82,488(1985)等に記載の部位特異的変異導入法を用いて、例えば配列番号1で表されるアミノ酸配列からなる蛋白質をコードするDNAに部位特異的変異を導入することにより取得することができる。欠失、置換、挿入および/もしくは付加されるアミノ酸の数は1個以上でありその数は特に限定されないが、上記の部位特異的変異導入法等の周知の技術により、欠失、置換、挿入および/もしくは付加できる程度の数であり、例えば、1〜数十個、好ましくは1〜20個、より好ましくは1〜10個、さらに好ましくは1〜5個である。また、本発明には、このような人為的に作成した変異体のみならず、自然界において生じた変異体も含まれる。
本発明の蛋白質である配列番号1で表されるアミノ酸配列において1以上のアミノ酸が欠失されたアミノ酸配列からなり、かつ配列番号1で表されるアミノ酸配列からなる蛋白質と実質的に同一の活性を有する蛋白質としては、例えば、シグナルペプチドが除去された蛋白質が挙げられる。
また、配列番号1に記載のアミノ酸配列からなる蛋白質と80%以上の相同性を有し、かつ配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有する蛋白質もまた本発明の蛋白質であり、配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有するためには、配列番号1に記載のアミノ酸配列との相同性が、少なくとも80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは97%以上、最も好ましくは99%以上であることが好ましい。
アミノ酸配列や塩基配列の同一性は、Karlin and AltschulによるアルゴリズムBLAST〔Proc.Natl.Acad.Sci.USA,90,5873−5877(1993)〕によって決定することができる。このアルゴリズムに基づいて、BLASTNやBLASTXと呼ばれるプログラムが開発されている〔Altschul et al.J.Mol.Biol.,215,403−410(1990)〕。BLASTに基づいてBLASTNによって塩基配列を解析する場合には、パラメーターはたとえばscore=100、wordlength=12とする。また、BLASTに基づいてBLASTXによってアミノ酸配列を解析する場合には、パラメーターはたとえばscore=50、wordlength=3とする。BLASTとGapped BLASTプログラムを用いる場合には、各プログラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知である(http://www.ncbi.nlm.nih.gov.)。
本発明の蛋白質は、上記本発明の蛋白質のアミノ酸配列中の部分アミノ酸配列からなる蛋白質であり、かつ配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有する蛋白質も含む。
配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有するには、配列番号1で表されるアミノ酸配列において、アミノ酸番号62〜69番のアミノ酸配列を含む部分アミノ酸配列からなる蛋白質であることが好ましい。
また該蛋白質に1以上のアミノ酸の欠失、置換、挿入および/または付加したアミノ酸配列からなる蛋白質もまた本発明の蛋白質である。該蛋白質としては、上述した周知の部位特異的変異導入法により導入可能な数のアミノ酸が欠失、置換または付加した蛋白質であり、かつ配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有する蛋白質をあげることができる。
また、上記(d)に記載の蛋白質と80%以上の相同性を有し、かつ配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有する蛋白質もまた本発明の蛋白質である。配列番号1に記載のアミノ酸配列からなる蛋白質と実質的に同一の活性を有するには、(d)に記載の蛋白質のアミノ酸配列との相同性が、上記BLASTのプログラムを利用して計算したときに、少なくとも80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは97%以上、最も好ましくは99%以上である。
本発明のDNAとしては、
(1)配列番号2の塩基配列のコード領域を含むDNA
(2)上記定義した(a)、(b)、(c)、(d)、(e)または(f)に記載の本発明の蛋白質をコードするDNA
(3)(2)に記載のDNA、または配列番号2で表される塩基配列を有するDNAとストリンジェントな条件下でハイブリダイズし、かつ配列番号1で表されるアミノ酸配列を有する蛋白質と実質的に同一な活性を有する蛋白質をコードするDNAをあげることができる。
ストリンジェントな条件下でハイブリダイズするDNAとは、例えば配列番号2で表される塩基配列を有するDNAなどの本発明のDNAまたはその一部のDNA断片をプローブとして、コロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法あるいはサザンブロットハイブリダイゼーション法等を用いることにより得られるDNAを意味し、具体的には、コロニーあるいはプラーク由来のDNAを固定化したフィルターを用いて、0.7〜1.0mol/Lの塩化ナトリウム存在下、65℃でハイブリダイゼーションを行った後、0.1〜2倍濃度のSSC溶液(1倍濃度のSSC溶液の組成は、150mmol/L塩化ナトリウム、15mmol/Lクエン酸ナトリウムよりなる)を用い、65℃条件下でフィルターを洗浄することにより同定できるDNAをあげることができる。ハイブリダイゼーションは、モレキュラー・クローニング第2版、カレント・プロトコールズ・イン・モレキュラー・バイオロジー、DNA Cloning 1:Core Techniques,A Practical Approach,Second Edition,Oxford University(1995)等に記載されている方法に準じて行うことができる。ハイブリダイズ可能なDNAとして具体的には、配列番号2で表される塩基配列と少なくとも60%以上の相同性を有するDNA、好ましくは70%以上、より好ましくは80%以上、さらに好ましくは90%以上、特に好ましくは95%以上、最も好ましくは98%以上の相同性を有するDNAをあげることができる。
以下、本発明を詳細に説明する。
1.本発明のDNAの調製
本発明のDNAは、ヒト組織由来のmRNA、例えばヒト脳由来のmRNAを単離し、そのcDNAライブラリーを作製し、次いで該cDNAライブラリーをスクリーニングして目的のクローンを得ることにより調製することができる。
ヒト脳mRNAは、市販のもの(例えば、Clontech社製)を用いてもよいし、以下のごとくヒト脳組織から調製してもよい。後者の場合には、まず、脳組織から全RNAを調製し、該全RNAからmRNAを単離することができる。
脳組織から全RNAを調製する方法としては、チオシアン酸グアニジン−トリフルオロ酢酸セシウム法〔Methods in Enzymology,154,3(1987)〕、酸性チオシアン酸グアニジン・フェノール・クロロホルム(AGPC)法〔Analytical Biochemistry,162,156(1987)、実験医学,9,1937(1991)〕などがあげられる。全RNAからpoly(A)+RNAとしてmRNAを調製する方法としては、オリゴ(dT)固定化セルロースカラム法(モレキュラー・クローニング第2版)等があげられる。あるいは、Fast Track mRNA Isolation Kit(Invitrogen社製)、Quick Prep mRNA Purification Kit(Pharmacia社製)などのキットを用いることによりmRNAを調製することができる。
調製したヒト脳組織mRNAからcDNAライブラリーを作製する。cDNAライブラリー作製法としては、モレキュラー・クローニング第2版、カレント・プロトコールズ・イン・モレキュラー・バイオロジー等に記載された方法、あるいは市販のキット、例えばSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Life Technologies社製)、ZAP−cDNA Synthesis Kit(STRATAGENE社製)を用いる方法などがあげられる。
cDNAライブラリーを作製するためのクローニングベクターとしては、大腸菌K12株中で自立複製できるものであれば、ファージベクター、プラスミドベクター等いずれでも使用できる。具体的には、ZAP Express〔STRATAGENE社、Strategies,5,58(1992)〕、pBluescript II SK(+)〔Nucleic Acids Research,17,9494(1989)〕、Lambda ZAP II(STRATAGENE社製)、λgt10、λgt11〔DNA cloning,A Practical Approach,1,49(1985)〕、λTriplEx(Clontech社製)、λExCell(Pharmacia社製)、pT7T318U(Pharmacia社製)、pcD2〔Mol.Cell.Biol.,3,280(1983)〕およびpUC18〔Gene,33,103(1985)〕等をあげることができる。
宿主微生物としては、エシュリヒア属(Escherichia)に属する微生物、特にエシュリヒア・コリ(Escherichia coli、以下、「大腸菌」ともいう)に属する微生物であればいずれでも用いることができる。具体的には、Escherichia coli XL1−Blue MRF’〔STRATAGENE社、Strategies,5,81(1992)〕、Escherichia coli C600〔Genetics,39,440(1954)〕、Escherichia coli Y1088〔Science,222,778(1983)〕、Escherichia coli Y1090〔Science,222,778(1983)〕、Escherichia coli NM522〔J.Mol.Biol.,166,1(1983)〕、Escherichia coli K802〔J.Mol.Biol.,16,118(1966)〕およびEscherichia coli JM105〔Gene,38,275(1985)〕等が用いられる。
このcDNAライブラリーを、そのまま以下の解析に用いてもよいが、不完全長cDNAの割合を下げ、完全長cDNAをできるだけ効率よく取得するために、菅野らが開発したオリゴキャップ法〔Gene,138,171(1994)、Gene,200,149(1997)、蛋白質核酸酵素,41,603(1996)、実験医学,11,2491(1993)、cDNAクローニング,羊土社(1996)、遺伝子ライブラリーの作製法,羊土社(1994)〕を用いて調製したcDNAライブラリーを以下の解析に用いてもよい。
作製したcDNAライブラリーから各クローンを単離し、それぞれのクローンについてcDNAの塩基配列を末端から、通常用いられる塩基配列解析方法、例えばサンガー(Sanger)らのジデオキシ法〔Proc.Natl.Acad.Sci.USA,74,5463(1977)〕あるいはABIPRISM377DNAシークエンサー(PE Biosystems社製)等の塩基配列分析装置を用いて分析することにより、該DNAの塩基配列を決定する。
それぞれのcDNAの塩基配列が新規な配列を有しているかどうかは、BLAST等の相同性検索プログラムを用いて、GenBank、EMBLおよびDDBJなどの塩基配列データベースを検索することにより、データベース中の既存の遺伝子の塩基配列と完全に一致すると考えられるような明らかな相同性を示す塩基配列がないことにより確認できる。
上記の方法で得られる新規な配列を含むcDNAの塩基配列として、例えば配列番号2で表される塩基配列があげられる。
配列番号2で表される塩基配列からなるDNAを翻訳して得られる蛋白質(配列番号1で表されるアミノ酸配列からなる蛋白質)は、BLAST2〔Nuc.Acid.Res.,25,3389(1997)〕を用いた相同性解析において、IGFBPスーパーファミリーに属する蛋白質ヒトIGFBP−7と39%の相同性を有する。
IGFBPスーパーファミリーにおいては、IGFあるいはインスリンとの結合に重要なインスリン様増殖因子結合モチーフ部分およびIGFBPモチーフを中心とした少なくとも10個のシステイン残基の位置が保存されていることが知られている。配列番号1で表されるアミノ酸配列からなる蛋白質においても、インスリン様増殖因子結合モチーフに相当する部分に、代表的なインスリン様増殖因子結合モチーフであるGly−Cys−Gly−Cys−Cys−X−X−Cys(Xは任意のアミノ酸を示す)からなるアミノ酸配列と非常に類似したGlu−Cys−Gly−Cys−Cys−Ala−Arg−Cysからなるアミノ酸配列が存在し、このモチーフ部分を中心に10個のシステイン残基がIGFBPスーパーファミリーに属する因子間で保存されている位置に存在する。従って、配列番号1で表されるアミノ酸配列からなる蛋白質がIGFBPスーパーファミリーに属する蛋白質としての活性を有することがわかる。
IGFBPスーパーファミリーに属する蛋白質と疾患の関係については、急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、悪性腫瘍、固形腫瘍等の異常な細胞増殖を伴う疾患、心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血、脈絡膜循環障害等の血管障害を伴う疾患、骨粗鬆症等の骨代謝の異常を伴う疾患、小人症、末端肥大症、小児慢性腎不全等のインスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、動脈硬化、気管支疾患、再狭窄等の異常な平滑筋細胞の分化増殖を伴う疾患、重症筋無力症等の異常な骨格筋細胞の分化増殖を伴う疾患、胃潰瘍等の胃酸分泌の異常を伴う疾患、微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏、自己免疫疾患等の異常なリンパ球浸潤を伴う炎症性の疾患等について報告があることから、本発明の蛋白質もまた、上記疾患と関連がある蛋白質であることが予想される。これら疾患は、例えば、本発明の蛋白質の機能や発現の異状に起因している可能性がある。本実施例において、本発明の蛋白質を発現させた細胞の培養上清が癌細胞株の増殖を抑制する活性を示したことから、本発明の蛋白質は、大腸癌などの異常な細胞増殖を伴う疾患に関係していると考えられる。
配列番号2で表される塩基配列を含む分子がcDNAライブラリー作製の際に人工的に生じたものではないことを確認するためには、ヒトゲノムライブラリーを、配列番号2で表される塩基配列に特異的な配列を用いてスクリーニングし、得られたゲノムクローンの塩基配列を決定することで判断することができる。
ヒトゲノムライブラリーは市販のもの(例えば、Research Genetics社製BACライブラリー)を用いてもよいし、ヒトの細胞や組織から自体公知の方法〔Genomics,29,413(1995)、Genomics,24,527(1994)等に記載の方法〕を用いて調製してもよい。
ヒトゲノムライブラリーを、配列番号2で表される塩基配列に特異的な配列を用いてスクリーニングする方法としては、配列番号2で表される塩基配列に特異的なプライマーを用いたPCR法〔PCR Protocols,Academic Press(1990)〕や、配列番号2で表される塩基配列に特異的なオリゴヌクレオチドを用いたコロニーハイブリダイゼーションやプラークハイブリダイゼーション法(モレキュラー・クローニング第2版)などがあげられる。
上記の方法で配列番号2で表される塩基配列の両方を含むゲノムDNAクローン(例えば、ヒトゲノムBACクローン)が得られる。このゲノムDNAの塩基配列を決定し配列番号2で表される塩基配列と比較すると、配列番号2で表される塩基配列は6つの部分に別れて同一ヒトゲノム上に約18kbにわたって存在していることが分かる。すなわち、配列番号1で表されるアミノ酸配列を有する蛋白質は6つのエクソンからから構成されており、cDNAライブラリー作製の際に人工的に生じたものではないことが分かる。
配列番号2で表される塩基配列からなるDNAが一旦取得され、その塩基配列が決定された後は、該塩基配列の5’端および3’端の塩基配列に基づいたプライマーを調製し、ヒトまたは非ヒト動物の脳等の組織または細胞に含まれるmRNAから合成したcDNAあるいはcDNAライブラリーを鋳型として、PCR法〔PCR Protocols,Academic Press(1990)〕を用いてDNAの増幅を行うことにより、本発明のDNAを取得することができる。
また、配列番号2で表されるDNAの全長あるいは一部をプローブとして、ヒトまたは非ヒト動物の脳等の組織または細胞に含まれるmRNAから合成したcDNAあるいはcDNAライブラリーに対してコロニーハイブリダイゼーションやプラークハイブリダイゼーション(モレキュラー・クローニング第2版)を行うことにより、本発明のDNAを取得することができる。
決定されたDNAの塩基配列に基づいて、フォスフォアミダイト法を利用したパーキン・エルマー社のDNA合成機model 392等のDNA合成機で化学合成することにより、本発明のDNAを取得することもできる。
取得したDNAについて、該DNAを含む組換えベクターを宿主細胞に導入して得られる形質転換体を用いて蛋白質を発現させることにより、または該DNAがコードするアミノ酸配列とヒトIGFBP−1、ヒトIGFBP−2、ヒトIGFBP−3、ヒトIGFBP−4、ヒトIGFBP−5、ヒトIGFBP−6、ヒトIGFBP−7、ヒトIGFBP−8、ヒトIGFBP−9若しくはヒトIGFBP−10のアミノ酸配列との相同性およびシステイン残基の位置を比較することにより、該DNAがIGFBPスーパーファミリーとしての活性を有する蛋白質をコードするDNAであることを確認することができる。
配列番号2で表される塩基配列またはその断片の塩基配列に関する情報に基づき、常法またはDNA合成機を用いることにより、本発明のDNAの塩基配列、例えば配列番号2で表される塩基配列のうち、連続した5〜60塩基、好ましくは10〜40塩基に相当する配列を有するオリゴヌクレオチドまたは該オリゴヌクレオチドと相補的な配列に相当するオリゴヌクレオチド(以下、アンチセンス・オリゴヌクレオチドという)を調製することがきる。
本発明のオリゴヌクレオチドとしては、オリゴDNA、オリゴRNA等のオリゴヌクレオチド、および該オリゴヌクレオチドの誘導体(以下、オリゴヌクレオチド誘導体という)等があげられる。
該オリゴヌクレオチドまたはアンチセンス・オリゴヌクレオチドとして、例えば、検出したいmRNAの一部の塩基配列において、5’末端側の塩基配列に相当するセンスプライマー、3’末端側の塩基配列に相当するアンチセンスプライマー等をあげることができる。ただし、mRNAにおいてウラシルに相当する塩基は、オリゴヌクレオチドプライマーにおいてはチミジンとなる。
センスプライマーおよびアンチセンスプライマーとしては、両者の融解温度(Tm)および塩基数が極端に変わることのないオリゴヌクレオチドで、5〜60塩基、好ましくは10〜50塩基数のものがあげられる。
オリゴヌクレオチド誘導体としては、オリゴヌクレオチド中のリン酸ジエステル結合がホスフォロチオエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリン酸ジエステル結合がN3’−P5’ホスフォアミデート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリボースとリン酸ジエステル結合がペプチド核酸結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のウラシルがC−5プロピニルウラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のウラシルがC−5チアゾールウラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のシトシンがC−5プロピニルシトシンで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のシトシンがフェノキサジン修飾シトシン(phenoxazine−modified cytosine)で置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリボースが2’−O−プロピルリボースで置換されたオリゴヌクレオチド誘導体、あるいはオリゴヌクレオチド中のリボースが2’−メトキシエトキシリボースで置換されたオリゴヌクレオチド誘導体等があげられる〔細胞工学,16,1463(1997)〕。
2.本発明の蛋白質の製造
本発明の蛋白質は、モレキュラー・クローニング第2版やカレント・プロトコールズ・イン・モレキュラー・バイオロジー等に記載された方法等を用い、例えば以下の方法により、本発明のDNAを宿主細胞中で発現させて、製造することができる。
全長cDNAをもとにして、必要に応じて、該蛋白質をコードする部分を含む適当な長さのDNA断片を調製する。
該DNA断片、または全長cDNAを適当な発現ベクターのプロモーターの下流に挿入することにより、組換えベクターを作製する。
該組換えベクターを、該発現ベクターに適合した宿主細胞に導入することにより、本発明の蛋白質を生産する形質転換体を得ることができる。
宿主細胞としては、細菌、酵母、動物細胞、昆虫細胞、植物細胞等、目的とする遺伝子を発現できるものであればいずれも用いることができる。
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中への組込が可能で、本発明の蛋白質をコードするDNAを転写できる位置にプロモーターを含有しているものが用いられる。
細菌等の原核生物を宿主細胞として用いる場合は、本発明の蛋白質をコードするDNAを含有してなる組換えベクターは原核生物中で自立複製可能であると同時に、プロモーター、リボソーム結合配列、本発明の蛋白質をコードする遺伝子、及び転写終結配列より構成されたベクターであることが好ましい。プロモーターを制御する遺伝子が含まれていてもよい。
発現ベクターとしては、例えば、pBTrp2、pBTac1、pBTac2(いずれもベーリンガーマンハイム社より市販)、pKK233−2(Pharmacia社製)、pSE280(Invitrogen社製)、pGEMEX−1(Promega社製)、pQE−8(QIAGEN社製)、pKYP10(特開昭58−110600)、pKYP200〔Agricultural Biological Chemistry,48,669(1984)〕、pLSA1〔Agric.Biol.Chem.,53,277(1989)〕、pGEL1〔Proc.Natl.Acad.Sci.USA,82,4306(1985)〕、pBluescript II SK(−)(Stratagene社製)、pTrs30〔Escherichia coli JM109/pTrS30(FERM BP−5407)より調製〕、pTrs32〔Escherichia coli JM109/pTrS32(FERM BP−5408)より調製〕、pGHA2〔Escherichia coli IGHA2(FERM B−400)より調製、特開昭60−221091〕、pGKA2〔Escherichia coli IGKA2(FERM BP−6798)より調製、特開昭60−221091〕、pTerm2(US4686191、US4939094、US5160735)、pSupex、pUB110、pTP5、pC194、pEG400〔J.Bacteriol.,172,2392(1990)〕、pGEX(Pharmacia社製)、pETシステム(Novagen社製)、pSupex等をあげることができる。
プロモーターとしては、宿主細胞中で発現できるものであればいかなるものでもよい。例えば、trpプロモーター(Ptrp)、lacプロモーター、PLプロモーター、PRプロモーター、T7プロモーター等の、大腸菌やファージ等に由来するプロモーターをあげることができる。またPtrpを2つ直列させたプロモーター(Ptrp×2)、tacプロモーター、lacT7プロモーター、let Iプロモーターのように人為的に設計改変されたプロモーター等も用いることができる。
リボソーム結合配列であるシャイン−ダルガノ(Shine−Dalgarno)配列と開始コドンとの間を適当な距離(例えば6〜18塩基)に調節したプラスミドを用いることが好ましい。
本発明の蛋白質をコードする部分の塩基配列を、宿主の発現に最適なコドンとなるように、塩基を置換することにより、目的とする蛋白質の生産率を向上させることができる。
本発明の組換えベクターにおいては、本発明のDNAの発現には転写終結配列は必ずしも必要ではないが、構造遺伝子の直下に転写終結配列を配置することが好ましい。
宿主細胞としては、エシェリヒア属、セラチア属、バチルス属、ブレビバクテリウム属、コリネバクテリウム属、ミクロバクテリウム属、シュードモナス属等に属する微生物、例えば、Escherichia coli XL1−Blue、Escherichia coli XL2−Blue、Escherichia coli DH1、Escherichia coli MC1000、Escherichia coli KY3276、Escherichia coli W1485、Escherichia coli JM109、Escherichia coli HB101、Escherichia coli No.49、Escherichia coli W3110、Escherichia coli NY49、Serratia ficaria、Serratia fonticola、Serratia liquefaciens、Serratia marcescens、Bacillus subtilis、Bacillus amyloliquefaciens、Brevibacterium immariophilum、ATCC14068、Brevibacterium saccharolyticum ATCC14066、Brevibacterium flavum ATCC14067、Brevibacterium ammoniagenes、Brevibacterium lactofermentum ATCC13869、Corynebacterium glutamicum ATCC13032、Corynebacterium acetoacidophilum ATCC13870、Microbacterium ammoniaphilum ATCC15354、Pseudomonas sp.D−0110等をあげることができる。
組換えベクターの導入方法としては、上記宿主細胞へDNAを導入する方法であればいずれも用いることができ、例えば、カルシウムイオンを用いる方法〔Proc.Natl.Acad.Sci.USA,69,2110(1972)〕、プロトプラスト法(特開昭63−248394)、またはGene,17,107(1982)やMolecular & General Genetics,168,111(1979)に記載の方法等をあげることができる。
酵母を宿主細胞として用いる場合には、発現ベクターとして、例えば、YEP13(ATCC37115)、YEp24(ATCC37051)、YCp50(ATCC37419)等をあげることができる。
プロモーターとしては、酵母菌株中で発現できるものであればいずれのものを用いてもよく、例えば、ヘキソースキナーゼ等の解糖系の遺伝子のプロモーター、PHO5プロモーター、PGKプロモーター、GAPプロモーター、ADHプロモーター、gal 1プロモーター、gal 10プロモーター、ヒートショック蛋白質プロモーター、MFα1プロモーター、CUP 1プロモーター等をあげることができる。
宿主細胞としては、サッカロミセス属、クリュイベロミセス属、トリコスポロン属、シュワニオミセス属等に属する微生物、例えば、Saccharomyces cerevisiae、Schizosaccharomyces pombe、Kluyveromyces lactis、Trichosporon pullulans、Schwanniomyces alluvius等をあげることができる。
組換えベクターの導入方法としては、酵母にDNAを導入する方法であればいずれも用いることができ、例えば、エレクトロポレーション法〔Methods.Enzymol.,194,182(1990)〕、スフェロプラスト法〔Proc.Natl.Acad.Sci.USA,84,1929(1978)〕、酢酸リチウム法〔J.Bacteriology,153,163(1983)〕、Proc.Natl.Acad.Sci.USA,75,1929(1978)記載の方法等をあげることができる。
動物細胞を宿主として用いる場合には、発現ベクターとして、例えば、pcDNAI、pcDM8(フナコシ社製)、pAGE107〔特開平3−22979;Cytotechnology,3,133,(1990)〕、pAS3−3(特開平2−227075)、pCDM8〔Nature,329,840,(1987)〕、pcDNAI/Amp(Invitrogen社製)、pREP4(Invitrogen社製)、pAGE103〔J.Biochemistry,101,1307(1987)〕、pAGE210等をあげることができる。
プロモーターとしては、動物細胞中で発現できるものであればいずれも用いることができ、例えば、サイトメガロウイルス(CMV)のIE(immediate early)遺伝子のプロモーター、SV40の初期プロモーター、レトロウイルスのプロモーター、メタロチオネインプロモーター、ヒートショックプロモーター、SRαプロモーター等をあげることができる。また、ヒトCMVのIE遺伝子のエンハンサーをプロモーターと共に用いてもよい。
宿主細胞としては、ヒトの細胞であるナマルバ(Namalwa)細胞、サルの細胞であるCOS細胞、チャイニーズ・ハムスターの細胞であるCHO細胞、HBT5637(特開昭63−299)等をあげることができる。
組換えベクターの導入方法としては、動物細胞にDNAを導入する方法であればいずれも用いることができ、例えば、エレクトロポレーション法〔Cytotechnology,3,133(1990)〕、リン酸カルシウム法(特開平2−227075)、リポフェクション法〔Proc.Natl.Acad.Sci.USA,84,7413(1987)〕等をあげることができる。
昆虫細胞を宿主として用いる場合には、例えばカレント・プロトコールズ・イン・モレキュラー・バイオロジー、Baculovirus Expression Vectors,A Laboratory Manual,W.H.Freeman and Company,New York(1992)、Bio/Technology,6,47(1988)等に記載された方法によって、蛋白質を発現することができる。
即ち、組換え遺伝子導入ベクターおよびバキュロウイルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウイルスを得た後、さらに組換えウイルスを昆虫細胞に感染させ、蛋白質を発現させることができる。
該方法において用いられる遺伝子導入ベクターとしては、例えば、pVL1392、pVL1393、pBlueBacIII(ともにInvitorogen社製)等をあげることができる。
バキュロウイルスとしては、例えば、夜盗蛾科昆虫に感染するウイルスであるアウトグラファ・カリフォルニカ・ヌクレアー・ポリヘドロシス・ウイルス(Autographa californica nuclear polyhedrosis virus)等を用いることができる。
昆虫細胞としては、Spodoptera frugiperdaの卵巣細胞であるSf9、Sf21〔Baculovirus Expression Vectors,A Laboratory Manual,W.H.Freeman and Company,New York(1992)〕、Trichoplusia niの卵巣細胞であるHigh 5(Invitrogen社製)等を用いることができる。
組換えウイルスを調製するための、昆虫細胞への上記組換え遺伝子導入ベクターと上記バキュロウイルスの共導入方法としては、例えば、リン酸カルシウム法(特開平2−227075)、リポフェクション法〔Proc.Natl.Acad.Sci.USA,84,7413(1987)〕等をあげることができる。
植物細胞を宿主細胞として用いる場合には、発現ベクターとして、例えば、Tiプラスミド、タバコモザイクウイルスベクター等をあげることができる。
プロモーターとしては、植物細胞中で発現できるものであればいずれのものを用いてもよく、例えば、カリフラワーモザイクウイルス(CaMV)の35Sプロモーター、イネアクチン1プロモーター等をあげることができる。
宿主細胞としては、タバコ、ジャガイモ、トマト、ニンジン、ダイズ、アブラナ、アルファルファ、イネ、コムギ、オオムギ等の植物細胞等をあげることができる。
組換えベクターの導入方法としては、植物細胞にDNAを導入する方法であればいずれも用いることができ、例えば、アグロバクテリウム(Agrobacterium)(特開昭59−140885、特開昭60−70080、WO94/00977)、エレクトロポレーション法(特開昭60−251887)、パーティクルガン(遺伝子銃)を用いる方法(特許第2606856、特許第2517813)等をあげることができる。
遺伝子の発現方法としては、直接発現以外に、モレキュラー・クローニング第2版に記載されている方法等に準じて、分泌生産、融合蛋白質発現等を行うことができる。
酵母、動物細胞、昆虫細胞または植物細胞により発現させた場合には、糖あるいは糖鎖が付加された蛋白質を得ることができる。
以上のようにして得られる形質転換体を培地に培養し、培養物中に本発明の蛋白質を生成蓄積させ、該培養物から採取することにより、本発明の蛋白質を製造することができる。本発明の形質転換体を培地に培養する方法は、宿主の培養に用いられる通常の方法に従って行うことができる。
大腸菌等の原核生物あるいは酵母等の真核生物を宿主として得られた形質転換体を培養する培地としては、該生物が資化し得る炭素源、窒素源、無機塩類等を含有し、形質転換体の培養を効率的に行える培地であれば天然培地、合成培地のいずれを用いてもよい。
炭素源としては、該生物が資化し得るものであればよく、グルコース、フラクトース、スクロース、これらを含有する糖蜜、デンプンあるいはデンプン加水分解物等の炭水化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノールなどのアルコール類等を用いることができる。
窒素源としては、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸アンモニウム等の無機酸もしくは有機酸のアンモニウム塩、その他の含窒素化合物、ならびに、ペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕および大豆粕加水分解物、各種発酵菌体およびその消化物等を用いることができる。
無機塩としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マン癌、硫酸銅、炭酸カルシウム等を用いることができる。
培養は、通常振盪培養または深部通気攪拌培養などの好気的条件下で行う。培養温度は15〜40℃がよく、培養時間は、通常16時間〜7日間である。培養中のpHは3.0〜9.0に保持する。pHの調整は、無機または有機の酸、アルカリ溶液、尿素、炭酸カルシウム、アンモニアなどを用いて行う。
また、培養中必要に応じて、アンピシリンやテトラサイクリン等の抗生物質を培地に添加してもよい。
プロモーターとして誘導性のプロモーターを用いた組換えベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、lacプロモーターを用いた組換えベクターで形質転換した微生物を培養するときにはイソプロピル−β−D−チオガラクトピラノシド等を、trpプロモーターを用いた組換えベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地に添加してもよい。
動物細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用されているRPMI1640培地〔The Journal of the American Medical Association,199,519(1967)〕、EagleのMEM培地〔Science,122,501(1952)〕、ダルベッコ改変MEM培地〔Virology,8,396(1959)〕、199培地〔Proceeding of the Society for the Biological Medicine,73,1(1950)〕またはこれら培地に牛胎児血清等を添加した培地等を用いることができる。
培養は、通常pH6〜8、30〜40℃、5%CO2存在下等の条件下で1〜7日間行う。
また、培養中必要に応じて、カナマイシン、ペニシリン等の抗生物質を培地に添加してもよい。
昆虫細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用されているTNM−FH培地(Pharmingen社製)、Sf−900 II SFM培地(Life Technologies社製)、ExCell400、ExCell405(いずれもJRH Biosciences社製)、Grace’s Insect Medium〔Grace,T.C.C.,Nature,195,788(1962)〕等を用いることができる。
培養は、通常pH6〜7、25〜30℃等の条件下で、1〜5日間行う。
また、培養中必要に応じて、ゲンタマイシン等の抗生物質を培地に添加してもよい。
植物細胞を宿主として得られた形質転換体は、細胞として、または植物の細胞や器官に分化させて培養することができる。該形質転換体を培養する培地としては、一般に使用されているムラシゲ・アンド・スクーグ(MS)培地、ホワイト(White)培地、またはこれら培地にオーキシン、サイトカイニン等、植物ホルモンを添加した培地等を用いることができる。
培養は、通常pH5〜9、20〜40℃の条件下で3〜60日間行う。
また、培養中必要に応じて、カナマイシン、ハイグロマイシン等の抗生物質を培地に添加してもよい。
上記のとおり、本発明の蛋白質をコードするDNAを組み込んだ組換え体ベクターを保有する微生物、動物細胞、あるいは植物細胞由来の形質転換体を、通常の培養方法に従って培養し、該蛋白質を生成蓄積させ、該培養物より該蛋白質を採取することにより、該蛋白質を製造することができる。
遺伝子の発現方法としては、直接発現以外に、モレキュラー・クローニング第2版に記載されている方法等に準じて、分泌生産、融合蛋白質発現等を行うことができる。
本発明の蛋白質の生産方法としては、宿主細胞内に生産させる方法、宿主細胞外に分泌させる方法、あるいは宿主細胞外膜上に生産させる方法があり、使用する宿主細胞や、生産させる蛋白質の構造を変えることにより、該方法を選択することができる。
本発明の蛋白質が宿主細胞内あるいは宿主細胞外膜上に生産される場合、ポールソンらの方法〔J.Biol.Chem.,264,17619(1989)〕、ロウらの方法〔Proc.Natl.Acad.Sci.,USA,86,8227(1989)、Genes Develop.,4,1288(1990)〕、または特開平5−336963、WO94/23021等に記載の方法を準用することにより、該蛋白質を宿主細胞外に積極的に分泌させることができる。
すなわち、遺伝子組換えの手法を用いて、本発明の蛋白質の活性部位を含む蛋白質の手前にシグナルペプチドを付加した形で発現させることにより、本発明の蛋白質を宿主細胞外に積極的に分泌させることができる。
また、特開平2−227075に記載されている方法に準じて、ジヒドロ葉酸還元酵素遺伝子等を用いた遺伝子増幅系を利用して生産量を上昇させることもできる。
さらに、遺伝子導入した動物または植物の細胞を再分化させることにより、遺伝子が導入された動物個体(トランスジェニック非ヒト動物)または植物個体(トランスジェニック植物)を造成し、これらの個体を用いて本発明の蛋白質を製造することもできる。
形質転換体が動物個体または植物個体の場合は、通常の方法に従って、飼育または栽培し、該蛋白質を生成蓄積させ、該動物個体または植物個体より該蛋白質を採取することにより、該蛋白質を製造することができる。
動物個体を用いて本発明の蛋白質を製造する方法としては、例えば公知の方法〔American Journal of Clinical Nutrition,63,639S(1996)、American Journal of Clinical Nutrition,63,627S(1996)、Bio/Technology,9,830(1991)〕に準じて遺伝子を導入して造成した動物中に本発明の蛋白質を生産する方法があげられる。
動物個体の場合は、例えば、本発明の蛋白質をコードするDNAを導入したトランスジェニック非ヒト動物を飼育し、該蛋白質を該動物中に生成・蓄積させ、該動物中より該蛋白質を採取することにより、該蛋白質を製造することができる。該動物中の生成・蓄積場所としては、例えば、該動物のミルク(特開昭63−309192)、卵等をあげることができる。この際に用いられるプロモーターとしては、動物で発現できるものであればいずれも用いることができるが、例えば、乳腺細胞特異的なプロモーターであるαカゼインプロモーター、βカゼインプロモーター、βラクトグロブリンプロモーター、ホエー酸性プロテインプロモーター等が好適に用いられる。
植物個体を用いて本発明の蛋白質を製造する方法としては、例えば本発明の蛋白質をコードするDNAを導入したトランスジェニック植物を公知の方法〔組織培養,20(1994)、組織培養,21(1995)、Trends in Biotechnology,15,45(1997)〕に準じて栽培し、該蛋白質を該植物中に生成・蓄積させ、該植物中より該蛋白質を採取することにより、該蛋白質を生産する方法があげられる。
本発明の形質転換体により製造された蛋白質は、例えば本発明の蛋白質が、細胞内に溶解状態で発現した場合には、培養終了後、細胞を遠心分離により回収し、水系緩衝液にけん濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、ダイノミル等により細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、通常の酵素の単離精製法、即ち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル(DEAE)−セファロース、DIAION HPA−75(三菱化成社製)等レジンを用いた陰イオン交換クロマトグラフィー法、S−Sepharose FF(Pharmacia社製)等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の手法を単独あるいは組み合わせて用い、精製標品を得ることができる。
また、該蛋白質が細胞内に不溶体を形成して発現した場合は、同様に細胞を回収後破砕し、遠心分離を行うことにより、沈殿画分として蛋白質の不溶体を回収する。回収した蛋白質の不溶体を蛋白質変性剤で可溶化する。該可溶化液を希釈または透析することにより、該蛋白質を正常な立体構造に戻した後、上記と同様の単離精製法により該蛋白質の精製標品を得ることができる。
本発明の蛋白質あるいはその糖修飾体等の誘導体が細胞外に分泌された場合には、培養上清に該蛋白質あるいはその糖鎖付加体等の誘導体を回収することができる。即ち、該培養物を上記と同様の遠心分離等の手法により処理することにより可溶性画分を取得し、該可溶性画分から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。
このようにして取得される蛋白質として、例えば、配列番号1で表されるアミノ酸配列を有する蛋白質をあげることができる。
また、本発明の蛋白質は、Fmoc法(フルオレニルメチルオキシカルボニル法)、tBoc法(t−ブチルオキシカルボニル法)等の化学合成法によっても製造することができる。また、Advanced ChemTech社、パーキン・エルマー社、Pharmacia社、Protein Technology Instrument社、Synthecell−Vega社、PerSeptive社、島津製作所等のペプチド合成機を利用して化学合成することもできる。
3.本発明の蛋白質を認識する抗体の調製
本発明の蛋白質、またそれら蛋白質の部分断片ポリペプチドの精製標品、あるいは本発明の蛋白質の一部のアミノ酸配列を有するペプチドを抗原として用いることにより、ポリクローナル抗体、モノクローナル抗体等、本発明の蛋白質を認識する抗体を作製することができる。
(1)ポリクローナル抗体の作製
本発明の蛋白質、またそれら蛋白質の部分断片ポリペプチドの精製標品、あるいは本発明の蛋白質の一部のアミノ酸配列を有するペプチドを抗原として用い、動物に投与することによりポリクローナル抗体を作製することができる。
投与する動物として、ウサギ、ヤギ、ラット、マウス、ハムスター等を用いることができる。
該抗原の投与量は動物1匹当たり50〜100μgが好ましい。
ペプチドを用いる場合は、ペプチドをスカシガイヘモシアニン(keyhole limpet haemocyanin)や牛チログロブリンなどのキャリア蛋白に共有結合させたものを抗原とするのが望ましい。抗原とするペプチドは、ペプチド合成機で合成することができる。
該抗原の投与は、1回目の投与の後1〜2週間おきに3〜10回行う。各投与後、3〜7日目に眼底静脈叢より採血し、該血清が免疫に用いた抗原と反応することを酵素免疫測定法〔酵素免疫測定法(ELISA法):医学書院刊(1976)、Antibodies−A Laboratory Manual,Cold Spring Harbor Laboratory(1988)〕等で確認する。
免疫に用いた抗原に対し、その血清が充分な抗体価を示した非ヒト哺乳動物より血清を取得し、該血清を分離、精製することによりポリクローナル抗体を取得することができる。
分離、精製する方法としては、遠心分離、40〜50%飽和硫酸アンモニウムによる塩析、カプリル酸沈殿〔Antibodies,A Laboratory manual,Cold Spring Harbor Laboratory(1988)〕、またはDEAE−セファロースカラム、陰イオン交換カラム、プロテインAまたはG−カラムあるいはゲル濾過カラム等を用いるクロマトグラフィー等を、単独または組み合わせて処理する方法があげられる。
(2)モノクローナル抗体の作製
(a)抗体産性細胞の調製
免疫に用いた本発明の蛋白質の部分断片ポリペプチドに対し、その血清が十分な抗体価を示したラットを抗体産生細胞の供給源として供する。
該抗体価を示したラットに抗原物質を最終投与した後3〜7日目に、脾臓を摘出する。
該脾臓をMEM培地(日水製薬社製)中で細断し、ピンセットでほぐし、1,200rpmで5分間遠心分離した後、上清を捨てる。
得られた沈殿画分の脾細胞をトリス−塩化アンモニウム緩衝液(pH7.65)で1〜2分間処理し赤血球を除去した後、MEM培地で3回洗浄し、得られた脾細胞を抗体産生細胞として用いる。
(b)骨髄腫細胞の調製
骨髄腫細胞としては、マウスまたはラットから取得した株化細胞を使用する。例えば、8−アザグアニン耐性マウス(BALB/c由来)骨髄腫細胞株P3−X63Ag8−U1(以下、P3−U1と略す)〔Curr.Topics.Microbiol.Immunol.,81,1(1978)、Europ.J.Immunol.,6,511(1976)〕、SP2/0−Ag14(SP−2)〔Nature,276,269(1978)〕、P3−X63−Ag8653(653)〔J.Immunol.,123,1548(1979)〕、P3−X63−Ag8(X63)〔Nature,256,495(1975)〕等を用いることができる。これらの細胞株は、8−アザグアニン培地〔RPMI−1640培地にグルタミン(1.5mmol/L)、2−メルカプトエタノール(5×10−5mol/L)、ジェンタマイシン(10μg/ml)および牛胎児血清(FCS)(CSL社製、10%)を加えた培地(以下、正常培地という)に、さらに8−アザグアニン(15μg/ml)を加えた培地〕で継代するが、細胞融合の3〜4日前に正常培地で培養し、融合には該細胞を2×107個以上用いる。
(c)ハイブリドーマの作製
(a)で取得した抗体産生細胞と(b)で取得した骨髄腫細胞をMEM培地またはPBS(リン酸二ナトリウム1.83g、リン酸一カリウム0.21g、食塩7.65g、蒸留水1リットル、pH7.2)でよく洗浄し、細胞数が、抗体産生細胞:骨髄腫細胞=5〜10:1になるよう混合し、1,200rpmで5分間遠心分離した後、上清を捨てる。
得られた沈澱画分の細胞群をよくほぐし、該細胞群に、攪拌しながら、37℃で、108抗体産生細胞あたり、ポリエチレングライコール−1000(PEG−1000)2g、MEM 2mlおよびジメチルスルホキシド(DMSO)0.7mlを混合した溶液を0.2〜1ml添加し、さらに1〜2分間毎にMEM培地1〜2mlを数回添加する。添加後、MEM培地を加えて全量が50mlになるように調製する。該調製液を900rpmで5分間遠心分離後、上清を捨てる。得られた沈殿画分の細胞を、ゆるやかにほぐした後、メスピペットによる吸込み、吹出しでゆるやかにHAT培地〔正常培地にヒポキサンチン(10−4mol/L)、チミジン(1.5×10−5mol/L)およびアミノプテリン(4×10−7mol/L)を加えた培地〕100ml中に懸濁する。
該懸濁液を96穴培養用プレートに100〜200μl/穴ずつ分注し、5%CO2インキュベーター中、37℃で7〜14日間培養する。
培養後、培養上清の一部をとりアンチボディイズ〔Antibodies,A Laboratory manual,Cold Spring Harbor Laboratory,Chapter 14(1988)〕等に述べられている酵素免疫測定法により、本発明の蛋白質の部分断片ポリペプチドに特異的に反応するハイブリドーマを選択する。
酵素免疫測定法の具体的例として、以下の方法をあげることができる。
免疫の際、抗原に用いた本発明の蛋白質の部分断片ポリペプチドを適当なプレートにコートし、ハイブリドーマ培養上清もしくは後述の(d)で得られる精製抗体を第一抗体として反応させ、さらに第二抗体としてビオチン、酵素、化学発光物質あるいは放射線化合物等で標識した抗ラットまたは抗マウスイムノグロブリン抗体を反応させた後に標識物質に応じた反応を行ない、本発明の蛋白質に特異的に反応するものを本発明のモノクローナル抗体を生産するハイブリドーマとして選択する。
該ハイブリドーマを用いて、限界希釈法によりクローニングを2回繰り返し〔1回目は、HT培地(HAT培地からアミノプテリンを除いた培地)、2回目は、正常培地を使用する〕、安定して強い抗体価の認められたものを本発明のモノクローナル抗体を産生するハイブリドーマ株として選択する。
本発明のモノクローナル抗体を産生するハイブリドーマ株の1例としては、ハイブリドーマ株KM3103が挙げられる。ハイブリドーマ株KM3103は、平成14年3月26日に独立行政法人産業技術総合研究所特許生物寄託センター(日本国茨城県つくば市東1丁目1番地1中央第6:郵便番号305−8566)にFERM BP−7977として寄託されている。
(d)モノクローナル抗体の調製
プリスタン処理〔2,6,10,14−テトラメチルペンタデカン(Pristane)0.5mlを腹腔内投与し、2週間飼育する〕した8〜10週令のマウスまたはヌードマウスに、(c)で取得した本発明の蛋白質モノクローナル抗体産生ハイブリドーマ細胞5〜20×106細胞/匹を腹腔内に注射する。10〜21日間でハイブリドーマは腹水癌化する。
該腹水癌化したマウスから腹水を採取し、3000rpmで5分間遠心分離して固形分を除去する。
得られた上清より、ポリクローナルで用いた方法と同様の方法でモノクローナル抗体を精製、取得することができる。
抗体のサブクラスの決定は、マウスモノクローナル抗体タイピングキットまたはラットモノクローナル抗体タイピングキットを用いて行う。蛋白質量は、ローリー法あるいは280nmでの吸光度より算出する。
4.本発明のDNA、蛋白質または抗体の利用
(1)本発明の蛋白質をコードする遺伝子の発現を検出・定量することによる疾患の判定方法
本発明のDNAまたはオリゴヌクレオチドを用い、ノーザンハイブリダイゼーション法(モレキュラー・クローニング第2版)、PCR法およびRT(reverse−transcribed)−PCR法〔ともにPCR Protocols、Academic Press(1990)〕(以上、併せてPCR法ともいう)等を行い、本発明の蛋白質をコードする遺伝子の発現を検出することで、急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、脳腫瘍、悪性腫瘍、固形腫瘍等の異常な細胞増殖を伴う疾患、心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血、脈絡膜循環障害等の血管障害を伴う疾患、骨粗鬆症等の骨代謝の異常を伴う疾患、小人症、末端肥大症、小児慢性腎不全等のインスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、動脈硬化、気管支疾患、再狭窄等の異常な平滑筋細胞の分化増殖を伴う疾患、重症筋無力症等の異常な骨格筋細胞の分化増殖を伴う疾患、胃潰瘍等の胃酸分泌の異常を伴う疾患、微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏、自己免疫疾患等の異常なリンパ球浸潤を伴う炎症性の疾患等のIGFBPスーパーファミリーに属する蛋白質の関与の報告がある疾患の判定を行うことができる。
RT−PCR法は簡便な方法であるため、遺伝子の発現の検出法として特に有用である。
例えば、配列番号2で表される塩基配列中の連続した100〜2881塩基と同じ配列を有するDNA、または該DNAと相補的な配列を有するDNAをプローブとしてノーザンハイブリダイゼーションを行い、配列番号2に記載の塩基配列を有する遺伝子の発現量を定量し、健常者と比較することにより上記疾患を判定することができる。
該判定の具体的方法として、例えば、以下の方法をあげることができる。
被験者および健常者の白血球細胞または組織由来全RNA(10〜20μg)、またはそれらのmRNA(1〜5μg)を、変性溶液〔50%(v/v)ホルムアミド、2.2mol/Lホルムアルデヒド、20mmol/L MOPS[3−(N−モルホリノ)プロパンスルホン酸](PH7.0)、5mmol/L 酢酸ナトリウム、1mmol/L EDTA〕中、65℃で5分間加熱し、変性させ、2.2mol/L ホルムアルデヒドを含む1%アガロースゲルで電気泳動する。
電気泳動後、ゲル中のRNAをニトロセルロースフィルター(Optimal BA−S85;Schleicher & Schuell社製)上にブロッティングし、減圧下80℃で1時間加熱し固定化する。
該フィルターをハイブリダイゼーション溶液〔5×SSPE(750mmol/L NaCl、50mmol/L NaH2PO4、5mmol/L EDTA;pH7.4)、5×デンハルト溶液(0.1%フィコール、0.1%ポリビニルピロリドン、0.1%ウシ血清アルブミン)、1%SDS(ドデシル硫酸ナトリウム)、0.2mg/mlのサケ***DNA(Pharmacia Biotech社製)〕中に浸漬しプレハイブリダイゼーションを行う。
プレハイブリダイゼーション後、該溶液にプローブを添加し、65℃でハイブリダイゼーションを行う。
プローブとしては、例えば配列番号2に記載のDNA断片をマルチプライムDNA標識システム(アマシャム社製)を用いて32Pで標識したものを使用できる。
ハイブリダイゼーション後のフィルターを、以下の順で洗浄する。
(a)0.1%SDSを含む2×SSC(300mmol/L NaCl、30mmol/L クエン酸ナトリウム)溶液中、室温で15分間洗浄する。これを数回繰り返す。
(b)0.1%SDSを含む1×SSC(150mmol/L NaCl、15mmol/L クエン酸ナトリウム)溶液中、50〜70℃で15分間洗浄する。これを数回繰り返す。
(c)50〜70℃の0.1%SDSを含む0.1×SSC(15mmol/L NaCl、1.5mmol/L クエン酸ナトリウム)溶液中、50〜70℃で15分間洗浄する。これを数回繰り返す。
フィルター洗浄後、イメージングプレートを用いてオートラジオグラフィーを行い、バイオイメージングアナライザーBAS2000(富士写真フィルム)で配列番号2に記載のDNAの発現を検出し定量することができる。
また、例えば、本発明の蛋白質をコードするcDNAに特異的な1組のオリゴヌクレオチドをプライマーとして用い、被験者および健常者の白血球細胞または組織由来全RNA、それらのmRNAまたはこれらRNAから調製したcDNAを鋳型としてPCRを行い、増幅断片を検出・定量し、被験者と健常者の本発明の蛋白質をコードする遺伝子の発現量を比較することにより上記疾患を判定することができる。
オリゴヌクレオチドとしては、本発明のオリゴヌクレオチドを用いることができる。
PCRの鋳型となるmRNAまたは全RNAは、血液より分離・取得した各種白血球細胞、または疾患の疑いがある組織等から抽出することができる。
白血球細胞としては、多形核白血球、単球、リンパ球、T細胞、B細胞等をあげることができる。
多形核白血球および単核球は、被験者の末梢血より、ナイコメッド・ファーマ(Nycomed Pharma)社製のキットであるPolymorphprepTMを用いることにより、分離・取得することができる。
取得した単核球より、J.Immunol.,130,706(1983)等に記載の方法により、単球およびリンパ球を、Tissue Antigen,9,153(1977)、J.Immunol.,11,273(1976)、Nycomed社の血球細胞の単離法に関するマニュアル等に記載の方法により、T細胞やB細胞を分離・取得することができる。
T細胞はナイロンウール法〔Eur.J.Immunol.,3,645(1973)〕を用いて取得することもできる。また、T細胞、B細胞、単球/マクロファージにそれぞれ特異的な抗体を結合した磁気ビーズ(例えば、Dynal社製のDynabeads)を用いて、各細胞を分離・取得することができる。
白血球細胞または組織から全RNAを調製する方法としては、チオシアン酸グアニジン−トリフルオロ酢酸セシウム法〔Methods in Enzymol.,154,3(1987)〕等をあげることができる。
全RNAからポリ(A)+RNAを調製する方法としては、オリゴ(dT)固定化セルロースカラム法(モレキュラー・クローニング第2版)等をあげることができる。
また、ファースト・トラック・mRNA・アイソレーション・キット〔Fast Track mRNA Isolation Kit;Invitrogen社製〕、クイック・プレップ・mRNA・ピュリフィケーション・キット(Quick Prep mRNA Purification Kit;ファルマシア社製)等のキットを用いてmRNAを調製することもできる。
一本鎖cDNAは、全RNAまたはmRNAから、一本鎖cDNA合成キットSuperscript preamplification system(BRL社製)を用いて、合成することができる。合成は、キットに添付のマニュアルに従って行うことができる。
上記のように調製できる全RNA、mRNAまたはcDNAを用いたRT−PCR法〔PCR Protocols、Academic Press(1990)〕により、本発明の蛋白質をコードする遺伝子の発現量を定量することができる。
(2)本発明の蛋白質をコードする遺伝子の変異を検出することによる疾患の判定方法
本発明のオリゴヌクレオチドをプローブとして、ゲノムDNAに対してサザンハイブリダイゼーション法(モレキュラー・クローニング第2版)、PCR法等を行うことにより、本発明の蛋白質をコードする遺伝子の変異を検出することができる。該検出方法は、IGFBPファミリーに属する蛋白質が関与しているとの報告がある急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、脳腫瘍、悪性腫瘍、固形腫瘍等の異常な細胞増殖を伴う疾患、心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血、脈絡膜循環障害等の血管障害を伴う疾患、骨粗鬆症等の骨代謝の異常を伴う疾患、小人症、末端肥大症、小児慢性腎不全等のインスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、動脈硬化、気管支疾患、再狭窄等の異常な平滑筋細胞の分化増殖を伴う疾患、重症筋無力症等の異常な骨格筋細胞の分化増殖を伴う疾患、胃潰瘍等の胃酸分泌の異常を伴う疾患、微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏、自己免疫疾患等の異常なリンパ球浸潤を伴う炎症性の疾患等の判定に利用することができる。
具体的には、例えば、患者が保有する本発明の蛋白質をコードするDNAの翻訳領域をPCR法で増幅させ、塩基配列を決定し、健常者が有する該DNAの塩基配列と比較することにより、翻訳領域における変異の有無を調べることができる。
PCR法に供する鋳型となるcDNAは(1)に記載した方法により取得できる。取得されたcDNAの塩基配列は、パーキンエルマー社のDNAシークエンサー377と反応キット(ABI PrismTM BigDyeTM Terminator Cycle Sequencing Ready Reaction kit:Applied Biosystems社製)を用いて決定できる。
(3)免疫学的検出法・定量法による疾患の判定法
本発明の抗体を用いて、被験者および健常者の血液中または組織における本発明の蛋白質の発現を免疫学的に検出または定量し、健常者と比較することで下記疾患を判定することができる。
免疫学的に検出する方法としては、マイクロタイタープレートを用いるELISA法、蛍光抗体法、ウェスタンブロット法、免疫組織染色法等をあげることができる。
免疫学的に定量する方法としては、例えば液相中で本発明のポリペプチドと反応する抗体のうちエピトープが異なる2種類のモノクローナル抗体を用いたサンドイッチELISA法、125I等の放射性同位体で標識した本発明の蛋白質と該蛋白質を認識する抗体を用いるラジオイムノアッセイ法等があげられる。
該免疫学的方法は、本発明の蛋白質の関与が報告されている急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、悪性腫瘍、固形腫瘍等の異常な細胞増殖を伴う疾患、心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血、脈絡膜循環障害等の血管障害を伴う疾患、骨粗鬆症等の骨代謝の異常を伴う疾患、小人症、末端肥大症、小児慢性腎不全等のインスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、動脈硬化、気管支疾患、再狭窄等の異常な平滑筋細胞の分化増殖を伴う疾患、重症筋無力症等の異常な骨格筋細胞の分化増殖を伴う疾患、胃潰瘍等の胃酸分泌の異常を伴う疾患、微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏、自己免疫疾患等の異常なリンパ球浸潤を伴う炎症性の疾患等の疾患の判定に利用することができる。
(4)本発明の蛋白質をコードする遺伝子の転写または翻訳を抑制することによる疾患の予防および治療
IGFBPの発現量の上昇と疾患の関係については、小児慢性腎不全はIGFBP−2や3の増加が原因であること〔Electroiyte Mrtab.,18,320(1992)〕、副甲状腺ホルモンの上昇を伴う老人女性の骨折患者ではIGFBP−4の発現が上昇すること、白血病患者では脳脊髄液中のIGFBP−7およびIGFBP−3の濃度が上昇すること〔J.Clin.Endocrinol.Metab.,84,1361(1996)〕、大腸癌では、大腸癌組織および大腸癌細胞株においてIGFBP−7の発現が上昇していること〔J.Gastroenterology,33,213(1998)〕等が報告されている。
また、TGF−β、PTHPGE2によってIGFBP−7の発現が上昇すること〔Endocrinology,140,1998(1999)〕、骨芽細胞をグルココルチコイドで処理するとIGF−Iの発現を抑制する一方でIGFBP−7の発現を上昇させること〔Endocrinology,140,228(1999)〕、IGFBP−7は骨格筋への分化にもIGFの分化促進作用を抑制することで影響していること〔Exp.Cell Res.,237,192(1997)、Endocrinology,141,100(2000)〕から、IGFBP−7は骨代謝および骨格筋分化に関わる疾患にも関与すると考えられている。
従って、IGFBP遺伝子の発現量の増大、あるいはIGFBPの機能亢進が一因となっている疾患については、本発明の蛋白質をコードするDNAの転写量若しくは翻訳量を減少させることが疾患の予防および治療に有効である。またIGFBPに直接起因しない疾患であっても、本発明の蛋白質をコードする遺伝子の転写量若しくは翻訳量を低下させる、または本発明の蛋白質の機能を抑制することにより対症療法的に上記疾患の予防または治療を行うこともできる。
また、IGFBPの発現増加が原因で受容体を介した生理作用が亢進している患者がいる場合、本発明のDNA、オリゴヌクレオチドまたはその誘導体を患者に投与することにより、該生理作用を抑制することができ、またIGFBPの発現増加が疾患の直接の原因でなくても本発明のDNA、オリゴヌクレオチドまたはその誘導体の投与により対症療法が可能な疾患に対して、本発明のDNA、オリゴヌクレオチドまたはその誘導体は、有効な予防薬および治療薬となり得る。
該予防薬または治療薬を使用する方法としては、例えばアンチセンスRNA/DNA技術〔バイオサイエンスとインダストリー,50,322(1992)、化学,46,681(1991)、Biotechnology,9,358(1992)、Trends in Biotechnology,10,87(1992)、Trends in Biotechnology,10,152(1992)、細胞工学,16,1463(1997)〕、トリプル・ヘリックス技術〔Trends in Biotechnology,10,132(1992)〕、リボザイム技術〔Current Opinion in Chemical Biology,3,274(1999)、FEMS Microbiology Reviews,23,257(1999)、Frontiers in Bioscience,4,D497(1999)、Chemistry & Biology,6,R33(1999)、Nucleic Acids Research,26,5237(1998)、Trends In Biotechnology,16,438(1998)〕、あるいはデコイDNA法〔Nippon Rinsho−Japanese Journal of Clinical Medicine,56,563(1998)、Circulation Research,82,1023(1998)、Experimental Nephrology,5,429(1997)、Nippon Rinsho−Japanese Journal of Clinical Medicine,54,2583(1996)〕をあげることができ、該方法を用いることにより任意の遺伝子の発現を抑制することができる。
本発明のDNA、オリゴヌクレオチドまたはその誘導体を上記予防薬および治療薬として使用する場合は、本発明のDNA、オリゴヌクレオチドまたはその誘導体を単独あるいはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当なベクターに挿入した後、下記に記載した常法に従って製剤化、処方および投与することができる。
レトロウィルス、アデノウィルス等のウィルスベクターやその他遺伝子治療用のベクターに組み込んだ遺伝子治療用ベクターを遺伝子治療薬または予防薬として用いるには、該遺伝子治療用ベクターと遺伝子治療剤に用いる基剤を調合することにより製造することができる〔Nature Genet.,8,42(1994)〕。
遺伝子治療剤に用いる基剤としては、通常注射剤に用いる基剤であればどのようなものでもよく、蒸留水、塩化ナトリウム又は塩化ナトリウムと無機塩との混合物等の塩溶液、マンニトール、ラクトース、デキストラン、グルコース等の糖溶液、グリシン、アルギニン等のアミノ酸溶液、有機酸溶液又は塩溶液とグルコース溶液との混合溶液等があげられる。また常法に従い、これらの基剤に浸透圧調整剤、pH調整剤、ゴマ油、ダイズ油等の植物油又はレシチンもしくは非イオン界面活性剤等の界面活性剤等の助剤を用いて、溶液、懸濁液、分散液として注射剤を調製してもよい。これらの注射剤を、粉末化、凍結乾燥等の操作により用時溶解用製剤として調製することもできる。本発明の遺伝子治療剤は、液体の場合はそのままで、個体の場合は必要により滅菌処理をした上記の基剤に遺伝子治療の直前に溶解して治療に使用することができる。本発明の遺伝子治療剤の投与方法としては、患者の治療部位に吸収されるように、局所的に投与する方法をあげることができる。
また、非ウイルス遺伝子移入法によっても目的とする治療部位にDNAを輸送することができる。
当該分野で公知の非ウイルス遺伝子移入法には、リン酸カルシウム共沈法〔Virology,52,456−467(1973);Science,209,1414−1422(1980)〕、マイクロインジェクション法〔Proc.Natl.Acad.Sci.USA,77,5399−5403(1980);Proc.Natl.Acad.Sci.USA,77,7380−7384(1980);Cell,27,223−231(1981);Nature,294,92−94(1981)〕、リポソームを介した膜融合−介在移入法〔Proc.Natl.Acad.Sci.USA,84,7413−7417(1987);
Biochemistry,28,9508−9514(1989);J.Biol.Chem.,264,12126−12129(1989);Hum.Gene Ther.,3,267−275,(1992);Science,249,1285−1288(1990);Circulation,83,2007−2011(1992)〕あるいは直接DNA取り込みおよび受容体−媒介DNA移入法〔Science,247,1465−1468(1990);J.Biol.Chem.,266,14338−14342(1991);Proc.Natl.Acad.Sci.USA,87,3655−3659(1991);J.Biol.Chem.,264,16985−16987(1989);BioTechniques,11,474−485(1991);Proc.Natl.Acad.Sci.USA,87,3410−3414(1990);Proc.Natl.Acad.Sci.USA,88,4255−4259(1991);Proc.Natl.Acad.Sci.USA,87,4033−4037(1990);Proc.Natl.Acad.Sci.USA,88,8850−8854(1991);Hum.Gene Ther.,3,147−154(1991)〕等をあげることができる。
リポソームを介した膜融合−介在移入法ではリポソーム調製物を標的とする組織に直接投与することにより、当該組織の局所的な遺伝子の取り込みおよび発現が可能であることが腫瘍に関する研究において報告されている〔Hum.Gene Ther.,3,399(1992)〕。
(5)本発明の蛋白質の発現量または機能が低下している疾患の予防および治療
IGFBPの発現減少と疾患の関係については、骨粗鬆症ではIGFBP−5の発現が減少していること、腎摘出後や小腸切除後の代償性肥大ではIGFBP−3の発現低下に伴う遊離IGF−Iの増加がみられること〔Baillieres Clin.Endocrinol.Metab.,8,165(1994)〕、子宮内膜の悪性腫瘍ではIGFBP−1の発現が低下していること〔Growth Regul.,3,74(1993)〕、乳癌組織においてはヒト染色体上のIGFBP−7の遺伝子座に50%以上の頻度でLOHが観察され、IGFBP−7の発現の減少が見られること〔Oncogene,16,2459(1996)〕、前立腺癌組織におけるIGFBP−7の発現のmRNAレベルでの減少、および悪性の前立腺癌由来細胞株においてIGFBP−7の発現が検出されないこと〔J.Clin.Endocrinol.Metab.,83,4355(1998)〕、大型の子宮平滑筋腫部位では、隣接する子宮平滑筋細胞や腫瘍容積が120cm3以下の小さな子宮平滑筋に比べIGFBP−7のmRNAの発現が低下していること〔Am.J.Reprod.Immunol.,43,53(2000)〕、SV40T抗原で誘導されたマウス肝臓癌細胞では、IGFBP−7遺伝子の5’上流領域がメチルかされ発現量が減少していること〔Biochem.Biophys.Res.Commun.,267,109(2000)〕、ストレプトゾトシン投与によるI型糖尿病モデルでは腎臓および血管障害部位でIGFBP−7の発現が低下していること〔Diabetes,45,S111(1996)、J.Diabetes & its Complications,12,252(1998)〕、II型糖尿病患者の冠状動脈平滑筋細胞においてもIGFBP−7の発現低下が蛋白質レベルで観察されていること〔Diabetes,46,1627(1997)〕、牛の動脈由来平滑筋細胞を高グルコース培地で培養するとIGFBP−7の発現量がmRNAおよび蛋白レベルで低下すること〔Diabetes,46,1627(1997)、Diabetologia,41,134(1998)〕、またIGFBP−7は血管壁でのPGI2産生を刺激すること〔Nature,271,549(1978)〕、および溶血性***症候群〔Lancet,2,871(1978)〕、血栓性血小板減少性紫斑病〔Lancet,2,748(1979)〕、鎌形赤血球性貧血症〔Br.J.Haematol.,48,545(1981)〕、急性心筋梗塞〔Coronary,2,49(1985)〕、糖尿病性血管障害〔Metabolism,38,837(1989)、Haemostasis,16,447(1986)、Diab.Res.Clin.Pract.,3,243(1987)〕、動脈硬化性疾患において血中レベルが低下していること等が報告されている。
また、TGF−β、PTHPGE2によってIGFBP−7の発現が上昇すること〔Endocrinology,140,1998(1999)〕、骨芽細胞をグルココルチコイドで処理するとIGF−Iの発現を抑制する一方でIGFBP−7の発現を上昇させること〔Endocrinology,140,228(1999)〕、IGFBP−7は骨格筋への分化にもIGFの分化促進作用を抑制することで影響していること〔Exp.Cell Res.,237,192(1997)、Endocrinology,141,100(2000)〕から、IGFBP−7が骨代謝および骨格筋分化に関わる疾患にも関与すると考えられている。
IGFBP−7の発現が減少した前立腺癌細胞株にIGFBP−7を強制発現させると、細胞***時間の延長、軟寒天培地中でのコロニー形成能の低下、ヌードマウス移植時の腫瘍形成能の低下、薬剤処理によるアポトーシス誘導率の上昇すること〔Cancer Res.,59,2370(1999)〕、p53の機能が失われているオステオザルコーマ細胞株にIGFBP−7を強制発現させると、p53遺伝子を導入したときと同様に細胞株の増殖を抑制することができることから〔Oncogene,12,1361(1996)〕、本発明のDNAおよび蛋白質の発現を増大させることで、IGFBPの発現減少が関与する上記疾患を予防または治療できる。またIGFBPに直接起因しない疾患であっても、本発明の蛋白質をコードする遺伝子の転写量若しくは翻訳量を増大させる、または本発明の蛋白質量を増大させることにより対症療法が可能な上記疾患の予防または治療を行うことも可能である。
従って、IGFBPの発現減少または機能低下により該蛋白質の生理作用が減退している患者がいる場合、本発明のDNA、オリゴヌクレオチドまたはその誘導体、あるいは本発明の蛋白質を患者に投与することにより、該生理作用を促進することができ、またIGFBPの発現減少、または機能低下が疾患の直接の原因でなくても対症療法が可能な疾患に対しては、本発明のDNA、オリゴヌクレオチドまたはその誘導体、あるいは本発明の蛋白質は、上記疾患の予防薬および治療薬として有用である。
該予防薬および治療薬の投与方法としては、例えば、本発明の蛋白質の発現低下や変異のために正常な生理作用が期待できない患者がいる場合に、(i)本発明の蛋白質をコードするDNAを該患者に投与し発現させること(ii)対象となる細胞に本発明の蛋白質をコードするDNAを挿入し発現させた後に、該細胞を該患者に移植すること、あるいは(iii)本発明の蛋白質を該患者に投与することなどによって、患者の体内における本発明の蛋白質の量を増加させ、該蛋白質の生理作用を充分に発揮させることができる。したがって、本発明の蛋白質をコードするDNAまたは本発明の蛋白質は、本発明の蛋白質の発現量または機能が低下した疾患、または該蛋白質の変異による機能異常に起因する疾患、あるいは本発明の蛋白質に直接起因しなくとも、本発明の蛋白質をコードするDNA、あるいは本発明の蛋白質の投与により対症療法的な治療が可能な疾患に対して安全で低毒性な予防薬および治療薬として有用である。
本発明の蛋白質をコードするDNAを上記予防薬および治療薬として使用する場合は、本発明のDNAを単独あるいはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当なベクターに挿入した後、上記(4)に記載した常法に従って製剤化、処方および投与することができる。
また、本発明の蛋白質を有効成分として含有する医薬は、該有効成分を単独で投与することも可能ではあるが、通常は該有効成分を薬理学的に許容される一つあるいはそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知られる任意の方法により製造した医薬製剤として提供するのが望ましい。好ましくは水、あるいは食塩、グリシン、グルコース、ヒトアルブミン等の水溶液等の水性担体に溶解した無菌的な溶液が用いられる。また、製剤溶液を生理的条件に近づけるための緩衝化剤や等張化剤のような、薬理学的に許容される添加剤、例えば、酢酸ナトリウム、塩化ナトリウム、乳酸ナトリウム、塩化カリウム、クエン酸ナトリウム等を添加することもできる。また、凍結乾燥して貯蔵し、使用時に適当な溶媒に溶解させて用いることもできる。
投与経路は、治療に際し最も効果的なものを使用するのが望ましく、経口投与、あるいは口腔内、気道内、直腸内、皮下、筋肉内および静脈内等の非経口投与をあげることができる。投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏、テープ剤等があげられる。
経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒剤等があげられる。例えば乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖等の糖類、ポリエチレングリコール、プロピレングリコール等のグリコール類、ごま油、オリーブ油、大豆油などの油類、p−ヒドロキシ安息香酸エステル類等の防腐剤、ストロベリーフレーバー、ペパーミント等のフレーバー類等を添加剤として用いて製造できる。カプセル剤、錠剤、散剤、顆粒剤等は、乳糖、ブドウ糖、ショ糖、マンニトール等の賦形剤、デンプン、アルギン酸ナトリウム等の崩壊剤、ステアリン酸マグネシウム、タルク等の滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤等を添加剤として用いて製造できる。
非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤等があげられる。例えば、注射剤は、塩溶液、ブドウ糖溶液、あるいは両者の混合物からなる担体等を用いて調製する。座剤はカカオ脂、水素化脂肪またはカルボン酸等の担体を用いて調製される。また、噴霧剤は該蛋白質そのもの、ないしは受容者の口腔および気道粘膜を刺激せず、かつ該蛋白質を微細な粒子として分散させ吸収を容易にさせる担体等を用いて調製する。担体として具体的には乳糖、グリセリン等が例示される。該蛋白質および用いる担体の性質により、エアロゾル、ドライパウダー等の製剤が可能である。また、これらの非経口剤においても経口剤で添加剤として例示した成分を添加することもできる。
投与量または投与回数は、目的とする治療効果、投与方法、治療期間、年齢、体重等により異なるが、通常成人1日当たり10μg/kg〜8mg/kgである。
(6)本発明の蛋白質をコードする遺伝子のプロモーター領域を取得する方法
本発明のDNAまたはオリゴヌクレオチドをプローブとして、公知の方法〔東京大学医科学研究所制癌研究部編、新細胞工学実験プロトコール、秀潤社(1993年)〕を用いて、該遺伝子のプロモーター領域を取得することができる。
プロモーター領域としては、哺乳動物細胞において本発明の蛋白質をコードする遺伝子の転写に関与するすべてのプロモーター領域があげられる。例えば、ヒトの脳組織において本発明の蛋白質をコードする遺伝子の転写に関与するプロモーター領域をあげることができる。該プロモーターは後述(7)に記載した本発明の蛋白質をコードする遺伝子の転写または翻訳を制御する物質のスクリーニング法に利用することができる。
(7)本発明の蛋白質をコードする遺伝子の転写または翻訳を制御する物質のスクリーニング法
急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、悪性腫瘍、固形腫瘍等の異常な細胞増殖を伴う疾患、心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血、脈絡膜循環障害等の血管障害を伴う疾患、骨粗鬆症等の骨代謝の異常を伴う疾患、小人症、末端肥大症、小児慢性腎不全等のインスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、動脈硬化、気管支疾患、再狭窄等の異常な平滑筋細胞の分化増殖を伴う疾患、重症筋無力症等の異常な骨格筋細胞の分化増殖を伴う疾患、胃潰瘍等の胃酸分泌の異常を伴う疾患、微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏、自己免疫疾患等の異常なリンパ球浸潤を伴う炎症性の疾患等の疾患は、IGFBPスーパーファミリーに属する蛋白質が関与していると報告されている。
上記疾患の一因として、IGFBPをコードするDNAの転写量の変動、または本発明の蛋白質の機能変化が考えられ、この場合、本発明の蛋白質をコードするDNAの転写量若しくは翻訳量を制御することが疾患の予防および治療に有効である。またIGFBPに直接起因しない疾患であっても、本発明の蛋白質をコードする遺伝子の転写量若しくは翻訳量を制御することにより対症療法的に上記疾患の予防または治療を行うことが可能である。
従って、本発明の蛋白質をコードする遺伝子の転写過程、あるいは転写産物から蛋白質への翻訳過程を促進または抑制する化合物は、該蛋白質の発現を制御することにより、該蛋白質を介して発揮される細胞機能を制御することが可能であり、安全で低毒性な医薬組成物として有用である。
該化合物は、以下(a)〜(c)に示す方法により取得できる。
(a)[i]本発明の蛋白質を発現する細胞と、[ii]試験物質の存在下、本発明の蛋白質を発現する細胞を、上記2に記載の培養法で2時間から1週間培養後、細胞中の該蛋白質量を、上記(3)で記載した本発明の抗体を用いて測定、比較し、該蛋白質量を増加または低下させる活性を有する化合物を選択し取得する方法。
本発明の抗体を用いた測定法としては、例えば、マイクロタイタープレートを用いるELISA法、蛍光抗体法、ウェスタンブロット法、免疫組織染色等を用いた検出法をあげることができる。
(b)[i]本発明の蛋白質を発現する細胞と、[ii]被験物質の存在下、本発明の蛋白質を発現する細胞を、上記2に記載の培養法で2時間から1週間培養後、細胞中の該蛋白質をコードするDNAの転写産物量を、上記(1)で記載したノーザンハイブリダイゼーション法またはPCR法等の方法を用いて測定、比較し、該転写産物量を増加または低下させる活性を有する化合物を選択し取得する方法。
(c)上記(6)で取得したプロモーターの下流にレポーター遺伝子を連結したDNAを組み込んだプラスミドを公知の方法により作製し、上記2に記載の方法に準じて動物細胞に導入し、形質転換体を取得する。[i]該形質転換体と、[ii]被験物質の存在下、該形質転換体を、上記2に記載の培養法で2時間から1週間培養し、細胞中のレポーター遺伝子の発現量を公知の方法〔東京大学医科学研究所 制癌研究部編,新細胞工学実験プロトコール,秀潤社(1993)、Biotechniques,20,914(1996)、J.Antibiotics,49,453(1996)、Trends in Biochemical Sciences,20,448(1995)、細胞工学,16,581(1997)〕を用いて測定、比較し、該発現量を増加または低下させる活性を有する化合物を選択し取得する方法。
レポーター遺伝子としては、例えば、クロラムフェニコール・アセチルトランスフェラーゼ遺伝子、β−ガラクトシダーゼ遺伝子、β−ラクタマーゼ遺伝子、ルシフェラーゼ遺伝子、グリーン・フルオレッセント・プロテイン(GFP)遺伝子等をあげることができる。
(8)本発明の蛋白質の機能を制御する物質のスクリーニング方法
急性骨髄性白血病、乳癌、前立腺癌、大腸癌、肝臓癌、骨髄腫、子宮平滑筋腫、悪性腫瘍、固形腫瘍等の異常な細胞増殖を伴う疾患、心筋梗塞、脳梗塞、末梢血管閉鎖症、狭心症、高血圧、高脂血症、糖尿病、糖尿病性網膜症、糸球体腎炎、動脈硬化、血栓、溶血性***症候群、血栓性血小板減少性紫斑病、虚血性心疾患、虚血性脳疾患、心不全、うっ血、脈絡膜循環障害等の血管障害を伴う疾患、骨粗鬆症等の骨代謝の異常を伴う疾患、小人症、末端肥大症、小児慢性腎不全等のインスリン様増殖因子や成長ホルモン作用の障害を伴う疾患、動脈硬化、気管支疾患、再狭窄等の異常な平滑筋細胞の分化増殖を伴う疾患、重症筋無力症等の異常な骨格筋細胞の分化増殖を伴う疾患、胃潰瘍等の胃酸分泌の異常を伴う疾患、微生物感染、慢性B型肝炎、慢性関節リウマチ、敗血症、移植片−対−宿主疾患、インスリン依存性糖尿病、腎炎、外傷性悩損傷、炎症性腸疾患、アレルギー、アトピー、喘息、花粉症、気道過敏、自己免疫疾患等の異常なリンパ球浸潤を伴う炎症性の疾患等の疾患は、IGFBPスーパーファミリーに属する蛋白質が関与していると報告されている。
上記疾患の一因として、IGFBPの機能変化が考えられ、この場合、本発明の蛋白質の機能を阻害または亢進させることが該疾患の予防および治療に有効である。またIGFBPに直接起因しない疾患であっても、本発明の蛋白質の機能を阻害または亢進させることにより対症療法的に上記疾患の予防または治療を行うことができる。
よって、本発明の蛋白質の機能変化が原因で、細胞の生理作用が減退または亢進している患者がいる場合、本発明の機能を制御する化合物を投与することにより該生理作用を制御することができ、また本発明の蛋白質の機能変化が疾患の直接の原因でなくても本発明の蛋白質の機能を制御することで対症療法が可能な疾患もまた、該化合物の投与により予防および治療できる。
該化合物は、例えば以下に示す方法により取得できる。
[i]本発明の蛋白質を発現する細胞と、[ii]被験物質の存在下、本発明の蛋白質を発現する細胞を、上記2に記載の培養法で2時間から1週間培養後、該蛋白質が機能することにより生じる細胞応答を検出、比較し、該蛋白質の機能を阻害または亢進させる活性を有する化合物を選択し取得する方法。
本発明の蛋白質が機能することにより生じる細胞応答を検出する方法としては、例えば、培地中に含まれるインスリンあるいはインスリン様増殖因子に依存的な細胞内情報伝達、遺伝子の転写、糖の取り込み、増殖などの変化を検出する公知の方法が挙げられる。また、プロスタグランジン産生の変化を測定する公知の方法も挙げられる〔Nature,263,663(1976)、臨床科学,17,958(1981)〕。
(9)本発明の蛋白質と特異的に結合する蛋白質をスクリーニングする方法
本発明の蛋白質と特異的に結合する蛋白質は、本発明の蛋白質に起因する疾患の予防薬または治療薬の開発に利用することができる。例えば、本発明の蛋白質の受容体は、本発明の蛋白質と結合して、細胞内に情報を伝達し、生理作用を発現する機能を有しているので、該受容体の活性、または該受容体をコードする遺伝子の転写若しくは翻訳を制御する物質は、本発明の蛋白質、または本発明の蛋白質をコードする遺伝子の転写若しくは翻訳を制御する物質と同様、本発明の蛋白質の機能異常に起因する疾患の予防薬および治療薬として有用である。
本発明の蛋白質と特異的に結合する蛋白質をスクリーニングする方法としては、モレキュラー・クローニング第2版、カレント・プロトコールズ・イン・モレキュラー・バイオロジー、Science,255,989(1992)等に記載の発現クローニングの方法を挙げることができ、本発明の蛋白質と被験試料を接触させたとき、本発明の蛋白質に特異的に結合する蛋白質を被験試料より選択することで取得できる。
具体的には、以下の方法があげられる。
組織よりcDNAを調製する。該cDNAを適当な発現ベクターのプロモーターの下流に挿入することにより、組換えベクターを作製しcDNAライブラリーを作製する。該組換えベクターを、該発現ベクターに適合した宿主細胞に導入することにより、組織で発現している遺伝子を発現する形質転換体を得る。標識した本発明の蛋白質が特異的に結合する蛋白質を産生する形質転換体を選択する。
選択した該形質転換体に導入したcDNAにコードされている遺伝子配列を決定することにより、本発明の蛋白質と特異的に結合する蛋白質を取得することができる。
cDNAライブラリーを調製する方法としては、上記1に記載した方法が挙げられる。
組換えベクターの導入方法、形質転換体を得る方法、および得られた形質転換体を培地に培養する方法としては、上記2に記載した方法が挙げられる。
放射性同位元素やビオチン化等公知の蛋白質を標識する方法で標識した本発明の蛋白質と形質転換体とを共培養することで、標識された本発明の蛋白質と特異的に結合する遺伝子産物を産生する形質転換体を選択することができる。
選択した形質転換体に導入したcDNAを単離する方法としては、モレキュラー・クローニング第2版、カレント・プロトコールズ・イン・モレキュラー・バイオロジー、Mol.Cell.Biol.,8,2837(1988)等に記載のファージベクターやプラスミドベクターを回収する方法あるいはHirt法を挙げることができる。
単離したcDNAの遺伝子配列を決定する方法としては、上記1に記載した方法が挙げられる。
(10)本発明の抗体を含有する医薬
本発明の抗体は、本発明の蛋白質に関与する疾患の予防薬および治療薬として有用である。
IGFBPの発現量の上昇と疾患の関係については、小児慢性腎不全はIGFBP−2や3の増加が原因であること〔Electroiyte Mrtab.,18,320(1992)〕、副甲状腺ホルモンの上昇を伴う老人女性の骨折患者ではIGFBP−4の発現の上昇すること、白血病患者では脳脊髄液中のIGFBP−7およびIGFBP−3の濃度が上昇すること〔J.Clin.Endocrinol.Metab.,84,1361(1996)〕、大腸癌では、大腸癌組織および大腸癌細胞株においてIGFBP−7の発現が上昇していること〔J.Gastroenterology,33,213(1998)〕等が報告されている。
また、TGF−β、PTHおよびPGE2によってIGFBP−7の発現が上昇すること〔Endocrinology,140,1998(1999)〕、骨芽細胞をグルココルチコイドで処理するとIGF−Iの発現を抑制する一方でIGFBP−7の発現を上昇させること〔Endocrinology,140,228(1999)〕、IGFBP−7は骨格筋への分化にもIGFの分化促進作用を抑制することで影響していること〔Exp.Cell Res.,237,192(1997)、Endocrinology,141,100(2000)〕から、IGFBP−7が骨代謝および骨格筋分化に関わる疾患にも関与すると考えられている。
よって、IGFBPの発現量の増大、あるいは機能の亢進が一因である疾患に対しては、本発明の蛋白質の発現量を抑制する、あるいは機能を阻害することが疾患の予防および治療に有効である。またIGFBPに直接起因しない疾患であっても、本発明の蛋白質の発現量を抑制、あるいは機能を阻害することにより対症療法が可能な上記疾患の予防または治療を行うこともできる。
従って、IGFBPの発現量の増大、あるいは機能亢進が原因で、細胞の生理作用が亢進している患者がいる場合、本発明の蛋白質と結合する抗体を投与することにより該生理作用を抑制することができ、また本発明の蛋白質が疾患の直接の原因でなくても本発明の蛋白質の機能を抑制することで対症療法が可能な疾患もまた、本発明の抗体の投与により予防および治療できる。
該抗体を有効成分として含有する医薬は、上記(5)に記載した本発明の蛋白質を含有する医薬の製造に準じ、製剤学の技術分野においてよく知られる任意の方法により製造した医薬製剤として提供することができる。
(11)(7)の探索法で得られた化合物を含有する医薬
本発明の蛋白質をコードする遺伝子の転写過程、あるいは転写産物から蛋白質への翻訳過程を促進または抑制する化合物は、安全で低毒性な医薬組成物として有用である。
(7)で得られた化合物は(5)に記載した本発明の蛋白質を含有する医薬の製造法に準じ、製剤学の技術分野においてよく知られる任意の方法により製造した医薬製剤として提供することができる。
(12)本発明のDNAを用いたノックアウト非ヒト動物の作製
本発明のDNAを含有してなる組換えベクターを用い、目的とする非ヒト動物、例えばウシ、ヒツジ、ヤギ、ブタ、ウマ、マウス、ニワトリ等の胚性幹細胞(embryonic stem cell)において、染色体上の本発明の蛋白質をコードする遺伝子を公知の相同組換えの手法〔例えば、Nature,326,6110,295(1987)、Cell,51,3,503(1987)等〕により不活化または任意の配列と置換した変異クローンを作製する〔例えば、Nature,350,6315,243(1991)〕。胚性幹細胞の変異クローンを用い、動物の受精卵の胚盤胞(blastcyst)への注入キメラ法または集合キメラ法等の手法により、胚性幹細胞クローンと正常細胞からなるキメラ個体を調製することができる。このキメラ個体と正常個体の掛け合わせにより、全身の細胞の染色体上の本発明の蛋白質をコードする遺伝子に任意の変異を有する個体を得ることができ、さらにその個体の掛け合わせにより相同染色体の双方に変異が入ったホモ個体の中から、本発明の蛋白質をコードする遺伝子の発現が一部または完全に抑制された個体としてノックアウト非ヒト動物を得ることができる。
また、染色体上の本発明の蛋白質をコードする遺伝子の任意の位置へ変異を導入することにより、ノックアウト非ヒト動物を作製することも可能である。例えば染色体上の本発明の蛋白質をコードする遺伝子の翻訳領域中へ塩基を置換、欠失、挿入等させて変異を導入することにより、その産物の活性を改変させることも可能である。また、その発現制御領域への同様な変異を導入することにより、発現の程度、時期、組織特異性等を改変させることも可能である。さらにCre−loxP系との組合せにより、より積極的に発現時期、発現部位、発現量等を制御することも可能である。このような例として、脳のある特定の領域で発現されるプロモータを利用して、その領域でのみ目的遺伝子を欠失させた例〔Cell,87,7,1317(1996)〕やCreを発現するアデノウィルスを用いて、目的の時期に、臓器特異的に目的遺伝子を欠失させた例〔Science,278,5335,(1997)〕が知られている。
従って、染色体上の本発明の蛋白質をコードする遺伝子についても、このように任意の時期や組織で発現を制御できる、または任意の挿入、欠失、置換をその翻訳領域や発現制御領域に有する、ノックアウト非ヒト動物を作製することができる。
ノックアウト非ヒト動物は、任意の時期、任意の程度または任意の部位で、本発明の蛋白質に起因する種々の疾患の症状を誘導することができる。
このように、本発明のノックアウト非ヒト動物は、本発明の蛋白質に起因する種々の疾患の治療や予防において極めて有用な動物モデルとなる。特にその治療薬、予防薬、また機能性食品、健康食品等の評価用モデルとして非常に有用である。
発明を実施するための最良の形態
以下の実施例により本発明をより具体的に説明するが、実施例は本発明の単なる例示を示すものにすぎず、本発明の範囲を限定するものではない。
[実施例1] ヒト神経前駆細胞NT−2由来cDNAライブラリーの作製
ヒト胎児精巣由来のテラトカルシノーマ細胞であって、レチノイン酸処理により神経細胞に分化可能なNT−2神経前駆細胞(Stratagene社より購入)を用いて以下の方法によりcDNAライブラリーを作製した。添付マニュアルに従って、NT−2細胞を培養後、レチノイン酸を添加して、さらに2週間培養した。その培養細胞を集めて、モレキュラー・クローニング第2版に記載の方法によりmRNAを抽出した。さらに、オリゴdTセルロースでpolyA(+)RNAを精製した。それぞれのpolyA(+)RNAよりオリゴキャプ法〔Gene,138,171(1994)〕によりcDNAライブラリーを作製した。Oligo−cap linker(配列番号3で表される塩基配列を有するRNA)及びOligo dT primer(配列番号4で表される塩基配列を有するDNA)を用いて文献〔蛋白質核酸酵素,41,197(1996)、Gene,200,149(1997)〕に記載の方法に従ってBAP(Bacterial Alkaline Phosphatase)処理、TAP(Tobacco Acid Phosphatase)処理、RNAライゲーション、第一鎖cDNAの合成とRNAの除去を行った。
次いで、5’末端側のセンスプライマー(配列番号5で表される塩基配列を有するDNA)と3’末端側のアンチセンスプライマー(配列番号6で表される塩基配列を有するDNA)の2種のPCRプライマーを用いるPCR(polymerase chain reaction)により二本鎖cDNAに変換し、SfiIで切断した。なお、このPCRは市販のキットであるGeneamp XL PCRキット(Perkin Elmer社製)を使用して、95℃で5分間熱処理後、95℃で1分間、58℃で1分間及び72℃で10分間の反応サイクルを12回繰り返し、その後4℃で保持することにより行った。次いで、DraIIIで切断したベクターpME18SFL3(GeneBank AB009864、発現ベクター、3392bp)にcDNAを一定方向で連結して組換え体DNAを作製し、該組換え体DNAを用いてEscherichia coli DH10Bを形質転換することによりcDNAライブラリーを作製した。得られた形質転換体が保持する各々のプラスミドDNAについて、cDNAの5’端と3’端の塩基配列を、DNAシークエンシング試薬(Dye Terminator Cycle Sequencing FS Ready Reaction Kit,dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit又はBigDye Terminator Cycle Sequencing FS Ready Reaction Kit,PE Biosystems社製)を用い、マニュアルに従ってシークエンシング反応を行った後、DNAシークエンサー(ABI PRISM 377,PE Biosystems社製)を用いて決定した。
[実施例2] IGFBPスーパーファミリーに属する新規因子の同定
作製したcDNAライブラリーの各クローンの塩基配列について、蛋白アミノ酸データベースSWISSPROTあるいは塩基配列データベースGenBankに登録されているIGFBPスーパーファミリーに属する既知蛋白質としてヒトIGFBP−1、ヒトIGFBP−2、ヒトIGFBP−3、ヒトIGFBP−4、ヒトIGFBP−5、ヒトIGFBP−6、ヒトIGFBP−7、ヒトIGFBP−8、ヒトIGFBP−9若しくはヒトIGFBP−10の10分子を用い、これら分子をコードする塩基配列と相同性を有するクローンC−NT2RI2004312を選択した。C−NT2RI2004312の塩基配列は配列番号2の塩基番号451〜2881の塩基配列に相当し、塩基番号451〜863の塩基配列部分はBLAST2を用いた相同性解析においてヒトIGFBP−7(UniGene Hs.119206)とP値6.0×10−14で59%の有意な相同性を示した。
C−NT2RI2004312は5’部分が欠出した不完全長cDNAであったことから、C−NT2RI2004312の塩基配列をもとに、GenomeWalkerTMkits(Clontech社製)を用い添付のプロトコールに従い、C−NT2RI2004312の塩基配列を含むヒトゲノムDNA領域の塩基配列情報を取得した。取得した、C−NT2RI2004312の5’末端から36塩基部分の塩基配列(配列番号2の塩基番号451〜486の塩基配列の部分)を含むクローンの塩基配列を配列番号7に示した。C−NT2RI2004312の5’末端から36塩基部分の塩基配列を含むゲノム領域の450塩基上流に開始コドンATGが、さらに28塩基上流付近にスプライシングのアクセプター配列であるAGが存在した。また、C−NT2RI2004312の5’末端から36塩基部分の塩基配列を含むゲノム領域直後にはスプライシングのドナー配列であるGTが存在することから、配列番号7の塩基番号9〜494の塩基配列の部分は一つのエクソンを構成しており、C−NT2RI2004312の完全長cDNAの塩基配列は配列番号2に示す塩基配列であることが分かった。配列番号1にC−NT2RI2004312の完全長cDNAがコードする蛋白質のアミノ酸配列を示した。
C−NT2RI2004312の完全長cDNAがコードする蛋白質のアミノ酸配列は、BLAST2を用いた相同性解析において、インスリン様増殖因子結合蛋白質ファミリーに属する蛋白質であるヒトIGFBP−7(GenBankアクセッションナンバー:I52825)とP値1.0×10−39で39%の有意な相同性を示した。インスリン様増殖因子結合蛋白質スーパーファミリーにおいては、IGF−1やIGF−2、インスリンとの結合に重要な10個のシステイン残基が存在し、そのうち4個のシステイン残基を含むインスリン様増殖因子結合モチーフ〔GCGCCXXC(G:グリシン残基、C:システイン残基、X:任意のアミノ酸残基)〕を中心に、このファミリーに属する因子間で高く保存されている。C−NT2RI2004312の完全長cDNAがコードする蛋白質のアミノ酸配列をインスリン様増殖因子結合蛋白質スーパーファミリーの各因子と比較してみると、配列番号1で表されるアミノ酸配列においても、システイン残基の位置と数が保存されており、インスリン様増殖因子結合モチーフ部分も高く保存されていることが分かった(図1)。また、N末端側のシグナル配列も、C−NT2RI2004312の完全長cDNAがコードする蛋白質とインスリン様増殖因子結合蛋白質スーパーファミリーに属する因子間で高く保存されていた(図1)。
以上の結果より、C−NT2RI2004312の完全長cDNAがコードする蛋白質がインスリン様増殖因子結合蛋白質スーパーファミリーに属するヒト新規蛋白質であり、インスリン様増殖因子結合蛋白質スーパーファミリーに属する蛋白質としての活性を有することが明らかとなった。
配列番号2に示した塩基配列をもとに、塩基配列データベースGenBank/EMBL/DDBJを、BLAST2を用いて検索したところ、機能が未知な遺伝子として登録されているUniGene Hs.21400と相同性が高いことが分かった。このHs.21400は他の遺伝子と有為な相同性を示さない32個のESTから構成されており、BLAST2を用いた相同性解析で配列番号2に示した塩基配列の3’部分とP値8.5×10−103、相同性93%であった。また、配列番号2に示した塩基配列と同一遺伝子由来と考えられるヒトESTが一つ存在したが、このEST塩基配列はC−NT2RI2004312の完全長cDNAをカバーしていなかった。このESTのGeneBankアクセッションナンバーはAW129076である。
プラスミドC−NT2RI2004312を含む大腸菌:Escherichia coli DH10B/pME18SFL3−NT2RI2004312は、FERM BP−7473として、平成13年3月1日付けで独立行政法人産業技術総合研究所特許生物寄託センター(日本国茨城県つくば市東1丁目1番地中央第6 郵便番号305−8566)(旧称経済産業省産業技術総合研究所生命工学工業技術研究所、旧住所 日本国茨城県つくば市東1丁目1番3号(郵便番号305−8566))に寄託されている。
[実施例3] C−NT2RI2004312の完全長cDNAの塩基配列の解析
配列番号2に示したC−NT2RI2004312の完全長cDNAの塩基配列をもとに、蛋白質の開始コドン予測プログラムATGPr〔Bioinformatics,14,384(1998)〕を用いて開始コドン周辺配列を解析した。配列番号2に示した塩基配列では、27〜29番目に位置するATGが開始コドン、861〜863番目に位置するTAGが終始コドンと特定され、ORFにコードされる蛋白質は278アミノ酸から構成されると推定された。配列番号1に示したC−NT2RI2004312の完全長cDNAのコードするアミノ酸配列をもとに、GENETYX−MAC 7.3(SOFTWARE DEVELOPMENT CO.,LTD製)を用いて疎水性プロットを作製した結果、N末端部分には分泌蛋白質に特徴的な疎水性の高い領域が存在した。
[実施例4] C−NT2RI2004312の完全長cDNAがコードする遺伝子のゲノム解析
配列番号2に示したC−NT2RI2004312の完全長cDNAの塩基配列をもとに、塩基配列データベースGenBank/EMBL/DDBJをBLAST2を用いて検索し、配列番号2で表される塩基配列を含むゲノムクローン(GenBankアクションナンバー:AL135785)の塩基配列情報を取得した。
取得したヒトゲノム遺伝子配列と配列番号2で表される塩基配列の比較から、配列番号2で表される塩基配列は6つの部分に別れて同一ヒト第9染色体p11−13.3のゲノム上に約18kbにわたって存在していることが分かった。
[実施例5] C−NT2RI2004312の完全長cDNAを発現している臓器
UniGene Hs.21400のNCBIに登録されているSAGEデータ(http//www.ncbi.nlm.nih.gov/UniGene/からのリンクデータ)を表1に示した。配列番号2に示したC−NT2RI2004312の完全長cDNAは、脳組織由来のcDNAライブラリーSAGE Duke 1273で高頻度存在し、脳組織に比較的選択的に発現していることが分かった。また、C−NT2RI2004312自身もレチノイン酸処理したNT−2神経前駆細胞より調製されたことから、脳組織における細胞増殖、細胞分化、組織再生に関わりの深い分子で有ることが推定された。
[実施例6] RT−PCR法を用いたC−NT2RI2004312の由来遺伝子の発現解析
クロンテック社より購入したヒト臓器polyA+RNA 4μgを鋳型とし、市販のSUPER SCRIPT Preamplification System for first strand cDNA Synthesis(ギブコ社製)を用い、添付マニュアルに従ってcDNAを合成した。
ヒト臓器polyA+RNAとしては、脳、下垂体、精巣、甲状腺、子宮、副腎、小脳、腎臓、膵臓、小腸、骨髄、心臓、肝臓、肺、リンパ節、乳腺、胎盤、前立腺、唾液腺、骨格筋、脊髄、脾臓、胃、胸腺、気管由来のpolyA+RNAを用いた。
次いで、合成したcDNAを鋳型とし、配列番号8および9で表される塩基配列を有するDNA、配列番号10および11で表される塩基配列を有するDNAをそれぞれプライマーセットとして、PCRを行った。PCRは、合成したcDNAを滅菌水を用いて50倍に希釈した溶液およびAdvantageR cDNA PCR kit(クロンテック社製)を用いて常法により反応液を調製後、94℃で7分間反応させ、次いで94℃で1分間、68℃で3分間のサイクルを32サイクル反復し、最後に68℃で7分間反応させる条件で行った。該反応液をアガロースゲル電気泳動し、それぞれのプライマーセットに特異的なDNA断片の濃さを比較することで発現量の比較を行った結果、C−NT2RI2004312は脳、下垂体、精巣、甲状腺、子宮で有意に高い発現が確認された。
なお、配列番号8および配列番号9で表される塩基配列からなるDNAをC−NT2RI2004312に特異的なプライマーセットとして、配列番号10および配列番号11で表される塩基配列からなるDNAをヒトβアクチンに特異的なプライマーセットとして用いた。
[実施例7] C−NT2RI2004312の完全長cDNAの取得
(1) ヒト脳由来全一本鎖cDNAの調製
ヒト脳由来全RNA(クロンテック社製)を45μlの滅菌水に溶解し、RQ1 RNase−Free DNase(Promega社製)1μl、付属の10×DNase buffer 5μl、RNasin Ribonuclease inhibitor(Promega社製)0.5μlをそれぞれに添加して、37℃で30分間反応させることにより、試料中に混入したゲノムDNAを分解した。反応後、RNAeasy(QIAGEN社製)により全RNAを再精製し、50μlの滅菌水に溶解した。
得られた各々の全RNA 3μlに対しSUPERSCRIPTTN First−Strand Synthesis System for RT−PCR(Life Technologies社製)を用いて添付の説明書に従い、オリゴ(dT)をプライマーとした20μlの系で逆転写反応を行うことにより、一本鎖cDNAを合成した。PCRクローニングには該反応液の50倍希釈水溶液を使用した。使用するまで−80℃で保管した。
(2) C−NT2RI2004312の完全長cDNAの取得
(a) 5’末端側のcDNA断片の取得
上記(1)で作製したヒト脳由来一本鎖cDNAに対し、配列番号16および配列番号17で表される塩基配列からなるDNAをプライマーセットとして用いてポリメラーゼ連鎖反応(PCR)を行った。PCR反応は、ExTaq(宝酒造社製)を用いてヒト脳由来一本鎖cDNAを1μl含む25μlの反応液(ExTaq buffer、0.2mmol/l dNTPs、0.5μmol/l上記プライマーセット)を調製し、94℃で5分間の加熱の後、94℃で1分、60℃で1分間、72℃で1分間からなる反応を1サイクルとして30サイクル繰り返した後、さらに72℃で5分間加熱する条件で行った。PCR終了後、反応液を1%アガロースゲル電気泳動に供し、約1100bpの増幅DNA断片をGENECLEAN SPIN Kit(BIO101)を用いて精製(以下、アガロースゲルからのDNA断片の精製にはこの方法を用いた。)し、滅菌水20μlで溶出した。該DNA断片9μlをLigation High(東洋紡)を用いた20μlの反応系でpGEM T vector(クロンテック社製)50ngとの連結反応を行い、該反応液2μlを用いて大腸菌DH5αをコーエンらの方法〔Proc.Natl.Acad.Sci.U.S.A,69,2110(1972)〕(以下、大腸菌の形質転換にはこの方法を用いた)により形質転換した。得られた複数のアンピシリン耐性コロニーから、公知の方法〔Nucleic Acids Research,7,1513(1979)〕(以下、プラスミドの単離方法にはこの方法を用いた)に従って、プラスミドDNA(以下、pGEM−C−NT2RI2004312nと称す)を単離した。
(b) 3’末端側のcDNA断片の取得
上記(1)で作製したヒト脳由来一本鎖cDNAに対し、配列番号18および配列番号19で表される塩基配列からなるDNAをプライマーセットとして用いて、上記(2)(a)と同様にポリメラーゼ連鎖反応(PCR)を行った。得られた増幅DNA断片をLigation Highを用いてpBluescript SK−のApaI−SacI断片と連結し、上記(2)(a)と同様の方法でプラスミドDNA(以下、pSK−C−NT2RI2004312cと称す)を単離した。
(c) 完全長cDNAの取得
上記(2)(a)で作製したpGEM−C−NT2RI2004312nのSacII−ApaI断片を、LigationHighを用いて本項(2)(b)で作製したpSK−C−NT2RI2004312cのSacII−ApaI断片と連結し、上記(2)(a)と同様の方法でプラスミドDNA(以下、pSK−C−NT2RI2004312tと称す)を単離した。
インサートの有無は、アガロースゲル電気泳動によるサイズ比較により確認し、塩基配列はDNAシーケンサー377(Perkin Elmer社)およびBig Dye Terminator Cycle Sequencing FS Raedy Reaction Kit(Perkin Elmer社)を使用して添付のマニュアルに従い決定した。PCRに伴う塩基の読み誤りを除いた後、決定された塩基配列と配列番号2で表される塩基配列を比較し、該プラスミドに挿入されたDNAは、配列番号2で表されるC−NT2RI2004312の完全長cDNAと同じであることを確認した。
[実施例8] 動物細胞を宿主としたC−NT2RI2004312の完全長cDNAがコードする蛋白質の発現
(1)組換えベクターの作製
実施例7で取得されたクローンC−NT2RI2004312tを鋳型とし、配列番号12および配列番号13で表される塩基配列からなるDNAをプライマーセットとして、または配列番号12および配列番号14で表される塩基配列からなるDNAをプライマーセットとしてKOD DNA Polymerase(TOYOBO社製)を用いて常法により反応液を調製後、98℃で15秒間、65℃で2秒間、74℃で30秒間のサイクルを25サイクル繰り返す条件でPCRを行い、各々の反応にて約840bpの増幅DNA断片としてC−NT2RI2004312を得た。
次に該C−NT2RI2004312の5’側がpBluescriptII SK−(Stratagene社製)のHindIII部位に、C−NT2RI2004312の3’側が、pBluescriptII SK−のXhoI部位側になるように挿入することにより、配列番号12および配列番号13で表される塩基配列からなるDNAをプライマーセットとして用いたPCRにより得られたDNA断片を有するプラスミドであるpSK−C−NT2RI2004312、配列番号12および配列番号14で表される塩基配列からなるDNAをプライマーセットとして用いたPCRにより得られたDNA断片を有するプラスミドであるpSK−C−NT2RI2004312hを作製した。
次に、pSK−C−NT2RI2004312およびpSK−C−NT2RI2004312hのHindIII−KpnI断片(約830bp)を動物細胞用発現ベクターpcDNA3(インビトロジェン社製)のHindIII−KpnI部位に挿入し、pcDNA3−C−NT2RI2004312およびpcDNA3−C−NT2RI2004312hを作製した。
なお、配列番号12および配列番号14で表される塩基配列からなるDNAのプライマーセットは、C−NT2RI2004312のC末端にHisマーカーペプチドが付加されるように設計した。
(2)細胞へのベクターの導入
Opti−MEM(ギブコ社製)100μlにFuGENETM6トランスフェクション試薬(ロシュ・ダイアグノスティックス社製)2μlを加え、室温で5分間放置した。次いでコントロールプラスミドpcDNA3、上記(1)で作製したC−NT2RI2004312発現プラスミドpcDNA3−C−NT2RI2004312またはpcDNA3−C−NT2RI2004312hを1μg添加し、さらに室温で15分間放置した。この間に6ウェルプレートに10%FCSを含むDMEM培地(ギブコ社製)を2ml/ウェルとなるように分注し、さらにCOS−1細胞(Riken Cell Bank;RCB0143)を3×105個/ウェルで播種した。15分間放置後、該プラスミドとFuGENETM6トランスフェクション試薬の混合液を25μl/ウェルずつ6ウェルプレートへ添加し、37℃のCO2インキュベーター中で72時間培養し、一過性形質転換株を取得した。以下、pcDNA3、pcDNA3−C−NT2RI2004312、およびpcDNA3−C−NT2RI2004312hを各々導入して作製した形質転換株を、それぞれCOS−1/mock株、COS−1/C−NT2RI2004312株、およびCOS−1/C−NT2RI2004312h株と称する。
(3)発現の確認
(a) 培養上清の調製
上記(2)で作製したCOS−1/mock株、COS−1/C−NT2RI2004312株、およびCOS−1/C−NT2RI2004312h株の培養上清を4ml回収し、1,200rpmで5分間遠心分離して固形物を除去し、培養上清を取得した。なお、取得した培養上清は−20℃で保存し、必要に応じて解凍後、以下の実験に用いた。
(b) 細胞破砕液の取得
上記(2)で作製したCOS−1/mock株、COS−1/C−NT2RI2004312株、およびCOS−1/C−NT2RI2004312h株の細胞をセルスクレイパー(住友ベークライト社製)で6ウェルプレートから剥離後、Phosphate Buffered Saline(pH7.2)〔以下、「PBS」と称す(ギブコ社製)〕2mlに懸濁し、1,200rpmで5分間遠心分離して上清を除去した。次いで、動物細胞用プロテアーゼインヒビターカクテル(シグマ社製)100μlを含むPBS1mlに懸濁した後、超音波破砕することにより、細胞破砕液を取得した。なお、取得した細胞破砕液は−20℃で保存し、必要に応じて解凍後、以下の実験に用いた。
(c) ウエスタンブロッティングによる検出
上記(3)(a)で調製したCOS−1/mock株、COS−1/C−NT2RI2004312h株の培養上清10μlを15%ポリアクリルアミドゲル電気泳動(Antibodies−A Laboratory Manual,Cold Spring Harbor Laboratory,1988)にて分画後、常法に従いPVDF膜(ミリポア社製)にブロッティングした。該膜を5% skim milk−PBS(以下、「ブロッキング液」と称す)でブロッキング後、該膜に抗Hisモノクローナル抗体(ロシュ・ダイアグノスティックス社製)を1μg/mlの濃度になるように添加し、室温で1時間放置した。該膜を0.05%ポリオキシエチレン(20)ソルビタンモノラウレート〔商品名:スパン20(ICI社商標Tween20相当品:和光純薬社製)〕/PBS(以下、「Tween−PBS」と略す)でよく洗浄した後、二次抗体として2,000倍希釈したペルオキシダーゼ標識ウサギ抗マウスイムノグロブリン(DAKO社製)を用いて検出した。
COS−1/C−NT2RI2004312h株の培養上清では、COS−1/mock株には観察されないバンドが検出された。従って、COS−1細胞において、C−NT2RI2004312がコードする蛋白質の発現が確認された(図2)。
[実施例9] C−NT2RI2004312の完全長cDNAがコードする蛋白質を認識するモノクローナル抗体の作製
(1)抗原ペプチドの選択
C−NT2RI2004312の完全長cDNAがコードする蛋白質のアミノ酸配列を解析し、親水性の高い部分、N末端、C末端、二次構造上ターン構造、ランダムコイル構造を有する部分の中から、抗原として適当と考えられる部分配列として、化合物1(配列番号15で表されるアミノ酸配列を有するポリペプチド)を選択した。
本発明において使用したアミノ酸およびその保護基に関する略号は、生化学命名に関するIUPAC−IUB委員会(IUPAC−IUB Joint Commission on Biochemical Nomenclature)の勧告〔ヨーロピアン・ジャーナル・オブ・バイオケミストリー(European Journal of Biochemistry),138巻,9頁(1984年)〕に従った。
以下の略号は、特に断わらない限り対応する下記のアミノ酸を表す。
Ala:L−アラニン
Arg:L−アルギニン
Asp:L−アスパラギン酸
Asx:L−アスパラギン酸またはL−アスパラギン
Cys:L−システイン
Gly:グリシン
His:L−ヒスチジン
Leu:L−ロイシン
Lys:L−リジン
Pro:L−プロリン
Thr:L−スレオニン
以下の略号は、対応する下記のアミノ酸の保護基および側鎖保護アミノ酸を表す。
Fmoc:9−フルオレニルメチルオキシカルボニル
tBu:t−ブチル
Trt:トリチル
Pmc:2,2,5,7,8−ペンタメチルクロマン−6−スルフォニル
Fmoc−Arg(Pmc)−OH:Nα−9−フルオレニルメチルオキシカルボニル−Ng−2,2,5,7,8−ペンタメチルクロマン−6−スルホニル−L−アルギニン
Fmoc−Asp(OtBu)−OH:Nα−9−フルオレニルメチルオキシカルボニル−L−アスパラギン酸−β−t−ブチルエステル
Fmoc−His(Trt)−OH:Nα−9−フルオレニルメチルオキシカルボニル−Nim−トリチル−L−システイン
Fmoc−Lys(Boc)−OH:Nα−9−フルオレニルメチルオキシカルボニル−Nε−t−ブチルオキシカルボニル−L−リジン
Fmoc−Thr(tBu)−OH:Nα−9−フルオレニルメチルオキシカルボニル−O−t−ブチル−L−スレオニン
以下の略号は、対応する下記の反応溶媒、反応試薬等を表す。
HBTU:2−(1H−ベンゾトリアゾール−1−イル)−1,1,3,3−テトラメチルウロニウム・ヘキサフルオロホスフェート
HOBt:N−ヒドロキシベンゾトリアゾール
DIEA:ジイソプロピルエチルアミン
DMF:N,N−ジメチルホルムアミド
TFA:トリフルオロ酢酸
以下の実施例において、化合物の理化学的性質は次の方法により測定した。
質量分析は、日本電子JMS−HX110Aを用いFAB−MS法により行った。アミノ酸分析は、コーエン(Cohen,S.A.)らの方法〔アナリティカル・バイオケミストリー(Analytical Biochemistry),222,19(1994)〕により行った。加水分解は塩酸蒸気中110℃で20時間行い、加水分解物のアミノ酸組成はウォーターズ・アキュ・タグ(Waters AccQ−Tag)アミノ酸分析計(Waters社製)を用い分析した。
(2)化合物1(配列番号15で表されるアミノ酸配列からなるペプチドAc−Arg−Ala−Arg−His−Thr−Pro−Arg−Ala−His−Pro−Gly−His−Leu−His−Lys−Ala−Arg−Asp−Gly−Pro−Cys−OH)の合成
Fmoc−Cys(Trt)、22.8μmolが結合した担体樹脂〔H−Cys(Trt)−2−ClTrt resin樹脂、ノバビオケム社製〕40mgを自動合成機(島津製作所)の反応容器に入れ、600μlのDMFを加えて3分間攪拌し溶液を排出した後、島津製作所の合成プログラムに従い次の操作を行った。
(a)30%ピペリジン−DMF溶液500μlを加えて混合物を4分間攪拌し、該溶液を排出し、この操作をもう1回繰り返した。
(b)担体樹脂を600μlのDMFで1分間洗浄し、該溶液を排出し、この操作を5回繰り返した。
(c)Fmoc−Pro−OH(228μmol)、HBTU(228μmol)、HOBt 1水和物(228μmol)およびDIEA(684μmol)をDMF(1.12ml)中で3分間攪拌し、得られた溶液を樹脂に加えて混合物を30分間攪拌し、溶液を排出した。
(d)担体樹脂を600μlのDMFで1分間洗浄後溶液を排出し、これを5回繰り返した。
上記の工程により、Fmoc−Pro−Cys(Trt)を担体上に合成した。
次に、(a)(b)の工程の後、(c)の工程でFmoc−Gly−OHを用いて縮合反応を行い、(d)の洗浄工程を経て、Fmoc−Gly−Pro−Cys(Trt)を担体上に合成した。
以下、工程(c)において、Fmoc−Asp(OtBu)−OH、Fmoc−Arg(Pmc)−OH、Fmoc−Ala−OH、Fmoc−Lys(Boc)−OH、Fmoc−His(Trt)−OH、Fmoc−Leu−OH、Fmoc−His(Trt)−OH、Fmoc−Gly−OH、Fmoc−Pro−OH、Fmoc−His(Trt)−OH、Fmoc−Ala−OH、Fmoc−Arg(Pmc)−OH、Fmoc−Pro−OH、Fmoc−Thr(tBu)−OH、Fmoc−His(Trt)−OH、Fmoc−Arg(Pmc)−OH、Fmoc−Ala−OH、Fmoc−Arg(Pmc)−OHを順次用いて、(a)〜(d)を繰り返した後、(a)(b)の脱保護、洗浄工程を経て、メタノール、ブチルエーテルで順次洗浄し、減圧下12時間乾燥して、側鎖保護ペプチドの結合した担体樹脂を得た。次に得られた担体樹脂に対し次の(e)〜(g)の操作を行った。
(e)担体樹脂を800μlのDMFで1分間洗浄し、該溶液を排出し、この操作を3回繰り返した。
(f)無水酢酸(456μmol)及びDMF(500μl)を樹脂に加えて混合物を2時間攪拌し、溶液を排出した。
(g)担体樹脂を800μlのDMFで1分間洗浄し、該溶液を排出し、この操作を3回繰り返した。
この後、メタノール、ブチルエーテルで順次洗浄し、減圧下12時間乾燥して、N末端がアセチル化された側鎖保護ペプチドの結合した担体樹脂を得た。これに、TFA(82.5%)、チオアニソール(5%)、水(5%)、エチルメチルスルフィド(3%)、1,2−エタンジチオール(2.5%)およびチオフェノール(2%)からなる混合溶液1mlを加えて室温で8時間放置し、側鎖保護基を除去するとともに樹脂よりペプチドを切り出した。樹脂を濾別後、得られた溶液にエーテル約10mlを加え、生成した沈澱を遠心分離およびデカンテーションにより回収し、粗ペプチドとして66.6mgを取得した。この粗生成物全量を酢酸水溶液に溶解後、逆相カラム(資生堂製、CAPCELL PAK C18 30mmI.D.X 250mm)を用いたHPLCで精製した。0.1% TFA水溶液に、TFA 0.1%を含む90%アセトニトリル水溶液を加えていく直線濃度勾配法で溶出し、220nmで検出し、化合物1を含む画分を得た。この画分を凍結乾燥して、化合物1を23.0mg得た。
質量分析[FABMS];m/z=2413.0(M+H+)
アミノ酸分析;Asx 1.0(1),Gly 2.0(2),His 4.3(4),Arg 3.8(4),Thr 1.0(1),Ala 2.9(3),Pro 2.9(3),Leu 1.0(1),Lys 1.0(1),Cys 1.3(1)
(3)免疫原の調製
実施例9(2)で得られた化合物1は、免疫原性を高める目的で以下の方法でKLH(カルビオケム社)とのコンジュゲートを作製し、免疫原とした。すなわち、KLHをPBSに溶解して10mg/mlに調整し、1/10容量の25mg/ml MBS〔N−(m−Maleimidobenzoyloxy)succinimide;ナカライテスク社〕を滴下して30分撹拌反応させた。あらかじめPBSで平衡化したセファデックスG−25カラムなどのゲルろ過カラムでフリーのMBSを除いて得られたKLH−MBS 2.5mgを0.1mol/lリン酸ナトリウムバッファー(pH7.0)に溶解したペプチド1mgと混合し、室温で3時間、攪拌反応した。反応後、PBSで透析したペプチドを免疫原として用いた。
(4)動物の免疫と抗体産生細胞の調整
実施例9(3)で調製した化合物1のKLHコンジュゲート100μgを水酸化アルミニウムアジュバント〔Antibodies−A Laboratory Manual,Cold Spring Harbor Laboratory,p99、1988〕2mgおよび百日咳ワクチン(千葉県血清研究所製)1×109細胞とともに6週令雌Balb/cマウス3匹に投与した。投与2週間後より、KLHコンジュゲート100μgを1週間に1回、計4回投与した。該マウスの眼底静脈叢より採血し、その血清抗体価を以下に示す酵素免疫測定法で調べ、十分な抗体価を示したマウスにKLHコンジュゲート100μgを最終免疫し、その3日後に脾臓を摘出した。
脾臓をMEM(Minimum Essential Medium)培地(日水製薬社製)中で細断し、ピンセットでほぐし、遠心分離(250×g、5分間)した。得られた沈殿画分にトリス−塩化アンモニウム緩衝液(pH7.6)を添加し、1〜2分間処理することにより赤血球を除去した。得られた沈殿画分(細胞画分)をMEM培地で3回洗浄し、細胞融合に用いた。
(5)酵素免疫測定法(バインディングELISA)
アッセイ用の抗原には実施例9(2)で得られた化合物1をサイログロブリン(以下、THYと略す。)とコンジュゲートしたものを用いた。作製方法は実施例9(3)に記した通りであるが、架橋剤にはMBSの代わりにSMCC〔4−(N−Maleimidomethyl)−cyclohexane−1−carboxylic acid N−hydroxysuccinimidoester;シグマ社〕を用いた。96穴のEIA用プレート(グライナー社)に、上記のように調製したコンジュゲートを5μg/ml、50μl/穴で分注し、4℃で一晩放置して吸着させた。該プレートを洗浄後、1% 牛血清アルブミン(BSA)/ダルベッコリン酸バッファー(Phosphate buffered saline:PBS)を100μl/穴加え、室温で1時間放置し、残っている活性基をブロックした。
放置後、1% BSA/PBSを捨て、該プレートに一次抗体として被免疫マウス抗血清、ハイブリドーマ培養上清を50μl/穴で分注し、2時間放置した。該プレートをTween−PBSで洗浄後、2次抗体としてペルオキシダーゼ標識ウサギ抗マウスイムノグロブリン(ダコ社)を50μl/穴で加えて室温、1時間放置した。該プレートをTween−PBSで洗浄後、ABTS基質液〔2.2−アジノビス(3−エチルベンゾチアゾール−6−スルホン酸)アンモニウム、1mmoL/L ABTS/0.1moL/Lクエン酸バッファー(pH4.2)〕を添加し、発色させOD415nmの吸光度をプレートリーダー(Emax;Molecular Devices社)を用いて測定した。
(6)マウス骨髄腫細胞の調製
8−アザグアニン耐性マウス骨髄腫細胞株P3−X63Ag8U.1(P3−U1:ATCCより購入)を正常培地(10%ウシ胎児血清添加RPMI培地)で培養し、細胞融合時に2×107個以上の細胞を確保し、細胞融合に親株として供した。
(7)ハイブリドーマの作製
実施例9(4)で得られたマウス脾細胞と実施例9(6)で得られた骨髄腫細胞とを10:1になるよう混合し、遠心分離(250×g、5分間)した。得られた沈澱画分の細胞群をよくほぐした後、攪拌しながら、37℃で、ポリエチレングリコール−1000(PEG−1000)1g、MEM培地1mlおよびジメチルスルホキシド0.35mlの混液を108個のマウス脾細胞あたり0.5ml加え、該懸濁液に1〜2分間毎にMEM培地1mlを数回加えた後、MEM培地を加えて全量が50mlになるようにした。
該懸濁液を遠心分離(900rpm、5分間)し、得られた沈澱画分の細胞をゆるやかにほぐした後、該細胞を、メスピペットによる吸込み吸出しでゆるやかにHAT培地〔10%ウシ胎児血清添加RPMI培地にHAT Media Supplement(インビトロジェン社製)を加えた培地〕100mL中に懸濁した。該懸濁液を96穴培養用プレートに200μl/穴ずつ分注し、5%CO2インキュベーター中、37℃で10〜14日間培養した。
培養後、培養上清を実施例9(5)に記載した酵素免疫測定法で調べ、化合物1に反応して陰性対照化合物に反応しない穴を選び、そこに含まれる細胞から限界希釈法によるクローニングを2回繰り返し、抗C−NT2RI2004312モノクローナル抗体産生ハイブリドーマ株KM3103を確立した。
抗C−NT2RI2004312モノクローナル抗体KM3103を産生するハイブリドーマ細胞株は、平成14年3月26日付けで独立行政法人産業技術総合研究所特許寄託センター(日本国茨城県つくば市東1丁目1番地1 中央第6:郵便番号305−8566)にFERM BP−7977として寄託されている。
(8)モノクローナル抗体の精製
プリスタン処理した8週令ヌード雌マウス(BALB/c)に実施例9(7)で得られたハイブリドーマ株を5〜20×106細胞/匹それぞれ腹腔内注射した。10〜21日後、ハイブリドーマが腹水癌化することにより腹水のたまったマウスから、腹水を採取(1〜8ml/匹)した。
該腹水を遠心分離(1200×g、5分間)し固形分を除去した。精製IgGモノクローナル抗体は、カプリル酸沈殿法〔Antibodies−A Laboratory Manual,Cold Spring Harbor Laboratory(1988)〕により精製することにより取得した。モノクローナル抗体のサブクラスはサブクラスタイピングキットを用いたELISA法により決定した。図3に示すようにKM3103のサブクラスはIgG1であった。
(9)モノクローナル抗体の反応性の検討(バインディングELISA)
実施例9(5)に示した方法に従って行なった。ただし、1次抗体には実施例9(8)で得られたKM3103を10μg/mlから10倍希釈で5段階に希釈したものを用いた。結果を図4に示す。KM3103は濃度依存的に化合物1に特異的な反応性を示した。
[実施例10] モノクローナル抗体KM3103を用いたC−NT2RI2004312の完全長cDNAがコードする蛋白質のウエスタンブロッティングによる検出
実施例8で調製したCOS−1/mock株、COS−1/C−NT2RI2004312株、およびCOS−1/C−NT2RI2004312h株の培養上清10μl、または細胞破砕液2.5μlを、実施例8(3)(c)の記載に従い、検出抗体として実施例9で取得したモノクローナル抗体KM3103を用いてウエスタンブロッティングを行った。
抗C−NT2RI2004312モノクローナル抗体KM3103は、実施例8に記載したCOS−1細胞で発現させたC−NT2RI2004312の完全長cDNAがコードする蛋白質を特異的に認識することが分かった(図5)。
[実施例11] C−NT2RI2004312の完全長cDNAがコードする蛋白質の癌細胞増殖に及ぼす効果
C−NT2RI2004312の完全長cDNAがコードする蛋白質の癌細胞増殖への作用を調べるため、実施例8(3)(a)で取得したCOS−1/mock株、COS−1/C−NT2RI2004312株、およびCOS−1/C−NT2RI2004312h株の培養上清を用い、ヒト大腸癌細胞株HT−29(ATCC HTB−38)のIGF依存的な増殖に与える影響を以下のように検討した。
ウシ血清アルブミン(ギブコ社製)200μg/ml、ヒトトランスフェリン(ギブコ社製)10μg/mlを添加したD−MEM/F−12培地(ギブコ社製)中に1×105個/mlに調製したHT−29細胞を、96ウェルプレートに50μl/wellずつ播種し、37℃のCO2インキュベーター中で3時間培養した。次いで、ヒトIGF−I(Peprotech社製)、またはヒトIGF−II(Peprotech社製)をそれぞれ10ng/mlとなるように添加した。続いてCOS−1/mock株、COS−1/C−NT2RI2004312株、またはCOS−1/C−NT2RI2004312h株の培養上清を5μl/wellずつ添加して、さらに37℃のCO2インキュベーター中で5日間培養した後、生細胞数をCell Proliferation Reagent WST−1(ロシュ・ダイアグノスティックス社製)により測定した。
COS−1/C−NT2RI2004312株およびCOS−1/C−NT2RI2004312h株の培養上清を加えた試験区では、ヒト大腸癌細胞株HT−29細胞のIGF依存的な増殖を有意に阻害することが示された(図6)。
産業上の利用の可能性
本発明によれば、新規インスリン様増殖因子結合蛋白質、該蛋白質をコードするDNA、該蛋白質を認識する抗体、および該蛋白質が関与する疾患の判定法、診断薬、予防薬および治療薬が提供できる。
配列表フリーテキスト
配列番号3−人工配列の説明:合成RNA
配列番号4−人工配列の説明:合成DNA
配列番号5−人工配列の説明:合成DNA
配列番号6−人工配列の説明:合成DNA
配列番号8−人工配列の説明:合成DNA
配列番号9−人工配列の説明:合成DNA
配列番号10−人工配列の説明:合成DNA
配列番号11−人工配列の説明:合成DNA
配列番号12−人工配列の説明:合成DNA
配列番号13−人工配列の説明:合成DNA
配列番号14−人工配列の説明:合成DNA
配列番号15−人工配列の説明:合成ペプチド
配列番号16−人工配列の説明:合成DNA
配列番号17−人工配列の説明:合成DNA
配列番号18−人工配列の説明:合成DNA
配列番号19−人工配列の説明:合成DNA
【配列表】
【図面の簡単な説明】
図1は、C−NT2RI2004312の完全長cDNAがコードする蛋白質とIGFBPファミリー因子とのアミノ酸配列の比較を示す図である。なお、図中のIGFBP−1、IGFBP−2、IGFBP−3、IGFBP−4、IGFBP−5、IGFBP−6、IGFBP−7は、それぞれヒトIGFBP−1、ヒトIGFBP−2、ヒトIGFBP−3、ヒトIGFBP−4、ヒトIGFBP−5、ヒトIGFBP−6、ヒトIGFBP−7を指す。また、C−NT2RI2004312の完全長cDNAがコードする蛋白質とインスリン様増殖因子結合蛋白質スーパーファミリー間で保存されているアミノ酸配列を白抜きで示し、ファミリー間で高く保存されているシステイン残基に*印を記した。
図2は、COS−1細胞を宿主としたC−NT2RI2004312がコードする蛋白質の発現を示す写真である。mockは対照を表す。
図3は、サブクラスタイピングキットを用いたELISA法によるモノクローナル抗体KM3103のサブクラスを示す図である。左カラムがP3−U1、右カラムがモノクローナル抗体KM3103の結果を示す。各カラムは左から、全サブクラス、G1、G2a、G2b、G3、Mを認識する抗体を用いた試験区を示す。
図4は、バインディングELISAによりモノクローナル抗体KM3103とモノクローナル抗体KM3103の抗原ペプチドとの反応性を示す図である。図中、●は化合物1を加えた試験区を、○はコントロール化合物を加えた試験区を示す。
図5は、KM3103を用いたウエスタンブロッティングによるC−NT2RI2004312がコードする蛋白質の検出を示す写真である。mockは対照を表す。
図6は、HT−29細胞のIGF依存的な増殖に及ぼすC−NT2RI2004312がコードするタンパク質の影響を示す図である。mockは対照を表す。 Technical field
The present invention relates to a novel insulin-like growth factor binding protein, a DNA encoding the protein, an antibody recognizing the protein, and a method for determining a disease associated with the protein, a diagnostic agent, a prophylactic agent or a therapeutic agent.
Background art
Insulin-like growth factor binding protein (hereinafter, referred to as "IGFBP") is a polymer complex of insulin-like growth factor (hereinafter, referred to as "IGF") in a body fluid. As a group of molecules discovered from the existence of the compound, 10 types of molecules from IGFBP-1 to IGFBP-1 to 10 have been reported to date, and it is known that they form a superfamily [Endocr]. . Rev .. ,18, 801 (1997), Prog. Growth Factor Res. ,3, 243 (1991), Mol. Reprod. Dev. ,35368 (1993), Proc. Natl. Acad. Sci. USA,94, 12981 (1997)].
As a function of IGFBP, it has been suggested that there is a direct action by binding to integrin, but the main action is exerted by binding to IGF or insulin and regulating its activity, distribution, metabolism, etc. [Endocr. Rev .. ,18, 801 (1997), BioScience Term Library Cytokine / Growth Factor Revised Edition, p14-17 (1988)], specifically, IGFBP suppresses the transport and degradation of IGF into and out of blood and to the receptor. It is thought that it exerts its function by regulating the binding of.
Of the IGFBP superfamily, six types of molecules, IGFBP-1 to IGFBP-6, are structurally similar and bind to IGF with higher affinity than insulin. [Mol. Endocrinol. ,2, 404 (1988); ,8, 2497 (1989), Mol. Endocrinol. ,2, 1176 (1989), Mol. Endocrinol. ,4, 1806 (1990), Biochem. Biophys. Res. Commun. ,176, 219 (1991); Biol. Chem. ,266, 9043 (1991); Biol. Chem. ,266, 10646 (1991)].
Amino acid sequence homology between IGF high affinity IGFBP subfamily molecules is 49% -60% in humans. In addition, 18 kinds of cysteine residues are conserved in 5 kinds of IGFBPs except for IGFBP-6. Of these, 3 of the N-terminals are represented by Gly-Cys-Gly-Cys-Cys-XX-Cys. [X represents an arbitrary amino acid, and the homologous sequence forms an insulin-like growth factor binding motif (hereinafter, also referred to as “IGFBP motif”)], and may be involved in IGF binding. [Prog. Growth Factor Res. ,3, 243 (1991)].
It is known that IGFBP-1 and 3 both suppress and promote the action of IGF. IGFBP-2, 4, and 6 are inhibitory, and IGFBP-5 is an promoting IGF-binding protein. IGF is produced in the liver and bone tissue in a growth hormone-dependent manner and functions as a growth factor to promote physical growth. IGF-I is expressed in large amounts in the central nervous system, bone tissue, etc. IGF-II, which is presumed to play an important role in the growth of anaphase, exists, but IGFBP-1, 3, 4 show the same binding activity to IGF-I and IGF-II, and IGFBP-2, It is known that 5 and 6 mainly show strong binding activity to IGF-II.
The tissue distribution of IGF high-affinity IGFBP subfamily molecules is different for each molecule. IGFBP-1 is mainly present in amniotic fluid and fetal serum, IGFBP-2 is mainly present in fetal liver and adult brain, and IGFBP -3 is mainly present in liver and serum, IGFBP-4 is mainly present in renal glomeruli, skin and intestinal epithelium, IGFBP-5 is mainly present in intestinal epithelium and bone, and IGFBP-6 is mainly present in skin and heart Is known to be.
Regarding the relationship between IGFBP and disease states, the following findings have been reported.
It is known that in patients with dwarfism and acromegaly, fluctuations in the expression of IGF and IGFBP are directly involved in the pathophysiology. Growth disorders often occur in children with chronic renal failure, but the expression levels of growth hormone and IGF are normal, and it is known that most of them are caused by impaired IGF function due to an increase in IGFBP-2 or 3. [Miner. Electrolyte Mrtab. ,18, 320 (1992)]. IGFBP-4 and 5 have important functions in bone metabolism, and the expression level of IGFBP-5 is reduced in osteoporosis, and the expression of IGFBP-4 is increased in elderly female fracture patients with elevated parathyroid hormone It has been reported that Although an increase in the paracrine action of IGF-I has been observed in compensatory hypertrophy after nephrectomy or small intestine resection, the amount of IGF-I mRNA does not change, and free IGFBP-3 is reduced due to a decrease in IGFBP-3 expression. It has been reported that IGF-I is increased [J. Fuller; Bailieres Clin. Endocrinol. Metab. ,8, 165 (1994)]. Furthermore, it has been reported that the expression of IGFBP-1 is lower in endometriotic malignant tumors than in benign tumors [Growth Regul. ,3, 74, (1993)].
On the other hand, among the GFBP superfamily, four types of molecules, IGFBP-7 to IGFBP-7, are considered to be structurally similar and have the common property of having a low affinity for IGF. Has been classified into another subfamily as sex IGFBP [Proc. Natl. Acad. Sci. USA,94, 12981 (1997), Cancer Res. ,59, 2787 (1999)].
Various physiological activities of IGFBP-7 have been reported, and many reports have been made especially on the relationship between cancer and its pathological condition.
IGFBP-7 is upregulated in aged human epithelial cells [J. Clin. Endocrinol. Metab. ,4, 715 (1993)] On the other hand, since expression is reduced in carcinoma cell lines and the like, it is thought that they may have a function as a tumor suppressor activity gene [Proc. Natl. Acad. Sci. USA,92, 4472 (1995)].
The locus of IGFBP-7 on the human chromosome is 4q12, and its expression is also known to increase when human epithelial cells are treated with retinoic acid [Proc. Natl. Acad. Sci. USA,92, 4472 (1995)].
In breast cancer tissue, LOH (loss of heterozygosity) is observed at a frequency of about 50% at the position of chromosome 4q12 to 13 and it has been confirmed that the expression of IGFBP-7 is reduced [Oncogene,16, 2459 (1998)]. It has been reported that the expression of IGFBP-7 is also reduced at the mRNA level in prostate cancer tissues and is not particularly detected in cell lines derived from malignant prostate cancer [J. Clin. Endocrinol. Metab. ,83, 4355 (1998)].
Furthermore, when IGFBP-7 was forcibly expressed in a prostate cancer cell line in which the expression of IGFBP-7 was reduced, the cell division time was prolonged, the ability to form colonies in a soft agar medium was reduced, and the ability to form tumors in nude mouse transplantation. And the increase in the apoptosis induction rate by drug treatment have been observed, suggesting a relationship between the expression of IGFBP-7 and the malignancy of prostate cancer [Cancer Res. ,59, 2370 (1999)].
In leukemia patients, it has been reported that the levels of IGFBP-7 and IGFBP-3 in the cerebrospinal fluid increase, and attention has been paid to the relationship with the pathology of leukemia [J. Clin. Endocrinol. Metab. ,84, 1283 (1999)].
In colorectal cancer, the expression of IGFBP-7 is increased in colorectal cancer tissues and colorectal cancer cell lines, and therefore, attention is paid to its relationship with the disease state [J. Gastroenterology,33, 213 (1998)].
It has been reported that IGFBP-7 expression is reduced in large uterine leiomyoma sites, and that IGFBP-7 expression is increased in patients who have been treated with gonadotropin-releasing hormone. J. Reprod. Immunol. ,43, 53 (2000)].
In mouse liver cancer cells induced by the SV40T antigen, since the 5 'upstream region of the IGFBP-7 gene is methylated and its expression level is reduced, the methylation of the gene is regulated by the regulation of IGFBP-7 expression accompanying canceration. Has been proposed [Biochem. Biophys. Res. Commun. ,267, 109 (2000)].
The relationship between IGFBP-7 and diabetes has also been reported.
It has been shown that a factor that acts on vascular endothelial cells and promotes the production of prostacyclin PGI2 (PGI2-stimulating factor, hereinafter abbreviated as “PSF”) is the same as IGFBP-7 [Biochem. J. ,303, 591, (1994)], and the factor expressed in vascular endothelial cells and smooth muscle cells [Throm Haemost. ,74, 1407 (1995)], and it has been reported that in a type I diabetes model by administration of streptozotocin, the expression is reduced in the kidney and sites of vascular injury [Diabetes,45, S111 (1996); Diabetes & its Complications,12, 252 (1998)]. Also, a decrease in IGFBP-7 expression has been observed at the protein level in coronary artery smooth muscle cells of patients with type II diabetes [Diabetes,46, 1627 (1997)]. Furthermore, it has been confirmed at the mRNA and protein levels that the expression level of IGFBP-7 decreases when bovine artery-derived smooth muscle cells are cultured in a high glucose medium [Diabetes,46, 1627 (1997), Diabetologia,41, 134 (1998)].
TGF-β (Transforming growth factor-β), parathyroid hormone (PTH) and prostaglandin E2 (PGE2) increase IGFBP-7 expression in osteoblasts, so that IGFBP-7 is physiologically expressed in osteoblasts. [Endocrinology,140, 1998 (1999)]. It has also been reported that treatment of osteoblasts with glucocorticoids suppresses IGF-I expression while increasing IGFBP-7 expression [Endocrinology,140, 228 (1999)].
In addition, it has been suggested that IGFBP-7 also affects differentiation into skeletal muscle by suppressing the differentiation promoting action of IGF [Exp. Cell Res. ,237, 192 (1997); Endocrinology,141, 100 (2000)].
Since IGFBP-7 was the same molecule as the factor PSF that promotes the production of prostacyclin PGI2, it is considered that some of the physiological functions of IGFBP-7 are exerted by using PGI2 as an effector molecule.
It is known that PGI2, a kind of prostaglandin, has a strong inhibitory action on platelet aggregation and a vasorelaxant action, and acts antagonistically with TXA2, which has an opposite action, and is involved in maintaining homeostasis in vivo. [Br. J. Pharmac. ,76, 3 (1982)], it is known that in thrombosis, arteriosclerosis, and the like, the imbalance in the production of TXA2 and PGI2, in particular, the production of PGI2 is reduced and vascular disorders occur [Br. J. Pharmac. ,76, 3 (1982)]. In the onset and progression of diabetic vascular disorders, in addition to enhancing the production of platelet-derived TXA2 [Thromb. Res. ,19, 211 (1980); Lab. Clin. Med. ,97, 87 (1981)], and it has been confirmed in diabetic patients and experimental diabetic animals that a decrease in the production of vascular-derived PGI2 causes an increase in platelet aggregation [Lancet,1, 325 (1979); Lancet,2, 1365 (1979); Engl. J. Med. ,300, 366 (1979), Life Sci. ,23, 351 (1978)].
PSF is a factor present in the bloodstream and stimulates PGI2 production in the blood vessel wall [Nature,271549 (1978)], and the factor is a hemolytic uremic syndrome [Lancet,2, 871 (1978)], thrombotic thrombocytopenic purpura [Lancet,2, 748 (1979)], sickle cell anemia [Br. J. Haematol. ,48545 (1981)], acute myocardial infarction [Coronary,2, 49 (1985)], diabetic vascular disorders [Metabolism,38, 837 (1989), Haemostasis,16, 447 (1986), Diab. Res. Clin. Pract. ,3, 243 (1987)], and it has been reported that blood levels are reduced in arteriosclerotic diseases.
That is, it has been clarified that IGFBP-7 exerts a platelet aggregation inhibitory action, a smooth muscle relaxing action, and a gastric acid secretion inhibitory action by promoting PGI2 production by vascular endothelial cells and increasing PGI2 concentration in blood.
As described above, factors belonging to the IGFBP superfamily regulate the functions of IGF and insulin, compensate for pregnancy and debilitating diseases, bone metabolism, differentiate skeletal muscle cells, promote PGI2 production on vascular endothelial cells, and mediate PGI2. It has been shown to be involved in various physiological phenomena such as reduced platelet assemblage, vascular smooth muscle relaxation, bronchial smooth muscle relaxation, and gastric acid secretion suppression. In addition, dwarfism, acromegaly, childhood chronic renal failure, osteoporosis, breast cancer, prostate cancer, acute leukemia, colorectal cancer, uterine leiomyoma, liver cancer, type I diabetes, type II diabetes, thrombosis, arteriosclerosis, It has also been shown to be involved in diseases such as hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, sickle cell anemia, acute myocardial infarction, and diabetic vascular disorder.
Therefore, a protein having IGFBP activity belonging to the IGFBP superfamily, a gene encoding the protein, an antisense DNA, and an antibody recognizing the protein can be used for diseases involving abnormal cell proliferation, diseases involving vascular disorders, and bone metabolism. Diseases with abnormalities, diseases with disorders of IGF or growth hormone action, diseases with abnormal smooth muscle cell differentiation and proliferation, diseases with abnormal skeletal muscle cell differentiation and proliferation, diseases with abnormal gastric acid secretion or abnormal It is thought that it can be used as a drug for determining, treating or preventing inflammatory diseases involving lymphocyte infiltration, and factors belonging to the IGFBP superfamily have attracted much attention as targets for the development of useful new drugs.
It is also recalled that there is a possibility that a novel factor belonging to the IGFBP superfamily exists. If a new IGFBP gene can be obtained, the amino acid sequence of the IGFBP can be compared with the amino acid sequence of a known IGFBP, or the transcript of the IGFBP gene can be obtained. By examining the expression distribution, the function of the IGFBP can be estimated and useful information for drug development can be obtained. Further, if a new IGFBP gene can be obtained, it will be possible to screen for a substance that suppresses the expression or function of the IGFBP. Compounds obtained by the screening are expected as useful pharmaceuticals.
Disclosure of the invention
The present invention provides a novel insulin-like growth factor binding protein, a DNA encoding the protein, an antibody recognizing the protein, a method for determining a disease involving the protein, a diagnostic agent, a prophylactic agent, and a therapeutic agent.
The present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in obtaining a novel insulin-like growth factor binding protein belonging to the IGFBP family and a DNA encoding the protein, thereby completing the present invention. Reached.
That is, the present invention provides the following (1) to (43).
(1) A protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
(2) In the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids consist of an amino acid sequence with deletion, substitution, insertion and / or addition, and are substantially identical to the protein of (1) Active protein.
(3) A protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having substantially the same activity as the protein of (1).
(4) A protein comprising a partial amino acid sequence including the amino acid sequence of amino acids 62 to 69 in the amino acid sequence represented by SEQ ID NO: 1, and having substantially the same activity as the protein of (1).
(5) In the amino acid sequence of the protein according to (4), the protein comprises an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added, and is substantially identical to the protein according to (1). A protein having the activity of
(6) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence of the protein of (4), and having substantially the same activity as the protein of (1).
(7) A DNA encoding the protein of any one of (1) to (6).
(8) DNA containing a coding region of the nucleotide sequence represented by SEQ ID NO: 2.
(9) It hybridizes under stringent conditions to a DNA encoding the protein according to any one of (1) to (6) or a DNA containing the coding region of the nucleotide sequence represented by SEQ ID NO: 2. And a DNA encoding a protein having substantially the same activity as the protein of (1).
(10) A recombinant DNA obtained by incorporating the DNA according to any one of (7) to (9) into a vector.
(11) A transformant obtained by introducing the recombinant DNA according to (10) into a host cell.
(12) The transformant according to (11), wherein the host cell is a cell selected from the group consisting of bacteria, yeast, insect cells, plant cells, and animal cells.
(13) The transformant according to (11) or (12) is cultured in a medium, and the protein according to any one of (1) to (6) is produced and accumulated in the culture, and the culture is cultured. The method for producing a protein according to any one of (1) to (6), wherein the protein is collected from the protein.
(14) An antibody that recognizes the protein according to any one of (1) to (6).
(15) A monoclonal antibody produced by a hybridoma having a deposit number of FERM BP-7977.
(16) Hybridoma FERM BP-7977.
(17) A method for immunologically detecting or quantifying the protein according to any one of (1) to (6), using the antibody according to (14) or (15).
(18) an oligonucleotide having a sequence consisting of 5 to 60 consecutive nucleotides in the nucleotide sequence of the DNA according to any one of (7) to (9), and an oligonucleotide having a sequence complementary to the oligonucleotide; Or derivatives of these oligonucleotides.
(19) (1) to (9) including performing hybridization using the DNA according to any one of (7) to (9) or the oligonucleotide or the oligonucleotide derivative according to (18) as a probe. A method for detecting or quantifying the expression of a gene encoding the protein according to any one of 6).
(20) The protein according to any one of (1) to (6), which comprises performing a polymerase chain reaction using the oligonucleotide or the oligonucleotide derivative according to (18) as a primer. A method for detecting the expression of a gene or a method for quantifying the amount of expression.
(21) (1) to (6), including performing hybridization using the DNA according to any one of (7) to (9) or the oligonucleotide or the oligonucleotide derivative according to (18). A method for detecting a mutation in a gene encoding the protein according to any one of the above.
(22) A gene encoding the protein according to any one of (1) to (6), which comprises performing a polymerase chain reaction using the oligonucleotide or the oligonucleotide derivative according to (18). A method for detecting mutations.
(23) A method for determining a disease according to the following (A) or (B).
(A) A method for determining a disease, comprising detecting a mutation in a DNA encoding the protein according to any one of (1) to (6) or measuring an expression level thereof, and comparing with a healthy person. .
(B) Using the antibody according to (14) or (15), detecting the mutation or measuring the expression level of the protein according to any one of (1) to (6), and comparing it with a healthy person. A method for determining a disease, comprising:
(24) The disease is a disease associated with abnormal cell proliferation, a disease associated with vascular disorders, a disease associated with abnormal bone metabolism, a disease associated with a disorder of insulin-like growth factor or growth hormone action, or abnormal differentiation and proliferation of smooth muscle cells. The method according to (23), wherein the disease is a disease associated with abnormal skeletal muscle cells, a disease associated with abnormal gastric acid secretion, or an inflammatory disease associated with abnormal lymphocyte infiltration.
(25) The disease associated with abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer, colorectal cancer, liver cancer, myeloma, uterine leiomyoma, brain tumor, malignant tumor or solid tumor, and a disease associated with vascular disorders. But myocardial infarction, cerebral infarction, peripheral vascular atresia, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenia Purpura, ischemic heart disease, ischemic brain disease, heart failure, congestion or choroidal circulatory disorder, a disease with abnormal bone metabolism is osteoporosis, and a disease with impaired insulin-like growth factor or growth hormone action is small. Patients with human disease, acromegaly or chronic renal failure in children; diseases with abnormal smooth muscle cell differentiation and proliferation are arteriosclerosis, bronchial disease or restenosis; diseases with abnormal skeletal muscle cell differentiation and proliferation are severe Myasthenia Yes, a disease with abnormal gastric acid secretion is gastric ulcer, an inflammatory disease with abnormal lymphocyte infiltration is microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin dependence (24) The method according to (24), which is diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway hyperreactivity or autoimmune disease.
(26) The determination method according to any one of (23) to (25), wherein the determination method is based on the method described in (A) or (B) below.
(A) The detection method according to (21) or (22).
(B) The detection method or the quantification method according to (17), (19) or (20).
(27) A medicament containing the protein according to any one of (1) to (6).
(28) A medicine comprising the DNA according to any one of (7) to (9), or the oligonucleotide or the oligonucleotide derivative according to (18).
(29) The medicament according to (28), wherein the medicament is a vector for gene prevention or a vector for gene therapy.
(30) A medicine containing the antibody according to (14) or (15).
(31) The medicine is a disease associated with abnormal cell proliferation, a disease associated with vascular disorders, a disease associated with abnormal bone metabolism, a disease associated with a disorder of insulin-like growth factor or growth hormone action, or abnormal proliferation and differentiation of smooth muscle cells (27) a diagnostic, prophylactic or therapeutic agent for a disease associated with abnormalities, a disease associated with abnormal differentiation and proliferation of skeletal muscle cells, a disease associated with abnormal gastric acid secretion, or an inflammatory disease associated with abnormal lymphocyte infiltration. -The medicine according to (30).
(32) The disease associated with abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer, colorectal cancer, liver cancer, myeloma, uterine leiomyoma, brain tumor, malignant tumor, or solid tumor. Myocardial infarction, cerebral infarction, peripheral vascular atresia, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura Disease, ischemic heart disease, ischemic brain disease, heart failure, congestion or choroidal circulatory disorder, a disease associated with abnormal bone metabolism is osteoporosis, and a disease associated with impaired insulin-like growth factor or growth hormone action is child. Disease, acromegaly or pediatric chronic renal failure, the disease with abnormal smooth muscle cell differentiation and proliferation is arteriosclerosis, bronchial disease or restenosis, and the disease with abnormal skeletal muscle cell differentiation and proliferation is severe muscle In asthenia And gastric ulcer disease associated with abnormal gastric acid secretion, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin dependence The medicament according to (31), which is inflammatory diabetes, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway hyperreactivity or an autoimmune disease.
(33) (i) bringing a test sample into contact with a cell expressing the protein according to any one of (1) to (6), and (ii) expressing the protein in a cell expressing the protein according to any one of (1) to (6); (1) selecting a compound that regulates the expression level of the protein according to any one of (1) to (6) from the test sample; A method for screening a compound that regulates the expression level of the protein according to any one of (1) to (6).
(34) (i) the function of a cell that expresses the protein according to any one of (1) to (6) and (ii) the function of a cell when a test sample is brought into contact with a cell that expresses the protein. And selecting a compound that controls the function of the protein according to any one of (1) to (6) from the test sample. A method for screening a compound that regulates the function of a protein described in the above item.
(35) A transformant transformed with a plasmid containing a reporter gene-linked DNA downstream of a region controlling transcription of the gene encoding the protein according to any one of (1) to (6). And a test sample, and selecting a compound that regulates the expression of the gene encoding the protein described in (1) to (6) from the test sample. A method for screening a compound that regulates the expression of a gene encoding the protein according to
(36) A compound obtained by the screening method according to any one of (33) to (35) or a pharmacologically acceptable salt thereof.
(37) A medicament comprising the compound according to (36) or a pharmacologically acceptable salt thereof.
(38) Diseases with abnormal cell proliferation, diseases with vascular disorders, diseases with abnormal bone metabolism, diseases with disorders of insulin-like growth factor or growth hormone action, diseases with abnormal smooth muscle cell differentiation and proliferation The medicament according to (37), which is a prophylactic or therapeutic agent for a disease associated with abnormal differentiation and proliferation of skeletal muscle cells, a disease associated with abnormal gastric acid secretion, or an inflammatory disease associated with abnormal lymphocyte infiltration.
(39) The disease associated with abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer, colorectal cancer, liver cancer, myeloma, uterine leiomyoma, brain tumor, malignant tumor, or solid tumor. Myocardial infarction, cerebral infarction, peripheral vascular atresia, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura Disease, ischemic heart disease, ischemic brain disease, heart failure, congestion or choroidal circulatory disorder, a disease associated with abnormal bone metabolism is osteoporosis, and a disease associated with impaired insulin-like growth factor or growth hormone action is child. Disease, acromegaly or pediatric chronic renal failure, disease with abnormal smooth muscle cell differentiation and proliferation is arteriosclerosis, bronchial disease or restenosis, and disease with abnormal skeletal muscle cell differentiation and proliferation is severe muscle In asthenia And gastric ulcer disease associated with abnormal gastric acid secretion, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin dependence The medicament according to (38), which is inflammatory diabetes, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway hyperreactivity or an autoimmune disease.
(40) The protein according to any one of (1) to (6) is brought into contact with a test sample, and a protein that specifically binds to the protein is selected from the test sample, (1). A method for screening a protein that specifically binds to the protein according to any one of (6) to (6).
(41) A protein that specifically binds to the protein according to any one of (1) to (6), which is obtained by the screening method according to (40).
(42) A knockout non-human animal in which the expression of the gene encoding the protein according to any one of (1) to (6) is reduced or completely suppressed.
(43) A knockout non-human animal in which the function of the protein according to any one of (1) to (6) is reduced or completely suppressed.
As the protein of the present invention,
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1
(B) an amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids are deleted, substituted, inserted and / or added, and are substantially identical to the protein described in (a). Active protein
(C) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having substantially the same activity as the protein described in (a)
(D) a protein comprising a partial amino acid sequence including the amino acid sequence of amino acids 62 to 69 in the amino acid sequence represented by SEQ ID NO: 1 and having substantially the same activity as the protein described in (a) above;
(E) an amino acid sequence representing the protein described in (d) above, wherein the amino acid sequence comprises an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added, and is substantially the same as the protein described in (a) above. Having the same activity
(F) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence representing the protein of (d) and having substantially the same activity as the protein of (a). be able to.
Factors belonging to the insulin-like growth factor binding protein superfamily are proteinaceous factors having an affinity for IGF or insulin and have an insulin-like growth factor binding motif (IGFBP motif). The motif is conserved in the N-terminal portion of a protein belonging to the insulin-like growth factor binding protein superfamily, and is a portion of a protein comprising an amino acid sequence represented by Gly-Cys-Gly-Cys-Cys-XX-Cys. (X represents any amino acid). Proteins belonging to the insulin-like growth factor binding protein superfamily have at least 10 cysteine residues conserved around the IGFBP motif and exhibit affinity for IGF or insulin via the IGFBP motif portion. In the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by amino acids 62 to 69 is an IGFBP motif.
Examples of the protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1 include a protein having the amino acid sequence represented by SEQ ID NO: 1 and a binding integrin, IGF, insulin or receptor. Proteins having the same protein in the body and the like can be mentioned. Substantially identical indicates that the activities are identical in nature. Therefore, quantitative factors such as the degree of binding activity and the molecular weight of the protein may be different.
The protein of the present invention comprises an amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, inserted and / or added, and comprises an amino acid sequence represented by SEQ ID NO: 1. Proteins having substantially the same activity as the protein are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular cloning, Molecular cloning, Color cloning, Second Edition). John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), ucleic Acids Research,10, 6487 (1982), Proc. Natl. Acad. Sci. , USA,79, 6409 (1982), Gene,34, 315 (1985), Nucleic Acids Research,Thirteen, 4431 (1985), Proc. Natl. Acad. Sci USA,82, 488 (1985), etc., for example, by introducing a site-specific mutation into a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. it can. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number thereof is not particularly limited. Deletion, substitution, and insertion can be performed by well-known techniques such as the site-directed mutagenesis described above. And / or a number that can be added, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. In addition, the present invention includes not only such artificially created mutants but also naturally occurring mutants.
The protein of the present invention has an amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been deleted, and has substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 Examples of the protein having (i) include a protein from which a signal peptide has been removed.
Also, a protein having 80% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 1 and having substantially the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1 is also the present invention. In order to have substantially the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1, the homology with the amino acid sequence of SEQ ID NO: 1 is at least 80% or more, preferably It is preferably at least 85%, more preferably at least 90%, further preferably at least 95%, particularly preferably at least 97%, most preferably at least 99%.
The identity of amino acid sequences and nucleotide sequences can be determined by the algorithm BLAST [Proc. Natl. Acad. Sci. USA,90, 5873-5877 (1993)]. Based on this algorithm, programs called BLASTN and BLASTX have been developed [Altschul et al. J. Mol. Biol. ,215, 403-410 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, parameters are set to, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence by BLASTX based on BLAST, parameters are set to, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).
The protein of the present invention is a protein having a partial amino acid sequence in the amino acid sequence of the protein of the present invention, and also includes a protein having substantially the same activity as the protein having the amino acid sequence of SEQ ID NO: 1.
In order to have substantially the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 1 comprises a partial amino acid sequence containing the amino acid sequence of amino acids 62 to 69. Preferably, it is a protein.
A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the protein is also a protein of the present invention. The protein is a protein in which the number of amino acids that can be introduced by the well-known site-directed mutagenesis method described above is deleted, substituted or added, and substantially the same as the protein having the amino acid sequence of SEQ ID NO: 1. Proteins having the same activity can be mentioned.
In addition, a protein having 80% or more homology with the protein described in (d) and having substantially the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1 is also a protein of the present invention. is there. In order to have substantially the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1, the homology with the amino acid sequence of the protein of (d) was calculated by using the BLAST program. In addition, it is at least 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.
As the DNA of the present invention,
(1) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 2
(2) DNA encoding the protein of the present invention described in (a), (b), (c), (d), (e) or (f) as defined above
(3) substantially hybridizing with the DNA of (2) or the DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and substantially having the protein of the amino acid sequence of SEQ ID NO: 1 And DNA encoding a protein having the same activity.
DNA that hybridizes under stringent conditions refers to, for example, a colony hybridization method, a plaque, using a DNA of the present invention such as a DNA having a base sequence represented by SEQ ID NO: 2 or a partial DNA fragment thereof as a probe. A DNA obtained by using a hybridization method, a Southern blot hybridization method, or the like, and specifically, 0.7 to 1.0 mol / mol using a filter on which DNA derived from colonies or plaques is immobilized. After performing hybridization at 65 ° C. in the presence of L sodium chloride, the SSC solution having a concentration of 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 times is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate) Filter at 65 ° C It can be mentioned DNA that can be identified by washing the. Hybridization is described in Molecular Cloning, Second Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Technologies, A Practical Approach, Second Edition, Oxford University (1995), and the like. It can be performed according to it. Specific examples of the hybridizable DNA include a DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 2, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. As described above, particularly preferred are DNAs having a homology of 95% or more, most preferably 98% or more.
Hereinafter, the present invention will be described in detail.
1. Preparation of DNA of the present invention
The DNA of the present invention can be prepared by isolating mRNA from human tissues, for example, mRNA from human brain, preparing a cDNA library thereof, and then screening the cDNA library to obtain a desired clone. it can.
Human brain mRNA may be a commercially available one (for example, manufactured by Clontech) or may be prepared from human brain tissue as described below. In the latter case, first, total RNA can be prepared from brain tissue, and mRNA can be isolated from the total RNA.
As a method for preparing total RNA from brain tissue, a guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology,154, 3 (1987)], guanidine acid thiocyanate-phenol-chloroform (AGPC) method [Analytical Biochemistry,162156 (1987), Experimental Medicine,9, 1937 (1991)]. Poly (A) from total RNA+Examples of a method for preparing mRNA as RNA include an oligo (dT) -immobilized cellulose column method (Molecular Cloning, 2nd edition) and the like. Alternatively, mRNA can be prepared by using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or Quick Prep mRNA Purification Kit (manufactured by Pharmacia).
A cDNA library is prepared from the prepared human brain tissue mRNA. As a method for preparing a cDNA library, a method described in Molecular Cloning, Second Edition, Current Protocols in Molecular Biology, or a commercially available kit, for example, a SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Lifestyle Cloning) Technologies), a method using ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE), and the like.
As a cloning vector for preparing a cDNA library, any phage vector, plasmid vector, or the like can be used as long as it can replicate autonomously in E. coli K12 strain. Specifically, ZAP Express [Stratagene, Strategies,5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research,17, 9494 (1989)], Lambda ZAP II (manufactured by STRATAGENE), λgt10, λgt11 [DNA cloning, A Practical Approach,1, 49 (1985)], λTriplEx (manufactured by Clontech), λExCell (manufactured by Pharmacia), pT7T318U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol. ,3, 280 (1983)] and pUC18 [Gene,33, 103 (1985)].
As the host microorganism, microorganisms belonging to the genus Escherichia (Escherichia), particularly Escherichia coli (Escherichia coliAny microorganism can be used as long as the microorganism belongs to E. coli. In particular,Escherichia coli XL1-Blue MRF '[STRATAGENE, Strategies,5, 81 (1992)],Escherichia coli C600 [Genetics,39, 440 (1954)],Escherichia coli Y1088 [Science,222, 778 (1983)],Escherichia coli Y1090 [Science,222, 778 (1983)],Escherichia coli NM522 [J. Mol. Biol. ,166, 1 (1983)],Escherichia coli K802 [J. Mol. Biol. ,16, 118 (1966)] andEscherichia coli JM105 [Gene,38, 275 (1985)].
This cDNA library may be used for the following analysis as it is, but in order to reduce the ratio of incomplete length cDNA and obtain full length cDNA as efficiently as possible, the oligocap method developed by Sugano et al. [Gene,138, 171 (1994), Gene,200149 (1997), protein nucleic acid enzymes,41, 603 (1996), Experimental Medicine,11, 2491 (1993), cDNA cloning, Yodosha (1996), method for preparing a gene library, Yodosha (1994)].
Each clone was isolated from the prepared cDNA library, and the base sequence of the cDNA of each clone was determined from the end by a conventional nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci. USA,74, 5463 (1977)] or an ABIPRISM 377 DNA sequencer (manufactured by PE Biosystems) to determine the base sequence of the DNA.
Whether the base sequence of each cDNA has a new sequence can be determined by searching a base sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST. This can be confirmed by the absence of a nucleotide sequence having a clear homology, which is considered to completely match the nucleotide sequence of the gene.
The nucleotide sequence of the cDNA containing the novel sequence obtained by the above method includes, for example, the nucleotide sequence represented by SEQ ID NO: 2.
A protein obtained by translating a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 (a protein consisting of the amino acid sequence represented by SEQ ID NO: 1) is BLAST2 [Nuc. Acid. Res. ,25, 3389 (1997)] and has a homology of 39% with human IGFBP-7, a protein belonging to the IGFBP superfamily.
In the IGFBP superfamily, it is known that the position of at least 10 cysteine residues centered on the IGFBP motif and the insulin-like growth factor binding motif portion important for binding to IGF or insulin are conserved. Also in the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, a portion corresponding to the insulin-like growth factor binding motif is Gly-Cys-Gly-Cys-Cys-X-, which is a typical insulin-like growth factor binding motif. There is an amino acid sequence consisting of Glu-Cys-Gly-Cys-Cys-Ala-Arg-Cys which is very similar to the amino acid sequence consisting of X-Cys (X represents an arbitrary amino acid). Ten cysteine residues are present in conserved positions among factors belonging to the IGFBP superfamily. Therefore, it can be seen that the protein having the amino acid sequence represented by SEQ ID NO: 1 has activity as a protein belonging to the IGFBP superfamily.
Regarding the relationship between proteins belonging to the IGFBP superfamily and diseases, diseases involving abnormal cell proliferation such as acute myeloid leukemia, breast cancer, prostate cancer, colorectal cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor, and solid tumor , Myocardial infarction, cerebral infarction, peripheral vascular obstruction, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenia Purpura, ischemic heart disease, ischemic brain disease, heart failure, congestion, diseases with vascular disorders such as choroidal circulatory disorders, diseases with abnormal bone metabolism such as osteoporosis, dwarfism, acromegaly, pediatric chronic kidney Insulin-like growth factors or disorders associated with impaired growth hormone action such as insufficiency, diseases involving abnormal proliferation and differentiation of abnormal smooth muscle cells such as arteriosclerosis, bronchial disease, restenosis, and abnormal skeletal muscle cells such as myasthenia gravis Increased differentiation Disease, gastric acid secretion abnormality such as gastric ulcer, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammation Since there have been reports on inflammatory diseases associated with abnormal lymphocyte infiltration such as sexual bowel disease, allergy, atopy, asthma, hay fever, airway irritability, and autoimmune disease, the protein of the present invention also has the above-mentioned disease. It is expected to be a relevant protein. These diseases may be caused, for example, by abnormal function or expression of the protein of the present invention. In this example, since the culture supernatant of cells expressing the protein of the present invention showed an activity of suppressing the growth of cancer cell lines, the protein of the present invention is associated with abnormal cell growth such as colorectal cancer. It is thought to be related to the disease.
In order to confirm that the molecule containing the nucleotide sequence represented by SEQ ID NO: 2 was not artificially generated during the production of the cDNA library, the human genomic library was subjected to the nucleotide sequence represented by SEQ ID NO: 2 Can be determined by screening using a sequence specific to the genomic clone and determining the nucleotide sequence of the obtained genomic clone.
As the human genome library, a commercially available one (for example, a BAC library manufactured by Research Genetics) may be used, or a method known per se from human cells or tissues [Genomics,29413 (1995); Genomics,24, 527 (1994), etc.].
As a method for screening a human genomic library using a sequence specific to the nucleotide sequence represented by SEQ ID NO: 2, a PCR method using primers specific to the nucleotide sequence represented by SEQ ID NO: 2 [PCR Protocols , Academic Press (1990)], colony hybridization using an oligonucleotide specific to the nucleotide sequence represented by SEQ ID NO: 2, and plaque hybridization method (Molecular Cloning, 2nd edition).
By the above method, a genomic DNA clone (for example, a human genomic BAC clone) containing both of the nucleotide sequences represented by SEQ ID NO: 2 is obtained. When the nucleotide sequence of this genomic DNA is determined and compared with the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide sequence represented by SEQ ID NO: 2 is divided into six parts and exists over about 18 kb on the same human genome. I understand. That is, it can be seen that the protein having the amino acid sequence represented by SEQ ID NO: 1 is composed of six exons, and was not artificially generated during the production of a cDNA library.
Once a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 has been obtained and its nucleotide sequence has been determined, primers based on the nucleotide sequences at the 5 ′ and 3 ′ ends of the nucleotide sequence are prepared, and human Alternatively, by using a cDNA or a cDNA library synthesized from mRNA contained in a tissue or a cell such as the brain of a non-human animal as a template, the DNA is amplified using a PCR method (PCR Protocols, Academic Press (1990)). The DNA of the present invention can be obtained.
Also, colony hybridization or cDNA library synthesis from mRNA contained in tissues or cells such as human or non-human animal brain using the full length or a part of the DNA represented by SEQ ID NO: 2 as a probe is performed. The DNA of the present invention can be obtained by performing plaque hybridization (Molecular Cloning, 2nd edition).
Based on the determined DNA base sequence, the DNA of the present invention can also be obtained by chemical synthesis using a DNA synthesizer such as Perkin-Elmer DNA synthesizer model 392 using the phosphoramidite method. .
The obtained DNA is expressed by using a transformant obtained by introducing a recombinant vector containing the DNA into a host cell, or by expressing the amino acid sequence encoded by the DNA with human IGFBP-1, human IGFBP. -2, homology with the amino acid sequence of human IGFBP-3, human IGFBP-4, human IGFBP-5, human IGFBP-6, human IGFBP-7, human IGFBP-8, human IGFBP-9 or human IGFBP-10; By comparing the positions of cysteine residues, it can be confirmed that the DNA is a DNA encoding a protein having activity as an IGFBP superfamily.
Based on the information on the nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 2 or a fragment thereof, the nucleotide sequence of the DNA of the present invention, for example, the nucleotide sequence represented by SEQ ID NO: 2 can be obtained by a conventional method or by using a DNA synthesizer. Among them, an oligonucleotide having a sequence corresponding to continuous 5 to 60 bases, preferably 10 to 40 bases or an oligonucleotide corresponding to a sequence complementary to the oligonucleotide (hereinafter referred to as antisense oligonucleotide) is prepared. I can do it.
Examples of the oligonucleotide of the present invention include oligonucleotides such as oligo DNA and oligo RNA, and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
Examples of the oligonucleotide or antisense oligonucleotide include, for example, a sense primer corresponding to a 5 ′ terminal nucleotide sequence and a 3 ′ terminal nucleotide sequence in a partial base sequence of mRNA to be detected. Etc. can be given. However, the base corresponding to uracil in mRNA becomes thymidine in the oligonucleotide primer.
Examples of the sense primer and the antisense primer include oligonucleotides whose melting temperature (Tm) and the number of bases of both do not extremely change and have 5 to 60 bases, preferably 10 to 50 bases.
Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphoric diester bond in an oligonucleotide converted to an N3′-P5 ′ phosphoramidate bond. Oligonucleotide derivative, an oligonucleotide derivative in which the ribose and phosphodiester bond in the oligonucleotide were converted to a peptide nucleic acid bond, an oligonucleotide derivative in which uracil in the oligonucleotide was substituted with C-5 propynyluracil, an oligonucleotide derivative in the oligonucleotide Oligonucleotide derivatives in which uracil is substituted with C-5 thiazoleuracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with C-5 propynylcytosine, oligonucleotide Is an oligonucleotide derivative in which cytosine is substituted by phenoxazine-modified cytosine, an oligonucleotide derivative in which ribose in the oligonucleotide is substituted by 2′-O-propyl ribose, or ribose in the oligonucleotide is 2 Oligonucleotide derivatives substituted with '-methoxyethoxyribose [cell engineering,16, 1463 (1997)].
2. Production of the protein of the present invention
The protein of the present invention can be obtained by expressing the DNA of the present invention in host cells by, for example, the following method using the method described in Molecular Cloning Second Edition, Current Protocols in Molecular Biology, and the like. Then, it can be manufactured.
Based on the full-length cDNA, if necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
A recombinant vector is prepared by inserting the DNA fragment or full-length cDNA downstream of the promoter of an appropriate expression vector.
By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant producing the protein of the present invention can be obtained.
As the host cell, any cell that can express the gene of interest, such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
As the expression vector, those which are capable of autonomous replication in the above-mentioned host cells or capable of being integrated into a chromosome, and which contain a promoter at a position where the DNA encoding the protein of the present invention can be transcribed are used.
When a prokaryote such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryote, and has a promoter, ribosome binding sequence, It is preferable that the vector be composed of a gene encoding the above protein and a transcription termination sequence. A gene that controls the promoter may be included.
Examples of expression vectors include pBTrp2, pBTac1, and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8. (Manufactured by QIAGEN), pKYP10 (Japanese Unexamined Patent Application Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry,48, 669 (1984)], pLSA1 [Agric. Biol. Chem. ,53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA,82, 4306 (1985)], pBluescript II SK (-) (manufactured by Stratagene), pTrs30 [Escherichia coli Prepared from JM109 / pTrS30 (FERM BP-5407)], pTrs32 [Escherichia coli Prepared from JM109 / pTrS32 (FERM BP-5408)], pGHA2 [Escherichia coli Prepared from IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [Escherichia coli Prepared from IGKA2 (FERM BP-6798), JP-A-60-221091], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. ,172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), pSupex, and the like.
Any promoter can be used as long as it can be expressed in a host cell. For example,trpPromoter (Ptrp),lacPromoter, PLPromoter, PRExamples of the promoter include promoters derived from Escherichia coli and phages, such as a promoter and a T7 promoter. Also PtrpPromoter (Ptrp× 2),tacArtificially designed and modified promoters such as a promoter, a lacT7 promoter, and a let I promoter can also be used.
It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
By substituting the base sequence of the portion encoding the protein of the present invention so that the codon is optimal for host expression, the production rate of the target protein can be improved.
In the recombinant vector of the present invention, a transcription termination sequence is not always necessary for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
As host cells, microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example,Escherichia coli XL1-Blue,Escherichia coli XL2-Blue,Escherichia coli DH1,Escherichia coli MC1000,Escherichia coli KY3276,Escherichia coli W1485,Escherichia coli JM109,Escherichia coli HB101,Escherichia coli No. 49,Escherichia coli W3110,Escherichia coli NY49,Serratia ficaria,Serratia fonticola,Serratia likefaciens,Serratia marcescens,Bacillus subtilis,Bacillus amyloliquefaciens,Brevibacterium immariophilum, ATCC14068,Brevibacterium saccharolyticum ATCC 14066,Brevibacterium flavum ATCC 14067,Brevibacterium amoniagenes,Brevibacterium lactofermentum ATCC 13869,Corynebacterium glutamicum ATCC 13032,Corynebacterium acetoacidophilum ATCC 13870,Microbacterium ammonophilum ATCC 15354,Pseudomonas sp. D-0110 and the like.
Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], the protoplast method (JP-A-63-248394), or Gene,17, 107 (1982) and Molecular & General Genetics,168, 111 (1979).
When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), and the like.
As the promoter, any promoter can be used as long as it can be expressed in a yeast strain. For example, promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter,
As the host cells, microorganisms belonging to the genera Saccharomyces, Kluyveromyces, Trichosporone, Schwaniomyces, for example,Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis,Trichosporon pullulans,Schwanniomyces alluviusEtc. can be given.
As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, an electroporation method [Methods. Enzymol. ,194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA,84, 1929 (1978)], lithium acetate method [J. Bacteriology,153163 (1983)], Proc. Natl. Acad. Sci. USA,75, 1929 (1978).
When an animal cell is used as a host, as an expression vector, for example, pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Unexamined Patent Application Publication No. 3-22979;3, 133, (1990)], pAS3-3 (Japanese Unexamined Patent Application Publication No. 2-227075), pCDM8 [Nature,329840, (1987)], pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry,101, 1307 (1987)] and pAGE210.
Any promoter can be used as long as it can be expressed in animal cells. Examples thereof include a cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, and metallothionein. Promoter, heat shock promoter, SRα promoter and the like. Further, the enhancer of the IE gene of human CMV may be used together with the promoter.
Examples of the host cell include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 (Japanese Patent Application Laid-Open No. 63-299).
As a method for introducing a recombinant vector, any method for introducing DNA into animal cells can be used. For example, an electroporation method [Cytotechnology,3, 133 (1990)], the calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), the lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)].
When insect cells are used as a host, for example, current protocols in molecular biology, Baculovirus Expression Vectors, A Laboratory Manual, W.M. H. Freeman and Company, New York (1992), Bio / Technology,6, 47 (1988) and the like.
That is, after a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus in an insect cell culture supernatant, the recombinant virus is further infected into insect cells to express proteins. it can.
Examples of the gene transfer vector used in this method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
As the baculovirus, for example, Autographa californica nuclea polyhedrosis virus (Autographa californica nuclear polyhedrosis virus), which is a virus that infects night moth insects, can be used.
As insect cells,Spodoptera frugiperdaAnd Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, W.C. H. Freeman and Company, New York (1992)],Trichoplusia niHigh 5 (manufactured by Invitrogen), which is an ovarian cell, can be used.
Examples of a method of co-introducing the above-described recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include, for example, a calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), a lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)].
When a plant cell is used as a host cell, examples of an expression vector include a Ti plasmid and a tobacco mosaic virus vector.
Any promoter may be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and
Examples of host cells include plant cells of tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, and the like.
As a method for introducing a recombinant vector, any method for introducing DNA into a plant cell can be used. For example, Agrobacterium (Agrobacterium(JP-A-59-140885, JP-A-60-70080, WO94 / 00977), an electroporation method (JP-A-60-251887), and a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813).
As a method for expressing a gene, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, etc., in addition to direct expression.
When expressed by yeast, animal cells, insect cells, or plant cells, a protein having a sugar or a sugar chain added thereto can be obtained.
The protein of the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the protein of the present invention in the culture, and collecting from the culture. The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture C.
The carbon source may be any assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, organic acids such as propionic acid, ethanol, and the like. Alcohols such as propanol can be used.
Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, a soybean meal hydrolyzate, various fermentation cells and digests thereof can be used.
As the inorganic salt, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, man sulfate, copper sulfate, calcium carbonate, and the like can be used.
The culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 40 ° C., and the culturing time is usually 16 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example,lacWhen culturing a microorganism transformed with a recombinant vector using a promoter, isopropyl-β-D-thiogalactopyranoside or the like is used.trpWhen culturing a microorganism transformed with a recombinant vector using a promoter, indoleacrylic acid or the like may be added to the medium.
As a medium for culturing a transformant obtained using animal cells as a host, a commonly used RPMI1640 medium [The Journal of the American Medical Association,199, 519 (1967)], Eagle's MEM medium [Science,122, 501 (1952)], Dulbecco's modified MEM medium [Virology,8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine,73, 1 (1950)], or a medium obtained by adding fetal bovine serum or the like to such a medium.
Culture is usually performed at pH 6-8, 30-40 ° C, and 5% CO2.2It is carried out for 1 to 7 days under conditions such as presence.
If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
As a medium for culturing a transformant obtained using an insect cell as a host, generally used TNM-FH medium (manufactured by Pharmingen), Sf-900II SFM medium (manufactured by Life Technologies), ExCell400, ExCell405 ( Both are manufactured by JRH Biosciences), Grace's Insect Medium [Grace, T .; C. C. , Nature,195, 788 (1962)].
The cultivation is usually performed under conditions of
If necessary, an antibiotic such as gentamicin may be added to the medium during the culturing.
A transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ. As a medium for culturing the transformant, a commonly used Murashige and Skoog (MS) medium, a white (White) medium, or a medium obtained by adding a plant hormone such as auxin or cytokinin to such a medium is used. be able to.
The cultivation is usually performed at pH 5 to 9, 20 to 40 ° C for 3 to 60 days.
If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
As described above, a transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector into which a DNA encoding the protein of the present invention has been incorporated is cultured according to a conventional culture method to produce and accumulate the protein. Then, the protein can be produced by collecting the protein from the culture.
As a method for expressing a gene, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, etc., in addition to direct expression.
The method of producing the protein of the present invention includes a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the host cell outer membrane. The method can be selected by changing.
When the protein of the present invention is produced in a host cell or on a host cell outer membrane, the method of Paulson et al. [J. Biol. Chem. ,26417619 (1989)] and the method of Lowe et al. [Proc. Natl. Acad. Sci. , USA,86, 8227 (1989), Genes Developer. ,4, 1288 (1990)], or the method described in JP-A-5-336963, WO94 / 23021, etc., can be used to positively secrete the protein out of host cells.
That is, the protein of the present invention is expressed in a form in which a signal peptide is added before the protein containing the active site of the protein of the present invention using a gene recombination technique, whereby the protein of the present invention is actively secreted out of host cells. be able to.
Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
Further, by redifferentiating the cells of the transgenic animal or plant, an animal (transgenic non-human animal) or plant (transgenic plant) into which the gene has been transduced is created, and these individuals are used to create a book. The protein of the invention can also be produced.
When the transformant is an animal or plant individual, the protein is produced by breeding or cultivating the protein, accumulating and producing the protein, and collecting the protein from the animal or plant individual according to a usual method. be able to.
As a method for producing the protein of the present invention using an animal individual, for example, a known method [American Journal of Clinical Nutrition,63, 639S (1996), American Journal of Clinical Nutrition,63, 627S (1996), Bio / Technology,9830 (1991)] to produce the protein of the present invention in an animal constructed by introducing a gene.
In the case of an animal individual, for example, breeding a transgenic non-human animal into which the DNA encoding the protein of the present invention has been introduced, producing and accumulating the protein in the animal, and collecting the protein from the animal Thus, the protein can be produced. Examples of the production / accumulation site in the animal include milk of the animal (JP-A-63-309192), eggs, and the like. Any promoter that can be used in this case can be used as long as it can be expressed in animals. For example, α casein promoter, β casein promoter, β lactoglobulin promoter, whey acid A protein promoter or the like is preferably used.
As a method for producing the protein of the present invention using a plant individual, for example, a transgenic plant into which DNA encoding the protein of the present invention has been introduced can be prepared by a known method [tissue culture,20(1994), tissue culture,21(1995), Trends in Biotechnology,Fifteen, 45 (1997)], producing and accumulating the protein in the plant, and collecting the protein from the plant to produce the protein.
The protein produced by the transformant of the present invention is, for example, when the protein of the present invention is expressed in a dissolved state in a cell, after completion of the culture, the cell is recovered by centrifugation, and suspended in an aqueous buffer. Thereafter, the cells are crushed using an ultrasonic crusher, a French press, a Mantongaulin homogenizer, a Dynomill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting out method with ammonium sulfate, a desalting method, a precipitation method with an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) Chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. Alternatively, they can be used in combination to obtain a purified preparation.
When the protein is expressed by forming an insoluble form in the cells, the cells are similarly recovered, crushed, and centrifuged to collect the protein insoluble form as a precipitate fraction. The insoluble form of the recovered protein is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution to return the protein to a normal three-dimensional structure, a purified sample of the protein can be obtained by the same isolation and purification method as described above.
When the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant. That is, the culture is treated by a technique such as centrifugation as described above to obtain a soluble fraction, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.
Examples of the protein thus obtained include a protein having the amino acid sequence represented by SEQ ID NO: 1.
The protein of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Chemical synthesis can also be performed using a peptide synthesizer such as Advanced ChemTech, Perkin-Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, etc.
3. Preparation of an antibody that recognizes the protein of the present invention
The protein of the present invention, such as a polyclonal antibody or a monoclonal antibody, can be obtained by using the protein of the present invention, a purified preparation of a partial fragment polypeptide of the protein, or a peptide having a partial amino acid sequence of the protein of the present invention as an antigen. Can be prepared.
(1) Preparation of polyclonal antibody
Using the protein of the present invention, a purified preparation of a partial fragment polypeptide of the protein, or a peptide having a partial amino acid sequence of the protein of the present invention as an antigen, a polyclonal antibody can be produced by administering the antibody to an animal. it can.
Rabbits, goats, rats, mice, hamsters and the like can be used as animals to be administered.
The dose of the antigen is preferably 50 to 100 μg per animal.
When a peptide is used, it is desirable that the antigen be a covalently bound peptide to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin. A peptide serving as an antigen can be synthesized by a peptide synthesizer.
The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus
A polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, and caprylic acid precipitation (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)), or a DEAE-Sepharose column, anion exchange column. , Chromatography using a protein A or G-column or a gel filtration column, etc., alone or in combination.
(2) Preparation of monoclonal antibody
(A) Preparation of antibody-producing cells
Rats whose serum shows a sufficient antibody titer against the partial fragment polypeptide of the protein of the present invention used for immunization are used as a source of antibody-producing cells.
Three to seven days after the final administration of the antigenic substance to the rat showing the antibody titer, the spleen is removed.
The spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
The spleen cells in the obtained precipitate fraction are treated with a Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, and then washed three times with a MEM medium. Use as cells.
(B) Preparation of myeloma cells
As myeloma cells, cell lines obtained from mice or rats are used. For example, an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol. ,81, 1 (1978), Europ. J. Immunol. ,6, 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature,276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol. ,123, 1548 (1979)], P3-X63-Ag8 (X63) [Nature,256, 495 (1975)]. These cell lines were prepared in an 8-azaguanine medium [glutamine (1.5 mmol / L), 2-mercaptoethanol (5 × 10 5 in RPMI-1640 medium).-5mol / L), gentamicin (10 μg / ml) and fetal calf serum (FCS) (manufactured by CSL, 10%) were further added with 8-azaguanine (15 μg / ml) in a medium (hereinafter referred to as a normal medium). Medium), and cultured in a
(C) Preparation of hybridoma
The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were combined with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of salt, and 1 liter of distilled water). , PH 7.2), and the cells are mixed so that the number of cells becomes 5-10: 1 (antibody-producing cells: myeloma cells). After centrifugation at 1,200 rpm for 5 minutes, the supernatant is discarded.
The cell group of the obtained precipitate fraction was loosened well, and the cell group was stirred at 37 ° C. for 10 hours.80.2 to 1 ml of a solution obtained by mixing 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM and 0.7 ml of dimethyl sulfoxide (DMSO) was added per antibody-producing cell, and the MEM medium was further added every 1 to 2 minutes. Add 1-2 ml several times. After the addition, a MEM medium is added to adjust the total volume to 50 ml. After centrifuging the preparation at 900 rpm for 5 minutes, the supernatant is discarded. The cells in the obtained precipitate fraction are loosened loosely, and then slowly sucked and blown out with a female pipette using a HAT medium [hypoxanthine (10% in normal medium).-4mol / L), thymidine (1.5 × 10-5mol / L) and aminopterin (4 × 10-7mol / L) to the medium.
The suspension was dispensed at 100 to 200 μl / well into a 96-well culture plate, and 5
After the cultivation, a part of the culture supernatant is taken and subjected to an enzyme immunoassay as described in Antibodies, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988), and the like to obtain a portion of the protein of the present invention. A hybridoma that specifically reacts with the fragment polypeptide is selected.
The following method can be given as a specific example of the enzyme immunoassay.
At the time of immunization, a partial fragment polypeptide of the protein of the present invention used as an antigen is coated on an appropriate plate, and the hybridoma culture supernatant or a purified antibody obtained in (d) described below is reacted as a first antibody. Two antibodies that react specifically with the protein of the present invention after reacting with an anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound, etc. Is selected as a hybridoma producing the monoclonal antibody of the present invention.
Using the hybridoma, cloning was repeated twice by the limiting dilution method (the first time, an HT medium (a medium obtained by removing aminopterin from the HAT medium), and the second time, a normal medium was used). Those whose valencies are recognized are selected as hybridoma strains producing the monoclonal antibody of the present invention.
One example of a hybridoma strain producing the monoclonal antibody of the present invention includes hybridoma strain KM3103. The hybridoma strain KM3103 was registered on March 26, 2002 by the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (1-1-1, Higashi, Tsukuba, Ibaraki, Japan, Central No. 6: Postal Code 305-8566) with FERM BP. It has been deposited as -7977.
(D) Preparation of monoclonal antibody
8 to 10-week-old mice or nude mice treated with pristane (0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane) administered intraperitoneally and bred for 2 weeks) were obtained in (c). 5-20 × 10 5 hybridoma cells producing the protein monoclonal antibody of the present invention6Cells / animal are injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites cancer.
Ascites is collected from the mouse with ascites tumor and centrifuged at 3000 rpm for 5 minutes to remove solids.
From the obtained supernatant, a monoclonal antibody can be purified and obtained by the same method as that used for polyclonal.
The subclass of the antibody is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein amount is calculated by the Lowry method or from the absorbance at 280 nm.
4. Use of the DNA, protein or antibody of the present invention
(1) A method for determining a disease by detecting and quantifying the expression of a gene encoding the protein of the present invention
Using the DNA or oligonucleotide of the present invention, Northern hybridization method (Molecular Cloning, 2nd edition), PCR method and reverse transcribed (RT) -PCR method (both PCR Protocols and Academic Press (1990)) And the like, and detecting the expression of the gene encoding the protein of the present invention, thereby detecting acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, and brain tumor. , Malignant tumors, diseases with abnormal cell proliferation such as solid tumors, myocardial infarction, cerebral infarction, peripheral vascular obstruction, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, artery Sclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic Insulin-like growth factors and growth hormones such as brain diseases, heart failure, congestion, diseases involving vascular disorders such as choroidal circulatory disorders, diseases involving abnormal bone metabolism such as osteoporosis, dwarfism, acromegaly, and chronic renal failure in children. Diseases with impaired action, diseases with abnormal smooth muscle cell differentiation and proliferation such as arteriosclerosis, bronchial disease, restenosis, diseases with abnormal skeletal muscle cell differentiation and proliferation such as myasthenia gravis, gastric ulcers, etc. Diseases with abnormal gastric acid secretion, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy To determine diseases that have been reported to involve proteins belonging to the IGFBP superfamily, such as inflammatory diseases associated with abnormal lymphocyte infiltration, such as asthma, hay fever, airway hyperreactivity, and autoimmune diseases It can be.
Since the RT-PCR method is a simple method, it is particularly useful as a method for detecting gene expression.
For example, Northern hybridization is carried out using a DNA having the same sequence as the continuous 100 to 2881 bases in the base sequence represented by SEQ ID NO: 2 or a DNA having a sequence complementary to the DNA as a probe, and The above-mentioned disease can be determined by quantifying the expression level of a gene having the described nucleotide sequence and comparing the expression level with a healthy subject.
As a specific method for the determination, for example, the following method can be mentioned.
Total RNA (10 to 20 μg) derived from white blood cells or tissues of test subjects and healthy subjects, or their mRNA (1 to 5 μg) was added to a denaturing solution [50% (v / v) formamide, 2.2 mol / L formaldehyde, 20 mmol / l In L MOPS [3- (N-morpholino) propanesulfonic acid] (PH 7.0), 5 mmol / L sodium acetate, 1 mmol / L EDTA], heated at 65 ° C. for 5 minutes to denature, 2.2 mol / L formaldehyde On a 1% agarose gel containing
After the electrophoresis, the RNA in the gel is blotted on a nitrocellulose filter (Optimal BA-S85; manufactured by Schleicher & Schuell), and heated at 80 ° C. under reduced pressure for 1 hour to be immobilized.
The filter was washed with a hybridization solution [5 × SSPE (750 mmol / L NaCl, 50 mmol / L NaH2PO45 mmol / L EDTA; pH 7.4), 5 × Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 1% SDS (sodium dodecyl sulfate), 0.2 mg / Ml of salmon sperm DNA (Pharmacia Biotech)] for prehybridization.
After prehybridization, a probe is added to the solution, and hybridization is performed at 65 ° C.
As the probe, for example, the DNA fragment shown in SEQ ID NO: 2 can be obtained by using a multi-prime DNA labeling system (Amersham).32Those labeled with P can be used.
The filter after hybridization is washed in the following order.
(A) Wash in a 2 × SSC (300 mmol / L NaCl, 30 mmol / L sodium citrate) solution containing 0.1% SDS at room temperature for 15 minutes. This is repeated several times.
(B) Wash in a 1 × SSC (150 mmol / L NaCl, 15 mmol / L sodium citrate) solution containing 0.1% SDS at 50 to 70 ° C. for 15 minutes. This is repeated several times.
(C) Wash at 50-70 ° C. for 15 minutes in a 0.1 × SSC (15 mmol / L NaCl, 1.5 mmol / L sodium citrate) solution containing 0.1% SDS at 50-70 ° C. This is repeated several times.
After washing the filter, autoradiography is performed using an imaging plate, and the expression of the DNA of SEQ ID NO: 2 can be detected and quantified using a bioimaging analyzer BAS2000 (Fuji Photo Film).
In addition, for example, using a set of oligonucleotides specific to cDNA encoding the protein of the present invention as primers, total RNAs derived from leukocyte cells or tissues of test subjects and healthy persons, mRNAs thereof, or cDNAs prepared from these RNAs are prepared. The above-mentioned disease can be determined by performing PCR as a template, detecting and quantifying the amplified fragment, and comparing the expression level of the gene encoding the protein of the present invention between the subject and a healthy subject.
As the oligonucleotide, the oligonucleotide of the present invention can be used.
MRNA or total RNA serving as a template for PCR can be extracted from various leukocyte cells separated and obtained from blood, or a tissue suspected of having a disease.
Examples of leukocyte cells include polymorphonuclear leukocytes, monocytes, lymphocytes, T cells, B cells, and the like.
Polymorphonuclear leukocytes and mononuclear cells were obtained from peripheral blood of a subject by using Polymorphprep, a kit manufactured by Nycomed Pharma.TMCan be separated and obtained.
From the obtained mononuclear cells, Immunol. ,130, 706 (1983), etc., monocytes and lymphocytes were converted to Tissue Antigen,9, 153 (1977); Immunol. ,11T cells and B cells can be separated and obtained by the method described in Nycomed's manual on the method of isolating blood cells, etc., 273 (1976).
T cells were prepared using the nylon wool method [Eur. J. Immunol. ,3, 645 (1973)]. In addition, each cell can be separated and obtained using magnetic beads (for example, Dynabeads manufactured by Dynal) to which antibodies specific to T cells, B cells, and monocytes / macrophages are respectively bound.
As a method for preparing total RNA from leukocyte cells or tissues, guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol. ,154, 3 (1987)].
Poly (A) from total RNA+Examples of a method for preparing RNA include an oligo (dT) -immobilized cellulose column method (Molecular Cloning, 2nd edition) and the like.
Also, kits such as a Fast Track mRNA Isolation Kit (Fast Track mRNA Isolation Kit; manufactured by Invitrogen) and a Quick Prep mRNA Purification Kit (Quick Prep mRNA Purification Kit; manufactured by Pharmacia) are available. Can also be used to prepare mRNA.
Single-stranded cDNA can be synthesized from total RNA or mRNA using a single-stranded cDNA synthesis kit, Superscript preamplification system (manufactured by BRL). Synthesis can be performed according to the manual attached to the kit.
The expression amount of the gene encoding the protein of the present invention can be quantified by the RT-PCR method (PCR Protocols, Academic Press (1990)) using the total RNA, mRNA or cDNA that can be prepared as described above.
(2) A method for determining a disease by detecting a mutation in a gene encoding the protein of the present invention
Using the oligonucleotide of the present invention as a probe, genomic DNA can be subjected to Southern hybridization (molecular cloning, second edition), PCR, or the like to detect mutations in the gene encoding the protein of the present invention. it can. The detection method is reported to involve proteins belonging to the IGFBP family, including acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, brain tumor, malignant tumor, and solid tumor. Diseases associated with abnormal cell proliferation, such as myocardial infarction, cerebral infarction, peripheral vascular obstruction, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolysis Uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure, congestion, diseases associated with vascular disorders such as choroidal circulatory disorders, diseases associated with abnormal bone metabolism such as osteoporosis, children Diseases with impaired action of insulin-like growth factors and growth hormone, such as dystrophy, acromegaly, and pediatric chronic renal failure; diseases with abnormal smooth muscle cell differentiation and proliferation, such as arteriosclerosis, bronchial disease, and restenosis; Abnormal such as asthenia Diseases involving differentiation and proliferation of skeletal muscle cells, diseases involving abnormal gastric acid secretion such as gastric ulcer, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, It can be used to determine inflammatory diseases associated with abnormal lymphocyte infiltration, such as traumatic injuries, inflammatory bowel diseases, allergies, atopy, asthma, hay fever, airway irritability, and autoimmune diseases.
Specifically, for example, by amplifying the translation region of the DNA encoding the protein of the present invention possessed by the patient by PCR, determining the nucleotide sequence, and comparing the nucleotide sequence with the nucleotide sequence of a healthy person, The presence or absence of a mutation in the translation region can be examined.
CDNA serving as a template to be used in the PCR method can be obtained by the method described in (1). The nucleotide sequence of the obtained cDNA was determined by using a DNA sequencer 377 of PerkinElmer and a reaction kit (ABI Prism).TM BigDyeTM It can be determined using a Terminator Cycle Sequencing Ready Reaction Kit (manufactured by Applied Biosystems).
(3) Disease determination method by immunological detection method and quantitative method
The following diseases can be determined by immunologically detecting or quantifying the expression of the protein of the present invention in blood or tissues of a subject and a healthy subject using the antibody of the present invention, and comparing the expression with that of a healthy subject.
Examples of the method for immunological detection include an ELISA method using a microtiter plate, a fluorescent antibody method, a Western blot method, and an immunohistological staining method.
Examples of the method for immunological quantification include, for example, a sandwich ELISA method using two kinds of monoclonal antibodies having different epitopes among antibodies reacting with the polypeptide of the present invention in a liquid phase,125Radioimmunoassay using the protein of the present invention labeled with a radioisotope such as I and an antibody recognizing the protein.
The immunological method may be used to report abnormal myelogenous leukemia, breast cancer, prostate cancer, colorectal cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor, solid tumor, etc. Diseases involving cell proliferation, myocardial infarction, cerebral infarction, peripheral vascular obstruction, angina, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, Thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic brain disease, heart failure, congestion, diseases with vascular disorders such as choroidal circulatory disorders, diseases with abnormal bone metabolism such as osteoporosis, dwarfism, and terminal hypertrophy Disorders such as insulin-like growth factors and growth hormone action, such as chronic renal failure in children, arteriosclerosis, bronchial disease, diseases associated with abnormal smooth muscle cell differentiation and proliferation, such as restenosis, and myasthenia gravis. Diseases associated with abnormal skeletal muscle cell differentiation and proliferation Diseases associated with abnormalities of gastric acid secretion such as gastric ulcer, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, It can be used to determine diseases such as inflammatory diseases accompanied by abnormal lymphocyte infiltration such as allergy, atopy, asthma, hay fever, airway hypersensitivity, and autoimmune diseases.
(4) Prevention and treatment of diseases by suppressing the transcription or translation of the gene encoding the protein of the present invention
Regarding the relationship between the increase in the expression level of IGFBP and the disease, the chronic renal failure in children is caused by an increase in IGFBP-2 or 3 [Electroyte Mrtab. ,18, 320 (1992)], IGFBP-4 expression is increased in elderly women with parathyroid hormone fractures, and IGFBP-7 and IGFBP-3 levels in cerebrospinal fluid are increased in leukemia patients. That [J. Clin. Endocrinol. Metab. ,84, 1361 (1996)], that in colon cancer, the expression of IGFBP-7 is increased in colon cancer tissues and colon cancer cell lines [J. Gastroenterology,33, 213 (1998)].
In addition, TGF-β and PHPGE2 increase the expression of IGFBP-7 [Endocrinology,140, 1998 (1999)] that treatment of osteoblasts with glucocorticoids suppresses IGF-I expression while increasing IGFBP-7 expression [Endocrinology, 1998].140, 228 (1999)] that IGFBP-7 also influences differentiation into skeletal muscle by suppressing the differentiation promoting action of IGF [Exp. Cell Res. ,237, 192 (1997); Endocrinology,141, 100 (2000)], IGFBP-7 is considered to be involved in diseases related to bone metabolism and skeletal muscle differentiation.
Therefore, for diseases caused by an increase in the expression level of the IGFBP gene or an increase in the function of IGFBP, reduction in the amount of transcription or translation of the DNA encoding the protein of the present invention can be prevented and treated. It is effective for In addition, even if the disease is not directly caused by IGFBP, the amount of transcription or translation of the gene encoding the protein of the present invention is reduced or the function of the protein of the present invention is suppressed to prevent the above disease symptomatically. Or treatment can be provided.
In addition, when there is a patient whose physiological activity via a receptor is enhanced due to an increase in the expression of IGFBP, the physiological activity is suppressed by administering the DNA, oligonucleotide or derivative thereof of the present invention to the patient. The present invention also provides a method for treating a disease which can be symptomatically treated by administration of the DNA, oligonucleotide or derivative thereof of the present invention even if the increase in IGFBP expression is not a direct cause of the disease. The derivatives can be effective prophylactic and therapeutic agents.
Methods for using the prophylactic or therapeutic agents include, for example, antisense RNA / DNA technology [Bioscience and Industry,50, 322 (1992), Chemistry,46, 681 (1991), Biotechnology,9358 (1992), Trends in Biotechnology,10, 87 (1992), Trends in Biotechnology,10, 152 (1992), cell engineering,16, 1463 (1997)], triple helix technology [Trends in Biotechnology,10, 132 (1992)], ribozyme technology [Current Opinion in Chemical Biology,3, 274 (1999); FEMS Microbiology Reviews,23, 257 (1999), Frontiers in Bioscience,4, D497 (1999), Chemistry & Biology,6, R33 (1999), Nucleic Acids Research,26, 5237 (1998), Trends In Biotechnology,16438 (1998)] or the decoy DNA method [Nippon Rinsho-Japanese Journal of Clinical Medicine,56, 563 (1998), Circulation Research,82, 1023 (1998), Experimental Nephrology,5, 429 (1997), Nippon Rinsho-Japanese Journal of Clinical Medicine,54, 2583 (1996)], and the expression of any gene can be suppressed by using this method.
When the DNA, oligonucleotide or derivative thereof of the present invention is used as the above-mentioned prophylactic or therapeutic agent, the DNA, oligonucleotide or derivative thereof of the present invention may be used alone or in a retrovirus vector, adenovirus vector, adenovirus associated virus vector. After insertion into an appropriate vector such as, for example, it can be formulated, formulated and administered according to the usual methods described below.
In order to use a virus vector such as a retrovirus or an adenovirus or a gene therapy vector incorporated in another gene therapy vector as a gene therapy drug or a prophylactic drug, the gene therapy vector and the base used for the gene therapy agent are prepared. [Nature Genet. ,8, 42 (1994)].
As the base used for the gene therapy agent, any base may be used as long as it is usually used for injections, and distilled water, salt solutions such as sodium chloride or a mixture of sodium chloride and an inorganic salt, mannitol, lactose, Examples include a saccharide solution such as dextran and glucose, an amino acid solution such as glycine and arginine, and a mixed solution of an organic acid solution or a salt solution and a glucose solution. In addition, according to a conventional method, using an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil or soybean oil, or an auxiliary agent such as a lecithin or a nonionic surfactant as a base, the solution, suspension, etc. An injection may be prepared as a suspension or dispersion. These injections can also be prepared as preparations for dissolution at the time of use by operations such as powdering and freeze-drying. The gene therapy agent of the present invention can be used for treatment by dissolving it immediately before gene therapy in the above-mentioned base which has been sterilized if necessary in the case of a liquid. Examples of the method of administering the gene therapy agent of the present invention include a method of locally administering the gene therapy agent so that it is absorbed at the treatment site of the patient.
DNA can also be transported to a target treatment site by a non-viral gene transfer method.
Non-viral gene transfer methods known in the art include calcium phosphate coprecipitation [Virology,52, 456-467 (1973); Science,209, 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA,77Proc., 5399-5403 (1980); Natl. Acad. Sci. USA,77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature,294, 92-94 (1981)], liposome-mediated membrane fusion-mediated transfer method [Proc. Natl. Acad. Sci. USA,84, 7413-7417 (1987);
Biochemistry,28, 9508-9514 (1989); Biol. Chem. ,264Hum., 12126-12129 (1989); Gene Ther. ,3, 267-275, (1992); Science,249, 1285-1288 (1990); Circulation,83, 2007-2011 (1992)] or direct DNA uptake and receptor-mediated DNA transfer [Science,2471465-1468 (1990); Biol. Chem. ,266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA,87, 3655-3659 (1991); Biol. Chem. ,264, 16985-16987 (1989); BioTechniques,11, 474-485 (1991); Proc. Natl. Acad. Sci. USA,87Proc., 3410-3414 (1990); Natl. Acad. Sci. USA,88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA,87Proc., 4033-4037 (1990); Natl. Acad. Sci. USA,88, 8850-8854 (1991); Hum. Gene Ther. ,3147-154 (1991)].
It has been reported in tumor studies that the liposome-mediated membrane fusion-mediated transfer method allows local uptake and expression of genes in the target tissue by directly administering the liposome preparation to the target tissue. [Hum. Gene Ther. ,3, 399 (1992)].
(5) Prevention and treatment of diseases in which the expression level or function of the protein of the present invention is reduced
Regarding the relationship between the decreased expression of IGFBP and the disease, the expression of IGFBP-5 was decreased in osteoporosis, and in the compensatory hypertrophy after nephrectomy or small intestine resection, free IGF-I was decreased due to decreased expression of IGFBP-3. Increase [Baillieres Clin. Endocrinol. Metab. ,8, 165 (1994)], that the expression of IGFBP-1 is reduced in endometrial malignant tumors [Growth Regul. ,3, 74 (1993)] In breast cancer tissues, LOH is observed at a frequency of 50% or more at the IGFBP-7 locus on human chromosomes, and a decrease in IGFBP-7 expression is observed [Oncogene,16, 2459 (1996)], decreased expression of IGFBP-7 in prostate cancer tissues at the mRNA level, and lack of detection of IGFBP-7 expression in malignant prostate cancer-derived cell lines [J. Clin. Endocrinol. Metab. ,83, 4355 (1998)], in a large uterine leiomyoma site, the adjoining uterine smooth muscle cells and tumor volume were 120 cm.3The expression of IGFBP-7 mRNA is lower than that of the following small uterine smooth muscles [Am. J. Reprod. Immunol. ,4353 (2000)], that in mouse liver cancer cells induced by the SV40T antigen, the 5 'upstream region of the IGFBP-7 gene is methylated and the expression level is reduced [Biochem. Biophys. Res. Commun. ,267, 109 (2000)], that the expression of IGFBP-7 is reduced in kidney and vascular lesions in a type I diabetes model by administration of streptozotocin [Diabetes,45, S111 (1996); Diabetes & its Complications,12, 252 (1998)], that a decrease in IGFBP-7 expression is also observed at the protein level in coronary artery smooth muscle cells of type II diabetic patients [Diabetes,46, 1627 (1997)] that culture of bovine artery-derived smooth muscle cells in a high glucose medium reduces the expression level of IGFBP-7 at the mRNA and protein levels [Diabetes,46, 1627 (1997), Diabetologia,41, 134 (1998)] and that IGFBP-7 stimulates PGI2 production in the vascular wall [Nature,271549 (1978)], and hemolytic uremic syndrome [Lancet,2, 871 (1978)], thrombotic thrombocytopenic purpura [Lancet,2, 748 (1979)], sickle cell anemia [Br. J. Haematol. ,48545 (1981)], acute myocardial infarction [Coronary,2, 49 (1985)], diabetic vascular disorders [Metabolism,38, 837 (1989), Haemostasis,16, 447 (1986), Diab. Res. Clin. Pract. ,3, 243 (1987)], and a decrease in blood levels in arteriosclerotic diseases has been reported.
In addition, TGF-β and PHPGE2 increase the expression of IGFBP-7 [Endocrinology,140, 1998 (1999)] that treatment of osteoblasts with glucocorticoids suppresses IGF-I expression while increasing IGFBP-7 expression [Endocrinology, 1998].140, 228 (1999)] that IGFBP-7 also influences differentiation into skeletal muscle by suppressing the differentiation promoting action of IGF [Exp. Cell Res. ,237, 192 (1997); Endocrinology,141, 100 (2000)], IGFBP-7 is considered to be involved in diseases related to bone metabolism and skeletal muscle differentiation.
When IGFBP-7 is forcibly expressed in a prostate cancer cell line in which the expression of IGFBP-7 is reduced, the cell division time is prolonged, the colony forming ability in soft agar medium is reduced, and the tumor forming ability in nude mouse transplantation is reduced. Increase in apoptosis induction rate by drug treatment [Cancer Res. ,59, 2370 (1999)], when IGFBP-7 is forcibly expressed in an osteosarcoma cell line in which the function of p53 is lost, the growth of the cell line can be suppressed in the same manner as when the p53 gene is introduced. [Oncogene,12, 1361 (1996)], and by increasing the expression of the DNA and protein of the present invention, the above-mentioned diseases associated with a decrease in IGFBP expression can be prevented or treated. In addition, even if the disease is not directly attributable to IGFBP, prevention of the above-mentioned diseases that can be palliatively treated by increasing the amount of transcription or translation of the gene encoding the protein of the present invention or increasing the amount of the protein of the present invention. Alternatively, treatment can be provided.
Therefore, when there is a patient in which the physiological action of the protein is reduced due to a decrease in the expression or function of IGFBP, the DNA, oligonucleotide or derivative thereof of the present invention, or the protein of the present invention is administered to the patient. The DNA, oligonucleotide or derivative thereof of the present invention can be used for a disease that can promote a physiological effect and can be symptomatically treated even if the decrease in IGFBP expression or function is not a direct cause of the disease. Alternatively, the protein of the present invention is useful as a prophylactic and therapeutic agent for the above diseases.
As a method for administering the prophylactic and therapeutic agents, for example, when there is a patient who cannot expect a normal physiological action due to decreased expression or mutation of the protein of the present invention, (i) DNA encoding the protein of the present invention (Ii) inserting a DNA encoding the protein of the present invention into a target cell and expressing it, and then transplanting the cell into the patient, or (iii) the present invention. By administering the protein to the patient, the amount of the protein of the present invention in the body of the patient can be increased, and the physiological action of the protein can be sufficiently exerted. Therefore, the DNA encoding the protein of the present invention or the protein of the present invention may be used as a disease in which the expression level or function of the protein of the present invention is reduced, or a disease caused by abnormal function due to mutation of the protein, or the protein of the present invention. Even if it is not directly caused, it is useful as a safe and low-toxic prophylactic or therapeutic drug against diseases that can be treated symptomatically by administration of the DNA encoding the protein of the present invention or the protein of the present invention.
When the DNA encoding the protein of the present invention is used as the above prophylactic or therapeutic agent, the DNA of the present invention is inserted alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. Thereafter, it can be formulated, formulated and administered according to the usual method described in the above (4).
In addition, a medicine containing the protein of the present invention as an active ingredient can be administered alone, but usually the active ingredient is one or more pharmacologically acceptable carriers. And provided as pharmaceutical preparations prepared by any of the methods well-known in the art of pharmacy. Preferably, a sterile solution dissolved in water or an aqueous carrier such as an aqueous solution of salt, glycine, glucose, human albumin or the like is used. Also, pharmacologically acceptable additives, such as buffering agents and tonicity agents to bring the formulation solution closer to physiological conditions, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, citric acid Sodium and the like can be added. Alternatively, it can be stored after lyophilization and dissolved in an appropriate solvent before use.
The administration route is desirably the one that is most effective in the treatment, and may be oral administration, or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous. Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, p-hydroxybenzoate. It can be produced using preservatives such as acid esters and flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, an injection is prepared using a carrier composed of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Sprays are prepared using a carrier or the like which does not irritate the protein itself or the mucous membrane of the recipient and the respiratory tract and which disperses the protein as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the protein and the carrier used. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
The dose or the number of administrations varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 8 mg / kg per adult per day.
(6) A method for obtaining a promoter region of a gene encoding the protein of the present invention
Using the DNA or oligonucleotide of the present invention as a probe and a known method [New Cell Engineering Experimental Protocol, Shujunsha (1993)] Can be obtained.
Examples of the promoter region include all promoter regions involved in transcription of a gene encoding the protein of the present invention in mammalian cells. For example, a promoter region involved in transcription of a gene encoding the protein of the present invention in human brain tissue can be mentioned. The promoter can be used in a method for screening a substance that controls transcription or translation of a gene encoding the protein of the present invention described in (7) below.
(7) A method for screening a substance that controls transcription or translation of a gene encoding the protein of the present invention
Diseases with abnormal cell proliferation such as acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor, solid tumor, myocardial infarction, cerebral infarction, peripheral vascular atresia, narrow Heart disease, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic brain disease, Diseases with vascular disorders such as heart failure, congestion, choroidal circulatory disorders, diseases with abnormal bone metabolism such as osteoporosis, dwarfism, acromegaly, pediatric chronic renal failure, and other disorders of insulin-like growth factors and growth hormone action Disease associated with abnormal proliferation and differentiation of smooth muscle cells such as arteriosclerosis, bronchial disease, restenosis, disease associated with abnormal differentiation and proliferation of skeletal muscle cells such as myasthenia gravis, and gastric acid secretion such as gastric ulcer. Diseases with abnormalities, microbial infections, chronic Hepatitis hepatitis, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway irritability, autoimmune disease, etc. It has been reported that diseases such as inflammatory diseases accompanied by abnormal lymphocyte infiltration involve proteins belonging to the IGFBP superfamily.
A possible cause of the above-mentioned diseases is a change in the transcription amount of DNA encoding IGFBP or a change in the function of the protein of the present invention. In this case, the transcription amount or translation amount of the DNA encoding the protein of the present invention is controlled. Is effective in preventing and treating diseases. Even for diseases that are not directly caused by IGFBP, it is possible to prevent or treat the above diseases symptomatically by controlling the amount of transcription or translation of the gene encoding the protein of the present invention.
Therefore, a compound that promotes or suppresses the transcription process of a gene encoding the protein of the present invention or the translation process from a transcript to a protein is a cell that is exerted through the protein by controlling the expression of the protein. Its function can be controlled and it is useful as a safe and low toxic pharmaceutical composition.
The compound can be obtained by the following methods (a) to (c).
(A) After culturing [i] a cell expressing the protein of the present invention and [ii] a cell expressing the protein of the present invention in the presence of a test substance by the culture method described in 2 above for 2 hours to 1 week A method of measuring and comparing the amount of the protein in cells using the antibody of the present invention described in (3) above, and selecting and obtaining a compound having an activity of increasing or decreasing the amount of the protein.
As a measurement method using the antibody of the present invention, for example, a detection method using an ELISA method using a microtiter plate, a fluorescent antibody method, a Western blot method, an immunohistological staining and the like can be mentioned.
(B) After culturing [i] cells expressing the protein of the present invention and [ii] cells expressing the protein of the present invention in the presence of the test substance by the culture method described in 2 above for 2 hours to 1 week The amount of the transcript of the DNA encoding the protein in the cells is measured and compared using the method such as the Northern hybridization method or the PCR method described in the above (1) to increase or decrease the amount of the transcript. A method for selecting and obtaining a compound having activity.
(C) A plasmid in which a reporter gene-linked DNA has been incorporated downstream of the promoter obtained in (6) above is prepared by a known method, and introduced into animal cells according to the method described in (2) above. To get. [I] The transformant is cultivated for 2 hours to 1 week by the culture method described in 2 above in the presence of the transformant and [ii] a test substance, and the expression level of the reporter gene in the cells is known. [New Cancer Engineering Protocol, Shujunsha (1993), Biotechniques, ed.20, 914 (1996); Antibiotics,49, 453 (1996), Trends in Biochemical Sciences,20, 448 (1995), Cell Engineering,16, 581 (1997)], and selecting and obtaining a compound having an activity of increasing or decreasing the expression level.
Examples of the reporter gene include a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a β-lactamase gene, a luciferase gene, a green fluorescent protein (GFP) gene and the like.
(8) A method for screening a substance that controls the function of the protein of the present invention
Diseases with abnormal cell proliferation such as acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor, solid tumor, myocardial infarction, cerebral infarction, peripheral vascular atresia, narrow Heart disease, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombus, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic brain disease, Diseases with vascular disorders such as heart failure, congestion, choroidal circulatory disorders, diseases with abnormal bone metabolism such as osteoporosis, dwarfism, acromegaly, pediatric chronic renal failure, and other disorders of insulin-like growth factors and growth hormone action Disease associated with abnormal proliferation and differentiation of smooth muscle cells such as arteriosclerosis, bronchial disease, restenosis, disease associated with abnormal differentiation and proliferation of skeletal muscle cells such as myasthenia gravis, and gastric acid secretion such as gastric ulcer. Diseases with abnormalities, microbial infections, chronic Hepatitis hepatitis, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway irritability, autoimmune disease, etc. It has been reported that diseases such as inflammatory diseases accompanied by abnormal lymphocyte infiltration involve proteins belonging to the IGFBP superfamily.
One of the causes of the above-mentioned disease is considered to be a change in the function of IGFBP. In this case, inhibiting or enhancing the function of the protein of the present invention is effective for the prevention and treatment of the disease. Even for diseases not directly caused by IGFBP, the above-mentioned diseases can be prevented or treated symptomatically by inhibiting or enhancing the function of the protein of the present invention.
Therefore, when there is a patient whose physiological function of cells is reduced or enhanced due to a change in the function of the protein of the present invention, it is possible to control the physiological function by administering a compound that controls the function of the present invention. In addition, even if a change in the function of the protein of the present invention is not a direct cause of the disease, a disease for which symptomatic treatment can be performed by controlling the function of the protein of the present invention can also be prevented and treated by administering the compound.
The compound can be obtained, for example, by the following method.
[I] culturing cells expressing the protein of the present invention and [ii] cells expressing the protein of the present invention in the presence of the test substance by the culture method described in 2 above for 2 hours to 1 week, A method for detecting and comparing the cellular response caused by the functioning of, and selecting and obtaining a compound having an activity of inhibiting or enhancing the function of the protein.
Methods for detecting a cellular response caused by the function of the protein of the present invention include, for example, intracellular signal transmission dependent on insulin or insulin-like growth factor contained in a medium, gene transcription, sugar uptake, and proliferation. And other known methods for detecting such changes. In addition, known methods for measuring changes in prostaglandin production [Nature,263, 663 (1976), Clinical Science,17, 958 (1981)].
(9) A method for screening for a protein that specifically binds to the protein of the present invention
The protein that specifically binds to the protein of the present invention can be used for the development of a preventive or therapeutic drug for a disease caused by the protein of the present invention. For example, since the receptor of the protein of the present invention has a function of binding to the protein of the present invention, transmitting information into cells, and expressing a physiological action, the activity of the receptor or the receptor The substance that regulates the transcription or translation of the gene encoding the body is caused by the dysfunction of the protein of the present invention, like the protein of the present invention or the substance that regulates the transcription or translation of the gene encoding the protein of the present invention. It is useful as a prophylactic and therapeutic agent for diseases.
Methods for screening for a protein that specifically binds to the protein of the present invention include Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology, Science,255, 989 (1992), etc., wherein a protein that specifically binds to the protein of the present invention when the protein of the present invention is brought into contact with the test sample is selected from the test sample. Can be obtained with
Specifically, the following method can be used.
A cDNA is prepared from the tissue. By inserting the cDNA into a suitable expression vector downstream of the promoter, a recombinant vector is prepared and a cDNA library is prepared. By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant expressing the gene expressed in the tissue is obtained. A transformant producing a protein to which the labeled protein of the present invention specifically binds is selected.
By determining the gene sequence encoded by the cDNA introduced into the selected transformant, a protein that specifically binds to the protein of the present invention can be obtained.
As a method for preparing a cDNA library, the method described in the above 1 can be mentioned.
Examples of the method for introducing a recombinant vector, the method for obtaining a transformant, and the method for culturing the obtained transformant in a medium include the methods described in 2 above.
By co-culturing a transformant with the protein of the present invention labeled by a known protein labeling method such as radioisotope or biotinylation, a gene product that specifically binds to the labeled protein of the present invention is produced. Can be selected.
Methods for isolating the cDNA introduced into the selected transformant include Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology, Mol. Cell. Biol. ,8, 2837 (1988), etc., or the Hirt method.
Methods for determining the gene sequence of the isolated cDNA include the method described in 1 above.
(10) A medicine containing the antibody of the present invention
The antibodies of the present invention are useful as preventive and therapeutic agents for diseases related to the protein of the present invention.
Regarding the relationship between the increase in the expression level of IGFBP and the disease, the chronic renal failure in children is caused by an increase in IGFBP-2 or 3 [Electroyte Mrtab. ,18, 320 (1992)], increased expression of IGFBP-4 in fractured elderly women with elevated parathyroid hormone, and increased levels of IGFBP-7 and IGFBP-3 in cerebrospinal fluid in leukemia patients. That [J. Clin. Endocrinol. Metab. ,84, 1361 (1996)], that in colon cancer, the expression of IGFBP-7 is increased in colon cancer tissues and colon cancer cell lines [J. Gastroenterology,33, 213 (1998)].
In addition, TGF-β, PTH and PGE2 increase the expression of IGFBP-7 [Endocrinology,140, 1998 (1999)] that treatment of osteoblasts with glucocorticoids suppresses IGF-I expression while increasing IGFBP-7 expression [Endocrinology, 1998].140, 228 (1999)] that IGFBP-7 also influences differentiation into skeletal muscle by suppressing the differentiation promoting action of IGF [Exp. Cell Res. ,237, 192 (1997); Endocrinology,141, 100 (2000)], IGFBP-7 is considered to be involved in diseases related to bone metabolism and skeletal muscle differentiation.
Therefore, for diseases in which an increase in the expression level of IGFBP or an increase in the function is one factor, suppressing the expression level or inhibiting the function of the protein of the present invention is effective for prevention and treatment of the disease. is there. In addition, even for diseases not directly caused by IGFBP, prevention or treatment of the above-mentioned diseases that can be symptomatically treated by suppressing the expression level of the protein of the present invention or inhibiting the function thereof can be performed.
Therefore, when there is a patient in whom the physiological activity of cells is enhanced due to an increase in the expression level of IGFBP or hyperactivity, the physiological activity is suppressed by administering an antibody that binds to the protein of the present invention. In addition, diseases in which symptomatic treatment can be achieved by suppressing the function of the protein of the present invention even if the protein of the present invention is not the direct cause of the disease can also be prevented and treated by administering the antibody of the present invention.
The pharmaceutical containing the antibody as an active ingredient is provided as a pharmaceutical preparation produced by any method well-known in the technical field of pharmaceutics according to the production of the pharmaceutical containing the protein of the present invention described in (5) above. can do.
(11) A drug containing the compound obtained by the search method of (7)
The compound that promotes or suppresses the transcription process of the gene encoding the protein of the present invention or the translation process from a transcript to a protein is useful as a safe and low-toxic pharmaceutical composition.
The compound obtained in (7) is to be provided as a pharmaceutical preparation produced by any method well-known in the technical field of pharmaceutics according to the method for producing a pharmaceutical containing the protein of the present invention described in (5). Can be.
(12) Preparation of knockout non-human animal using DNA of the present invention
Using a recombinant vector containing the DNA of the present invention, a target non-human animal, for example, embryonic stem cells (embryonic stem cells) such as cows, sheep, goats, pigs, horses, mice, chickens, etc. A known homologous recombination method [for example, Nature,326, 6110, 295 (1987); Cell,51, 3, 503 (1987)] to produce a mutant clone inactivated or substituted with an arbitrary sequence [for example, Nature,350, 6315, 243 (1991)]. Using a mutant clone of an embryonic stem cell, a chimeric individual consisting of an embryonic stem cell clone and normal cells can be prepared by a technique such as an injection chimera method or a collective chimera method of injecting a fertilized egg of an animal into a blastocyst (blastcyst). it can. By crossing the chimeric individual with a normal individual, it is possible to obtain an individual having an arbitrary mutation in the gene encoding the protein of the present invention on the chromosome of whole body cells. A knockout non-human animal can be obtained as an individual in which the expression of the gene encoding the protein of the present invention is partially or completely suppressed from among homozygotes having the above-mentioned mutation.
In addition, a knockout non-human animal can be prepared by introducing a mutation into any position of the gene encoding the protein of the present invention on the chromosome. For example, by introducing a mutation by substituting, deleting, or inserting a base into the translation region of the gene encoding the protein of the present invention on the chromosome, the activity of the product can be modified. Also, by introducing a similar mutation into the expression control region, the degree, timing, tissue specificity, etc. of the expression can be altered. Furthermore, by combining with the Cre-loxP system, it is possible to more positively control the expression timing, expression site, expression amount, and the like. As such an example, an example in which a promoter expressed in a specific region of the brain is used to delete a target gene only in that region [Cell,87, 7, 1317 (1996)] and an example in which a gene of interest is specifically deleted at an intended time using an adenovirus expressing Cre [Science,278, 5335, (1997)].
Therefore, for the gene encoding the protein of the present invention on the chromosome, the expression can be controlled at any time or in such a manner, or any insertion, deletion, substitution has its translation region or expression control region, Knockout non-human animals can be produced.
The knockout non-human animal can induce the symptoms of various diseases caused by the protein of the present invention at any time, any degree, or any site.
Thus, the knockout non-human animal of the present invention becomes an extremely useful animal model in the treatment and prevention of various diseases caused by the protein of the present invention. In particular, it is very useful as a model for evaluating therapeutic and prophylactic agents, functional foods, health foods, and the like.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described more specifically with reference to the following examples, which are merely illustrative of the present invention and do not limit the scope of the present invention.
[Example 1] Preparation of cDNA library derived from human neural progenitor cell NT-2
A cDNA library was prepared by the following method using NT-2 neural progenitor cells (purchased from Stratagene), which are teratocarcinoma cells derived from human fetal testis and can be differentiated into neural cells by retinoic acid treatment. After culturing NT-2 cells according to the attached manual, retinoic acid was added, and the cells were further cultured for 2 weeks. The cultured cells were collected, and mRNA was extracted by the method described in Molecular Cloning, Second Edition. Furthermore, polyA (+) RNA was purified using oligo dT cellulose. The oligocap method [Gene,138, 171 (1994)]. Using the Oligo-cap linker (RNA having the nucleotide sequence represented by SEQ ID NO: 3) and Oligo dT primer (DNA having the nucleotide sequence represented by SEQ ID NO: 4), the literature [protein nucleic acid enzyme,41197 (1996); Gene,200149 (1997)], BAP (Bacterial Alkaline Phosphatase) treatment, TAP (Tobacco Acid Phosphatase) treatment, RNA ligation, synthesis of first-strand cDNA and removal of RNA were performed.
Next, two types of 5′-terminal sense primer (DNA having the nucleotide sequence represented by SEQ ID NO: 5) and 3′-terminal antisense primer (DNA having the nucleotide sequence represented by SEQ ID NO: 6) Conversion into double-stranded cDNA by PCR (polymerase chain reaction) using PCR primers,SfiI cut. The PCR was performed using a commercially available Geneamp XL PCR kit (manufactured by Perkin Elmer) at 95 ° C. for 5 minutes, followed by 1 minute at 95 ° C., 1 minute at 58 ° C., and 10 minutes at 72 ° C. Was repeated 12 times and then kept at 4 ° C. ThenDraThe cDNA was ligated in a fixed direction to the vector pME18SFL3 (GeneBank AB009864, expression vector, 3392 bp) cut with III to prepare a recombinant DNA, and the recombinant DNA was used.Escherichia coli A cDNA library was prepared by transforming DH10B. For each plasmid DNA retained in the obtained transformant, the nucleotide sequence of the 5 'end and 3' end of the cDNA was determined by using a DNA sequencing reagent (Dye Terminator Cycle Sequencing FS Ready Reaction Kit, dRhodamine Terminator Cycle Recycler Cycle Reactor Cycle Recycler Cycle Recycler Cycle Recycler Cycle Reactor Cycle Recycler) Using Kit or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems), a sequencing reaction was performed according to the manual, and then a DNA sequencer (ABI PRISM 377, manufactured by PE Biosystems using ABI PRISM, PE Biosystems).
[Example 2] Identification of a novel factor belonging to the IGFBP superfamily
Regarding the nucleotide sequence of each clone of the prepared cDNA library, human IGFBP-1, human IGFBP-2, human IGFBP-3 as a known protein belonging to the IGFBP superfamily registered in the protein amino acid database SWISSPROT or the nucleotide sequence database GenBank, Using 10 molecules of human IGFBP-4, human IGFBP-5, human IGFBP-6, human IGFBP-7, human IGFBP-8, human IGFBP-9 or human IGFBP-10, homology to the base sequence encoding these molecules Was selected. The nucleotide sequence of C-NT2RI20041212 corresponds to the nucleotide sequence of nucleotide numbers 451 to 2881 of SEQ ID NO: 2, and the nucleotide sequence portion of nucleotide numbers 451 to 863 is the same as that of human IGFBP-7 (UniGene Hs. 119206) in the homology analysis using BLAST2. ) And P value 6.0 × 10-14Showed significant homology of 59%.
Since C-NT2RI2004312 was an incomplete-length cDNA lacking the 5 'portion, GenomeWalker was determined based on the nucleotide sequence of C-NT2RI2004312.TMUsing kits (manufactured by Clontech), the nucleotide sequence information of the human genomic DNA region including the nucleotide sequence of C-NT2RI2004312 was obtained according to the attached protocol. SEQ ID NO: 7 shows the nucleotide sequence of the clone containing the obtained nucleotide sequence of 36 nucleotides from the 5 'end of C-NT2RI2004312 (the nucleotide sequence of nucleotides 451 to 486 in SEQ ID NO: 2). The start codon ATG was located 450 bases upstream of the genomic region containing the base sequence of 36 bases from the 5 'end of C-NT2RI2004312, and the splicing acceptor sequence AG was found near 28 bases upstream. Further, since GT which is a donor sequence of splicing is present immediately after the genomic region containing the base sequence of 36 bases from the 5 'end of C-NT2RI2004312, a part of the base sequence of bases 9 to 494 of SEQ ID NO: 7 Constitutes one exon, and the nucleotide sequence of the full-length cDNA of C-NT2RI2004312 was found to be the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 1 shows the amino acid sequence of the protein encoded by the full-length cDNA of C-NT2RI2004312.
In the homology analysis using BLAST2, the amino acid sequence of the protein encoded by the full-length cDNA of C-NT2RI2004312 was compared with that of human IGFBP-7 (GenBank Accession Number: I52825) which is a protein belonging to the insulin-like growth factor binding protein family. P value 1.0 × 10−39Showed a significant homology of 39%. In the insulin-like growth factor binding protein superfamily, there are ten cysteine residues important for binding to IGF-1, IGF-2, and insulin, and insulin-like growth factor binding containing four cysteine residues among them. The motif [GCCGCXXC (G: glycine residue, C: cysteine residue, X: any amino acid residue)] is highly conserved among the factors belonging to this family. When comparing the amino acid sequence of the protein encoded by the full-length cDNA of C-NT2RI2004312 with each of the factors of the insulin-like growth factor binding protein superfamily, the position of the cysteine residue in the amino acid sequence represented by SEQ ID NO: 1 was also found. And the number were conserved, indicating that the insulin-like growth factor binding motif portion was also highly conserved (FIG. 1). The N-terminal signal sequence was also highly conserved between the protein encoded by the full-length cDNA of C-NT2RI200412 and the factor belonging to the insulin-like growth factor binding protein superfamily (FIG. 1).
From the above results, it is clear that the protein encoded by the full-length cDNA of C-NT2RI2004312 is a novel human protein belonging to the insulin-like growth factor binding protein superfamily and has activity as a protein belonging to the insulin-like growth factor binding protein superfamily. Became clear.
When the base sequence database GenBank / EMBL / DDBJ was searched using BLAST2 based on the base sequence shown in SEQ ID NO: 2, UniGene Hs. The homology was found to be high with 21400. This Hs. 21400 is composed of 32 ESTs that show no significant homology with other genes. The 3 ′ portion of the base sequence shown in SEQ ID NO: 2 and a P value of 8.5 by homology analysis using BLAST2 were 8.5. × 10−103And the homology was 93%. In addition, there was one human EST which is considered to be derived from the same gene as the nucleotide sequence shown in SEQ ID NO: 2, but this EST nucleotide sequence did not cover the full-length cDNA of C-NT2RI2004312. The GeneBank accession number for this EST is AW129076.
Escherichia coli containing plasmid C-NT2RI2004312: Escherichia coli DH10B / pME18SFL3-NT2RI2004312 is referred to as FERM BP-7473 on March 1, 2001, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (Tsukuba, Ibaraki, Japan) 1-chome, Higashi 1-City,
[Example 3] Analysis of base sequence of full-length cDNA of C-NT2RI2004312
Based on the base sequence of the full-length cDNA of C-NT2RI2004312 shown in SEQ ID NO: 2, a protein start codon prediction program ATGPr [Bioinformatics,14384 (1998)] to analyze the sequence around the initiation codon. In the nucleotide sequence shown in SEQ ID NO: 2, ATG located at
[Example 4] Genome analysis of the gene encoded by the full-length cDNA of C-NT2RI2004312
Based on the base sequence of the full-length cDNA of C-NT2RI2004312 shown in SEQ ID NO: 2, a base sequence database GenBank / EMBL / DDBJ was searched using BLAST2, and a genomic clone containing the base sequence represented by SEQ ID NO: 2 (GenBank action number: AL135785).
From the comparison between the obtained human genome gene sequence and the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide sequence represented by SEQ ID NO: 2 is divided into six parts and is located on the genome of the same human chromosome 9 p11-13.3. It was found to be present over 18 kb.
[Example 5] Organs expressing full-length cDNA of C-NT2RI2004312
UniGene Hs. Table 1 shows the SAGE data (link data from http // www.ncbi.nlm.nih.gov / UniGene /) registered in the NCBI of 21400. The full-length cDNA of C-NT2RI2004312 shown in SEQ ID NO: 2 was found to occur at a high frequency in the brain tissue-derived cDNA library SAGE Duke 1273, and was found to be relatively selectively expressed in brain tissue. In addition, since C-NT2RI2004312 itself was prepared from retinoic acid-treated NT-2 neural progenitor cells, it was presumed that C-NT2RI2004312 was a molecule deeply involved in cell proliferation, cell differentiation and tissue regeneration in brain tissue.
[Example 6] Expression analysis of C-NT2RI2004312-derived gene using RT-PCR method
Human organ polyA purchased from Clontech+Using 4 μg of RNA as a template, cDNA was synthesized in accordance with the attached manual using a commercially available SUPER SCRIPT Preamplification System for first-strand cDNA Synthesis (manufactured by Gibco).
Human organ polyA+RNA includes brain, pituitary, testis, thyroid, uterus, adrenal gland, cerebellum, kidney, pancreas, small intestine, bone marrow, heart, liver, lung, lymph node, mammary gland, placenta, prostate, salivary gland, skeletal muscle, spinal cord, spleen , Stomach, thymus, trachea derived polyA+RNA was used.
Next, PCR was performed using the synthesized cDNA as a template and a DNA having the nucleotide sequence represented by SEQ ID NOS: 8 and 9 and a DNA having the nucleotide sequence represented by SEQ ID NOS: 10 and 11 as primer sets. In PCR, a solution obtained by diluting the synthesized cDNA 50-fold with sterile water and Advantage was used.R After preparing a reaction solution by a conventional method using cDNA PCR kit (manufactured by Clontech), the reaction was carried out at 94 ° C. for 7 minutes, and then a cycle of 94 ° C. for 1 minute and 68 ° C. for 3 minutes was repeated 32 times. The reaction was performed at 68 ° C. for 7 minutes. The reaction solution was subjected to agarose gel electrophoresis, and the expression levels were compared by comparing the concentrations of DNA fragments specific to the respective primer sets. As a result, C-NT2RI2004312 showed brain, pituitary, testis, thyroid, Significantly higher expression was confirmed in the uterus.
The DNA consisting of the nucleotide sequence represented by SEQ ID NO: 8 and SEQ ID NO: 9 was used as a primer set specific to C-NT2RI20041212, and the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 10 and SEQ ID NO: 11 was substituted by human β-actin. Was used as a primer set specific to.
[Example 7] Acquisition of full-length cDNA of C-NT2RI2004312
(1) Preparation of human brain-derived single-stranded cDNA
Human brain-derived total RNA (manufactured by Clontech) is dissolved in 45 μl of sterilized water, RQ1 RNase-Free DNase (manufactured by Promega) 1 μl, attached 10 × DNase buffer 5 μl, RNasin Ribonuclease inhibitor (manufactured by Promega) 0.5 μm Was added to each, and the mixture was reacted at 37 ° C. for 30 minutes to decompose genomic DNA mixed in the sample. After the reaction, the total RNA was repurified by RNAeasy (manufactured by QIAGEN) and dissolved in 50 μl of sterilized water.
SUPERSCRIPT was performed on 3 μl of each obtained total RNA.TN A single-stranded cDNA is synthesized by performing a reverse transcription reaction in a 20 μl system using oligo (dT) as a primer according to the attached instruction using First-Strand Synthesis System for RT-PCR (manufactured by Life Technologies). did. A 50-fold diluted aqueous solution of the reaction solution was used for PCR cloning. Stored at -80 ° C until use.
(2) Acquisition of full-length cDNA of C-NT2RI2004312
(A) Obtaining a cDNA fragment at the 5 'end
The polymerase chain reaction (PCR) was performed on the human brain-derived single-stranded cDNA prepared in (1) above, using a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 16 or SEQ ID NO: 17 as a primer set. In the PCR reaction, 25 μl of a reaction solution (ExTaq buffer, 0.2 mmol / l dNTPs, 0.5 μmol / l the above primer set) containing 1 μl of human brain-derived single-stranded cDNA was prepared using ExTaq (Takara Shuzo). After heating at 94 ° C. for 5 minutes, a reaction consisting of 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 1 minute is repeated as 30 cycles, followed by heating at 72 ° C. for 5 minutes. I went in. After completion of the PCR, the reaction solution is subjected to 1% agarose gel electrophoresis, and an amplified DNA fragment of about 1100 bp is purified using GENECLEAN SPIN Kit (BIO101) (hereinafter, this method is used for purification of a DNA fragment from agarose gel). And eluted with 20 μl of sterile water. A ligation reaction was carried out with 9 μl of the DNA fragment and 50 ng of pGEM T vector (Clontech) in a 20 μl reaction system using Ligation High (Toyobo). . Natl. Acad. Sci. U. S. A, 69, 2110 (1972)] (hereinafter, this method was used for transformation of Escherichia coli). From a plurality of the obtained ampicillin-resistant colonies, a known method [Nucleic Acids Research,7, 1513 (1979)] (hereinafter, this method was used for plasmid isolation) to isolate plasmid DNA (hereinafter, referred to as pGEM-C-NT2RI200412n).
(B) Acquisition of cDNA fragment at 3 'end
The human brain-derived single-stranded cDNA prepared in the above (1) was prepared in the same manner as in the above (2) (a), using a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 18 and SEQ ID NO: 19 as a primer set. The polymerase chain reaction (PCR) was performed. The obtained amplified DNA fragment was ligated to the ApaI-SacI fragment of pBluescript SK- using Ligation High, and plasmid DNA (hereinafter, referred to as pSK-C-NT2RI200412c) was obtained in the same manner as in the above (2) (a). Isolated.
(C) Obtaining full-length cDNA
PGEM-C-NT2RI200412n prepared in (2) (a) aboveSacII−ApaIThe fragment was obtained from pSK-C-NT2RI200412c prepared in (2) and (b) of this section using Ligation High.SacII−ApaIThe resulting fragment was ligated to the fragment, and a plasmid DNA (hereinafter, referred to as pSK-C-NT2RI200412t) was isolated in the same manner as in the above (2) (a).
The presence or absence of the insert was confirmed by size comparison by agarose gel electrophoresis, and the nucleotide sequence was determined using a DNA sequencer 377 (Perkin Elmer) and a Big Dye Terminator Cycle Sequencing FS Raedy Reaction Kit (manufactured by Perkin Elmer according to the manual provided by Perkin Elmer). Were determined. After eliminating the base reading error accompanying the PCR, the determined base sequence was compared with the base sequence represented by SEQ ID NO: 2, and the DNA inserted into the plasmid was identified as C-NT2RI200004312 represented by SEQ ID NO: 2. Was confirmed to be the same as the full-length cDNA.
[Example 8] Expression of a protein encoded by full-length cDNA of C-NT2RI200412 using animal cells as a host
(1) Preparation of recombinant vector
Using the clone C-NT2RI200412t obtained in Example 7 as a template, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 12 and SEQ ID NO: 13 as a primer set, or the nucleotide sequence represented by SEQ ID NO: 12 and SEQ ID NO: 14 A reaction solution is prepared by a conventional method using KOD DNA Polymerase (manufactured by TOYOBO) as a primer set using DNA consisting of: and a cycle of 15 seconds at 98 ° C., 2 seconds at 65 ° C., and 30 seconds at 74 ° C. is repeated 25 times. PCR was carried out under the conditions, and C-NT2RI2004312 was obtained as an amplified DNA fragment of about 840 bp in each reaction.
Next, the 5 'side of the C-NT2RI2004312 is pBluescript II SK- (Stratagene).HinAt the dIII site, the 3 'side of C-NT2RI2004312 is the pBluescriptII SK-XhoPlasmid pSK-C- is a plasmid having a DNA fragment obtained by PCR using a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 12 or SEQ ID NO: 13 as a primer set by inserting it so as to be on the I site side. PSK-C-NT2RI2004312h, which is a plasmid having a DNA fragment obtained by PCR using DNAs consisting of the nucleotide sequences represented by NT2RI200412, SEQ ID NO: 12 and SEQ ID NO: 14 as a primer set, was prepared.
Next, pSK-C-NT2RI200412h and pSK-C-NT2RI200412hHindIII-KpnThe I fragment (approximately 830 bp) was isolated from an expression vector pcDNA3 for animal cells (manufactured by Invitrogen)HindIII-KpnInsertion into the I site produced pcDNA3-C-NT2RI2004312 and pcDNA3-C-NT2RI2004312h.
The DNA primer set consisting of the nucleotide sequences represented by SEQ ID NO: 12 and SEQ ID NO: 14 was designed so that a His marker peptide was added to the C-terminal of C-NT2RI200412.
(2) Introduction of vector into cells
FuGENE in 100 μl of Opti-MEM (manufactured by Gibco)TM6 2 μl of transfection reagent (manufactured by Roche Diagnostics) was added and left at room temperature for 5 minutes. Next, 1 μg of the control plasmid pcDNA3, the C-NT2RI2004312 expression plasmid pcDNA3-C-NT2RI2004312 or pcDNA3-C-NT2RI2004312h prepared in the above (1) were added, and the mixture was further left at room temperature for 15 minutes. During this time, a DMEM medium (manufactured by Gibco) containing 10% FCS was dispensed into a 6-well plate at 2 ml / well, and COS-1 cells (Riken Cell Bank; RCB0143) were further added to 3 × 10 6 cells.5Seeded per well. After standing for 15 minutes, the plasmid and FuGENETMA mixture of 6 transfection reagents was added to a 6-well plate at 25 μl / well and CO2The cells were cultured in an incubator for 72 hours to obtain transient transformants. Hereinafter, transformed strains prepared by introducing pcDNA3, pcDNA3-C-NT2RI2004312, and pcDNA3-C-NT2RI2004312h were respectively transformed into COS-1 / mock strain, COS-1 / C-NT2RI2004312 strain, and COS-1 / Designated as C-NT2RI200412h strain.
(3) Confirmation of expression
(A) Preparation of culture supernatant
4 ml of the culture supernatant of the COS-1 / mock strain, the COS-1 / C-NT2RI20041212 strain, and the COS-1 / C-NT2RI200412h strain prepared in the above (2) were collected and centrifuged at 1,200 rpm for 5 minutes. The solids were removed to obtain a culture supernatant. The obtained culture supernatant was stored at −20 ° C., thawed if necessary, and used for the following experiments.
(B) Obtain cell lysate
After the cells of the COS-1 / mock strain, COS-1 / C-NT2RI20041212 strain, and COS-1 / C-NT2RI20031212h strain prepared in the above (2) are detached from the 6-well plate with a cell scraper (Sumitomo Bakelite). Phosphate Buffered Saline (pH 7.2) [hereinafter referred to as “PBS” (manufactured by Gibco)] in 2 ml, and centrifuged at 1,200 rpm for 5 minutes to remove the supernatant. Next, the suspension was suspended in 1 ml of PBS containing 100 μl of a protease inhibitor cocktail for animal cells (manufactured by Sigma), and sonicated to obtain a cell lysate. The obtained cell lysate was stored at −20 ° C., thawed if necessary, and used for the following experiments.
(C) Detection by Western blotting
10 μl of the culture supernatant of the COS-1 / mock strain and the COS-1 / C-NT2RI200412h strain prepared in (3) (a) above was subjected to 15% polyacrylamide gel electrophoresis (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988), and blotted on a PVDF membrane (Millipore) according to a conventional method. After blocking the membrane with 5% skim milk-PBS (hereinafter, referred to as “blocking solution”), the membrane is coated with an anti-His monoclonal antibody (manufactured by Roche Diagnostics) at a concentration of 1 μg / ml. Was added and left at room temperature for 1 hour. The membrane is made up of 0.05% polyoxyethylene (20) sorbitan monolaurate [trade name: Span 20 (equivalent to the trade name of
In the culture supernatant of the COS-1 / C-NT2RI200412h strain, a band not observed in the COS-1 / mock strain was detected. Therefore, in COS-1 cells, expression of the protein encoded by C-NT2RI2004312 was confirmed (FIG. 2).
Example 9 Preparation of Monoclonal Antibody Recognizing Protein Encoded by Full-length cDNA of C-NT2RI2004312
(1) Selection of antigenic peptide
Analyzing the amino acid sequence of the protein encoded by the full-length cDNA of C-NT2RI2004312, suitable as an antigen from a portion having a high hydrophilicity, an N-terminus, a C-terminus, a portion having a secondary turn structure, and a random coil structure. Compound 1 (a polypeptide having an amino acid sequence represented by SEQ ID NO: 15) was selected as a partial sequence considered to be supposed.
The abbreviations for the amino acids and their protecting groups used in the present invention can be found in the recommendation of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (European Journal of Biochemistry, Europe). 138, 9 (1984)].
The following abbreviations represent the corresponding amino acids below unless otherwise specified.
Ala: L-alanine
Arg: L-arginine
Asp: L-aspartic acid
Asx: L-aspartic acid or L-asparagine
Cys: L-cysteine
Gly: glycine
His: L-histidine
Leu: L-leucine
Lys: L-lysine
Pro: L-proline
Thr: L-threonine
The following abbreviations represent the corresponding amino acid protecting groups and side chain protected amino acids below.
Fmoc: 9-fluorenylmethyloxycarbonyl
tBu: t-butyl
Trt: Trityl
Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl
Fmoc-Arg (Pmc) -OH: Nα-9-Fluorenylmethyloxycarbonyl-Ng-2,2,5,7,8-pentamethylchroman-6-sulfonyl-L-arginine
Fmoc-Asp (OtBu) -OH: Nα-9-Fluorenylmethyloxycarbonyl-L-aspartic acid-β-t-butyl ester
Fmoc-His (Trt) -OH: Nα-9-Fluorenylmethyloxycarbonyl-Nim-Trityl-L-cysteine
Fmoc-Lys (Boc) -OH: Nα-9-Fluorenylmethyloxycarbonyl-Nε-T-butyloxycarbonyl-L-lysine
Fmoc-Thr (tBu) -OH: Nα-9-Fluorenylmethyloxycarbonyl-Ot-butyl-L-threonine
The following abbreviations represent the corresponding reaction solvents, reagents and the like described below.
HBTU: 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
HOBt: N-hydroxybenzotriazole
DIEA: diisopropylethylamine
DMF: N, N-dimethylformamide
TFA: trifluoroacetic acid
In the following examples, the physicochemical properties of the compounds were measured by the following methods.
Mass spectrometry was performed by FAB-MS method using JEOL JMS-HX110A. Amino acid analysis is performed according to the method of Cohen, SA [Analytical Biochemistry,222, 19 (1994)]. The hydrolysis was performed in hydrochloric acid vapor at 110 ° C. for 20 hours, and the amino acid composition of the hydrolyzate was analyzed using a Waters AccQ-Tag amino acid analyzer (manufactured by Waters).
(2) Compound 1 (peptide Ac-Arg-Ala-Arg-His-Thr-Pro-Arg-Ala-His-Pro-Gly-His-Leu-His-Lys- consisting of the amino acid sequence represented by SEQ ID NO: 15) Synthesis of Ala-Arg-Asp-Gly-Pro-Cys-OH)
40 mg of a carrier resin [H-Cys (Trt) -2-ClTrt resin, manufactured by Novaviochem Co., Ltd.] to which Fmoc-Cys (Trt) and 22.8 μmol are bound is placed in a reaction vessel of an automatic synthesizer (Shimadzu Corporation), and 600 μl of After adding DMF and stirring for 3 minutes to discharge the solution, the following operation was performed according to a synthesis program of Shimadzu Corporation.
(A) 500 μl of a 30% piperidine-DMF solution was added, the mixture was stirred for 4 minutes, the solution was discharged, and this operation was repeated once.
(B) The carrier resin was washed with 600 μl of DMF for 1 minute, the solution was discharged, and this operation was repeated 5 times.
(C) Fmoc-Pro-OH (228 μmol), HBTU (228 μmol), HOBt monohydrate (228 μmol) and DIEA (684 μmol) were stirred in DMF (1.12 ml) for 3 minutes, and the obtained solution was resinified. And the mixture was stirred for 30 minutes and the solution was drained.
(D) After the carrier resin was washed with 600 μl of DMF for 1 minute, the solution was discharged, and this was repeated five times.
Through the above steps, Fmoc-Pro-Cys (Trt) was synthesized on the carrier.
Next, after the steps (a) and (b), in the step (c), a condensation reaction is performed using Fmoc-Gly-OH, and after the washing step (d), Fmoc-Gly-Pro-Cys ( Trt) was synthesized on the support.
Hereinafter, in step (c), Fmoc-Asp (OtBu) -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH, Fmoc-His (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-His (Trt) -OH, Fmoc-Ala-OH, Fmoc-Alg (Pmc) -OH , Fmoc-Pro-OH, Fmoc-Thr (tBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Ala-OH and Fmoc-Arg (Pmc) -OH After repeating steps (a) to (d), the deprotection and washing steps of (a) and (b) were carried out, Successively washed with ether and dried under reduced pressure for 12 hours to obtain the bound carrier resin in side-chain protected peptide. Next, the following operations (e) to (g) were performed on the obtained carrier resin.
(E) The carrier resin was washed with 800 μl of DMF for 1 minute, the solution was discharged, and this operation was repeated three times.
(F) Acetic anhydride (456 μmol) and DMF (500 μl) were added to the resin, the mixture was stirred for 2 hours, and the solution was drained.
(G) The carrier resin was washed with 800 μl of DMF for 1 minute, the solution was discharged, and this operation was repeated three times.
Thereafter, the resin was washed successively with methanol and butyl ether, and dried under reduced pressure for 12 hours to obtain a carrier resin to which an N-terminal acetylated side chain protective peptide was bonded. This includes TFA (82.5%), thioanisole (5%), water (5%), ethyl methyl sulfide (3%), 1,2-ethanedithiol (2.5%) and thiophenol (2% ) Was added, and the mixture was allowed to stand at room temperature for 8 hours to remove side chain protecting groups and to cut out peptides from the resin. After filtering the resin, about 10 ml of ether was added to the obtained solution, and the resulting precipitate was collected by centrifugation and decantation to obtain 66.6 mg as a crude peptide. After dissolving the whole amount of the crude product in an aqueous acetic acid solution, the crude product was purified by HPLC using a reversed-phase column (manufactured by Shiseido,
Mass spectrometry [FABMS]; m / z = 2413.0 (M + H+)
Amino acid analysis; Asx 1.0 (1), Gly 2.0 (2), His 4.3 (4), Arg 3.8 (4), Thr 1.0 (1), Ala 2.9 (3) , Pro 2.9 (3), Leu 1.0 (1), Lys 1.0 (1), Cys 1.3 (1)
(3) Preparation of immunogen
(4) Immunization of animals and preparation of antibody-producing cells
100 mg of the KLH conjugate of
The spleen was shredded in a MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, and centrifuged (250 × g, 5 minutes). Erythrocytes were removed by adding a Tris-ammonium chloride buffer (pH 7.6) to the obtained precipitate fraction and treating for 1-2 minutes. The obtained precipitate fraction (cell fraction) was washed three times with MEM medium and used for cell fusion.
(5) Enzyme immunoassay (binding ELISA)
As the antigen for the assay, a compound obtained by conjugate of the
After standing, 1% BSA / PBS was discarded, and the anti-serum of the mouse to be immunized and the hybridoma culture supernatant were dispensed as primary antibodies into the plate at 50 μl / well and left for 2 hours. After washing the plate with Tween-PBS, peroxidase-labeled rabbit anti-mouse immunoglobulin (Dako) was added as a secondary antibody at 50 μl / well and left at room temperature for 1 hour. After washing the plate with Tween-PBS, the ABTS substrate solution [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium, 1 mmol / L ABTS / 0.1 mol / L citrate buffer (pH 4.2) )] Was added to develop a color, and the absorbance at OD 415 nm was measured using a plate reader (Emax; Molecular Devices).
(6) Preparation of mouse myeloma cells
8-azaguanine resistant mouse myeloma cell line P3-X63Ag8U. 1 (P3-U1: purchased from ATCC) was cultured in a normal medium (RPMI medium supplemented with 10% fetal bovine serum), and 2 × 107More than one cell was obtained and used as a parent strain for cell fusion.
(7) Preparation of hybridoma
The mouse splenocytes obtained in Example 9 (4) and the myeloma cells obtained in Example 9 (6) were mixed at a ratio of 10: 1, and centrifuged (250 × g, 5 minutes). After the cells of the obtained precipitate fraction were well loosened, a mixture of 1 g of polyethylene glycol-1000 (PEG-1000), 1 ml of MEM medium and 0.35 ml of dimethyl sulfoxide was added at 37 ° C. with stirring.80.5 ml was added per mouse spleen cell, 1 ml of MEM medium was added several times to the suspension every 1 to 2 minutes, and then MEM medium was added to make the
The suspension was centrifuged (900 rpm, 5 minutes), and the cells in the obtained precipitate fraction were loosened gently. Then, the cells were gently sucked and sucked with a mesipette to make HAT medium [10% fetal bovine serum]. HAT Media Supplement (manufactured by Invitrogen) in an added RPMI medium]. The suspension was dispensed into a 96-well culture plate at 200 μl / well, and 5
After the culture, the culture supernatant was examined by the enzyme immunoassay described in Example 9 (5), and a well which did not react with the
The hybridoma cell line producing the anti-C-NT2RI2004312 monoclonal antibody KM3103 was obtained from the National Institute of Advanced Industrial Science and Technology Patent Depositary on March 26, 2002 (1-1 1-1 Higashi, Tsukuba, Ibaraki, Japan, Chuo No. 6). : Zip code 305-8566) as FERM BP-7977.
(8) Purification of monoclonal antibody
Eight weeks old nude female mice (BALB / c) treated with pristane were mixed with the hybridoma strain obtained in Example 9 (7) in an amount of 5 to 20 × 10 66Cells / animal were injected intraperitoneally. After 10 to 21 days, ascites was collected (1 to 8 ml / animal) from the mice that had accumulated ascites due to the ascites cancer of the hybridoma.
The ascites was centrifuged (1200 × g, 5 minutes) to remove solids. The purified IgG monoclonal antibody was obtained by purifying by the caprylic acid precipitation method [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. The subclass of the monoclonal antibody was determined by an ELISA method using a subcluster typing kit. As shown in FIG. 3, the subclass of KM3103 was IgG1.
(9) Examination of reactivity of monoclonal antibody (binding ELISA)
It carried out according to the method shown in Example 9 (5). However, as the primary antibody, KM3103 obtained in Example 9 (8) diluted in 10 steps from 10 μg / ml in 5 times was used. FIG. 4 shows the results. KM3103 showed specific reactivity to
[Example 10] Detection of protein encoded by full-length cDNA of C-NT2RI2004312 using Western blotting using monoclonal antibody KM3103
10 μl of the culture supernatant or 2.5 μl of the cell lysate of the COS-1 / mock strain, the COS-1 / C-NT2RI20041212 strain, and the COS-1 / C-NT2RI200412h strain prepared in Example 8 was used in Example 8 ( 3) According to the description of (c), Western blotting was performed using the monoclonal antibody KM3103 obtained in Example 9 as a detection antibody.
The anti-C-NT2RI200412 monoclonal antibody KM3103 was found to specifically recognize the protein encoded by the full-length cDNA of C-NT2RI2004312 expressed in COS-1 cells described in Example 8 (FIG. 5).
[Example 11] Effect of protein encoded by full-length cDNA of C-NT2RI2004312 on cancer cell growth
In order to examine the effect of the protein encoded by the full-length cDNA of C-NT2RI200412 on cancer cell growth, the COS-1 / mock strain, COS-1 / C-NT2RI200412 strain obtained in Example 8 (3) (a), Using the culture supernatant of the COS-1 / C-NT2RI200412h strain and the effect on the IGF-dependent proliferation of the human colon cancer cell line HT-29 (ATCC HTB-38) was examined as follows.
1 × 10 5 in D-MEM / F-12 medium (manufactured by Gibco) supplemented with 200 μg / ml of bovine serum albumin (manufactured by Gibco) and 10 μg / ml of human transferrin (manufactured by Gibco)5HT-29 cells adjusted to 50 cells / ml were seeded at 50 μl / well in a 96-well plate, and2The cells were cultured in an incubator for 3 hours. Next, human IGF-I (manufactured by Peprotech) or human IGF-II (manufactured by Peprotech) was added at a concentration of 10 ng / ml. Subsequently, 5 μl / well of the culture supernatant of the COS-1 / mock strain, the COS-1 / C-NT2RI200412 strain, or the COS-1 / C-NT2RI200412h strain was added, and the CO at 37 ° C. was further added.2After culturing for 5 days in an incubator, the number of viable cells was measured using Cell Proliferation Reagent WST-1 (manufactured by Roche Diagnostics).
In the test group to which the culture supernatant of the COS-1 / C-NT2RI20041212 strain and the culture supernatant of the COS-1 / C-NT2RI200412h strain was added, the IGF-dependent growth of the human colon cancer cell line HT-29 cells was significantly inhibited. As shown (FIG. 6).
Industrial potential
According to the present invention, a novel insulin-like growth factor binding protein, a DNA encoding the protein, an antibody recognizing the protein, a method for determining a disease involving the protein, a diagnostic agent, a prophylactic agent, and a therapeutic agent can be provided. .
Sequence listing free text
SEQ ID NO: 3—Description of Artificial Sequence: Synthetic RNA
SEQ ID NO: 4—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 5—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 6—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 9-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 10—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 11—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 12—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 13-Description of artificial sequence: synthetic DNA
SEQ ID NO: 14-Description of artificial sequence: synthetic DNA
SEQ ID NO: 15-Description of Artificial Sequence: Synthetic Peptide
SEQ ID NO: 16-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 17—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 18—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 19-Description of Artificial Sequence: Synthetic DNA
[Sequence list]
[Brief description of the drawings]
FIG. 1 is a diagram showing a comparison of the amino acid sequences of a protein encoded by the full-length cDNA of C-NT2RI2004312 and an IGFBP family factor. In the figures, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6 and IGFBP-7 are human IGFBP-1, human IGFBP-2 and human IGFBP-3, respectively. Refers to human IGFBP-4, human IGFBP-5, human IGFBP-6, human IGFBP-7. In addition, the amino acid sequences conserved between the protein encoded by the full-length cDNA of C-NT2RI2004312 and the insulin-like growth factor binding protein superfamily are outlined, and the cysteine residues highly conserved between the families are marked with *. Was noted.
FIG. 2 is a photograph showing the expression of a protein encoded by C-NT2RI200412 using COS-1 cells as a host. mock represents a control.
FIG. 3 is a diagram showing a subclass of the monoclonal antibody KM3103 by an ELISA method using a subcluster typing kit. The left column shows the results of P3-U1, and the right column shows the results of the monoclonal antibody KM3103. Each column shows, from the left, a test plot using an antibody that recognizes all subclasses, G1, G2a, G2b, G3, and M.
FIG. 4 is a diagram showing the reactivity of the monoclonal antibody KM3103 with the antigen peptide of the monoclonal antibody KM3103 by binding ELISA. In the figure, ● indicates a test group to which
FIG. 5 is a photograph showing the detection of a protein encoded by C-NT2RI2004312 by Western blotting using KM3103. mock represents a control.
FIG. 6 is a diagram showing the effect of the protein encoded by C-NT2RI2004312 on IGF-dependent proliferation of HT-29 cells. mock represents a control.
Claims (43)
(A)請求項1〜6のいずれか1項に記載の蛋白質をコードするDNAの変異の検出または発現量の測定を行ない、健常人と比較することを特徴とする、疾患の判定方法。
(B)請求項14または請求項15に記載の抗体を用いて、請求項1〜6のいずれか1項に記載の蛋白質の変異の検出または発現量の測定を行ない、健常人と比較することを特徴とする、疾患の判定方法。A method for determining a disease described in the following (A) or (B).
(A) A method for determining a disease, comprising detecting a mutation in a DNA encoding the protein according to any one of claims 1 to 6 or measuring an expression level thereof, and comparing with a healthy person.
(B) using the antibody according to claim 14 or claim 15, detecting the mutation of the protein according to any one of claims 1 to 6 or measuring the expression level thereof, and comparing it with a healthy person. A method for determining a disease, comprising:
(A) 請求項21または22に記載の検出方法。
(B) 請求項17、19または20に記載の検出方法、または定量方法。The determination method according to any one of claims 23 to 25, wherein the determination method is based on the method described in the following (A) or (B).
(A) The detection method according to claim 21 or 22.
(B) The detection method or the quantification method according to claim 17, 19 or 20.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2001104766 | 2001-04-03 | ||
JP2001104766 | 2001-04-03 | ||
PCT/JP2002/003343 WO2002081515A1 (en) | 2001-04-03 | 2002-04-03 | Insulin-like growth factor-binding protein |
Publications (1)
Publication Number | Publication Date |
---|---|
JPWO2002081515A1 true JPWO2002081515A1 (en) | 2004-07-29 |
Family
ID=18957578
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2002579900A Withdrawn JPWO2002081515A1 (en) | 2001-04-03 | 2002-04-03 | Insulin-like growth factor binding protein |
Country Status (2)
Country | Link |
---|---|
JP (1) | JPWO2002081515A1 (en) |
WO (1) | WO2002081515A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2010286559B2 (en) * | 2009-08-28 | 2015-01-22 | Astute Medical, Inc. | Methods and compositions for diagnosis and prognosis of renal injury and renal failure |
CN102153641B (en) * | 2010-12-17 | 2013-06-12 | 浙江大学 | Preparation method and application of mutain of human insulin-like growth factor binding protein 7 (IGFBP7) |
DE102012108598A1 (en) * | 2012-09-14 | 2014-04-10 | Forschungszentrum Jülich GmbH | New D3-D enantiomeric peptides derived from D3 and their use |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07132095A (en) * | 1993-06-11 | 1995-05-23 | Nawata Arata | Dna and protein coded thereby |
AU2001255214B2 (en) * | 2000-03-31 | 2006-06-08 | Arca Biopharma, Inc. | Method and materials relating to insulin-like growth factor binding protein-like polypeptides and polynucleotides |
US6436703B1 (en) * | 2000-03-31 | 2002-08-20 | Hyseq, Inc. | Nucleic acids and polypeptides |
WO2001094416A2 (en) * | 2000-06-07 | 2001-12-13 | Curagen Corporation | Human proteins and nucleic acids encoding same |
-
2002
- 2002-04-03 JP JP2002579900A patent/JPWO2002081515A1/en not_active Withdrawn
- 2002-04-03 WO PCT/JP2002/003343 patent/WO2002081515A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
WO2002081515A1 (en) | 2002-10-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7858091B2 (en) | Antibodies to insulin-like growth factor binding protein and uses thereof | |
JP4798663B2 (en) | Novel angiogenesis inhibitor | |
JP4112627B2 (en) | Novel polypeptide, novel DNA and novel antibody | |
JP2003523207A (en) | LIV-1-related proteins, polynucleotides encoding them, and their use in treating cancer | |
EP1225224A1 (en) | Shear stress-response dna | |
JPWO2002033094A1 (en) | Antibodies that inhibit VPLF activity | |
WO2000018805A1 (en) | Novel antibodies, drugs containing these antibodies and methods for screening compounds by using these antibodies | |
JP2001231578A (en) | Protein belonging to il-1 family | |
EP1308509A1 (en) | Novel physiologically active peptide and use thereof | |
US7358348B2 (en) | β-catenin nuclear localized protein | |
JPWO2002081515A1 (en) | Insulin-like growth factor binding protein | |
WO2007037245A1 (en) | Polypeptide having anti-angiogenic activity | |
JP4429269B2 (en) | Apoptosis-inducing genes and their use | |
US20110130335A1 (en) | Therapeutic agent and test agent for disease with myocardial necrosis | |
JP4474496B2 (en) | IgA nephropathy-related DNA | |
JP3929744B2 (en) | Novel polypeptide, novel DNA, novel antibody and novel genetically modified animal | |
WO2001083705A1 (en) | Myocardial cell proliferation-associated genes | |
JP2001017188A (en) | New vegf/pdgf-like factor | |
US20050048602A1 (en) | Kidney repairing factor | |
WO2000042180A1 (en) | Novel protein | |
JP4306865B2 (en) | Novel bioactive peptides and uses thereof | |
US20070128186A1 (en) | Proliferative glomerulonephritis-related gene | |
JP2000041681A (en) | New protein | |
JP2005021001A (en) | Abca7 splicing variant | |
JP2003093072A (en) | New protease inhibitor-like polypeptide and its dna |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A300 | Withdrawal of application because of no request for examination |
Free format text: JAPANESE INTERMEDIATE CODE: A300 Effective date: 20050607 |