JPS63157984A - Amino acid sequence and gene fragment of human alpha2-plasmin inhibitor - Google Patents
Amino acid sequence and gene fragment of human alpha2-plasmin inhibitorInfo
- Publication number
- JPS63157984A JPS63157984A JP30175386A JP30175386A JPS63157984A JP S63157984 A JPS63157984 A JP S63157984A JP 30175386 A JP30175386 A JP 30175386A JP 30175386 A JP30175386 A JP 30175386A JP S63157984 A JPS63157984 A JP S63157984A
- Authority
- JP
- Japan
- Prior art keywords
- human
- amino acid
- plasmin inhibitor
- acid sequence
- dna
- 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.)
- Pending
Links
- 239000012634 fragment Substances 0.000 title claims abstract description 22
- 125000003275 alpha amino acid group Chemical group 0.000 title claims abstract 6
- 108090000623 proteins and genes Chemical group 0.000 title claims description 13
- 101000783712 Homo sapiens Alpha-2-antiplasmin Proteins 0.000 title abstract description 37
- 150000001413 amino acids Chemical class 0.000 claims description 23
- 229940122791 Plasmin inhibitor Drugs 0.000 claims description 9
- 239000002806 plasmin inhibitor Substances 0.000 claims description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 abstract description 12
- 210000003494 hepatocyte Anatomy 0.000 abstract description 10
- 108020004999 messenger RNA Proteins 0.000 abstract description 7
- 239000000523 sample Substances 0.000 abstract description 4
- 238000009396 hybridization Methods 0.000 abstract description 2
- 230000036961 partial effect Effects 0.000 abstract description 2
- 239000003112 inhibitor Substances 0.000 abstract 2
- 108020003215 DNA Probes Proteins 0.000 abstract 1
- 239000003298 DNA probe Substances 0.000 abstract 1
- 239000013599 cloning vector Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 17
- 108091008146 restriction endonucleases Proteins 0.000 description 12
- 230000029087 digestion Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 7
- 108010088842 Fibrinolysin Proteins 0.000 description 7
- 229940012957 plasmin Drugs 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 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 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- KHWCHTKSEGGWEX-RRKCRQDMSA-N 2'-deoxyadenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 KHWCHTKSEGGWEX-RRKCRQDMSA-N 0.000 description 1
- NCMVOABPESMRCP-SHYZEUOFSA-N 2'-deoxycytosine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 NCMVOABPESMRCP-SHYZEUOFSA-N 0.000 description 1
- LTFMZDNNPPEQNG-KVQBGUIXSA-N 2'-deoxyguanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 LTFMZDNNPPEQNG-KVQBGUIXSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 101710196208 Fibrinolytic enzyme Proteins 0.000 description 1
- MCGNJCNXIMQCMN-DCAQKATOSA-N Glu-Met-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCC(O)=O MCGNJCNXIMQCMN-DCAQKATOSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000658545 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Type I restriction enyme HindI endonuclease subunit Proteins 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- GPAHWYRSHCKICP-GUBZILKMSA-N Met-Glu-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GPAHWYRSHCKICP-GUBZILKMSA-N 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 201000000667 alpha-2-plasmin inhibitor deficiency Diseases 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 1
- KHWCHTKSEGGWEX-UHFFFAOYSA-N deoxyadenylic acid Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(O)=O)O1 KHWCHTKSEGGWEX-UHFFFAOYSA-N 0.000 description 1
- LTFMZDNNPPEQNG-UHFFFAOYSA-N deoxyguanylic acid Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1CC(O)C(COP(O)(O)=O)O1 LTFMZDNNPPEQNG-UHFFFAOYSA-N 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
a、産業上の利用分野
本発明はヒトα2−プラスミンインヒビター(α2−p
lasmin tnhibitor ; a2−a
nNplasmin >を構成するアミノ酸配列及びそ
れをコードする遺伝子断片に関するものである。DETAILED DESCRIPTION OF THE INVENTION a. Field of Industrial Application The present invention relates to human α2-plasmin inhibitor (α2-p
lasmin tnhibitor; a2-a
This relates to the amino acid sequence constituting nNplasmin> and the gene fragment encoding it.
b、従来技術
ヒトのα2−プラスミンインヒビターは、青水と緒井に
よって最初に単離・精製され、線維素溶解酵素のプラス
ミン(plasmin )のエステラーゼ活性を瞬間的
に阻害する強力なプラスミンインヒビタ−であり、11
.7%の糖鎖を含む分子量約67.000の1本鎖の糖
蛋白質であることが知られている[M、 Moroi
& N、 Aoki :TheJ ournal
of B iological Chemis
try、 251゜5956−5965 、(197
6)参照]。b. Prior Art Human α2-plasmin inhibitor was first isolated and purified by Aomizu and Oi, and is a strong plasmin inhibitor that instantly inhibits the esterase activity of the fibrinolytic enzyme plasmin. 11
.. It is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 containing 7% sugar chains [M, Moroi
& N, Aoki: TheJ internal
of Biological Chemistry
try, 251°5956-5965, (197
6)].
一方、ヒト C2−プラスミンインヒビターには3種類
の活性部位があることが知られている。On the other hand, it is known that human C2-plasmin inhibitor has three types of active sites.
第1は、プラスミンの線維素溶解作用阻害部位(以下こ
れをパリアクティブサイト″ということがある> [
8,Wiman & D、 Co11en :Th
eJ ournal of Biological
Chemistry、 254゜9291−92
97 (1979)参照]であり、第2はカルボキシ末
端側のプラスミン結合部位[3,Wiman& D、
Co11en ;European Journa
l ofBiochemistry 、 84. 5
73−578(1978)参照]であり、第3はアミン
末端側のフィブリン結合部位テアル[Y、 5akat
a et al、 ;Thrombosis
Re5earch 、 16. 279−282(19
79)参照コ。The first is the site that inhibits the fibrinolytic action of plasmin (hereinafter referred to as the "pariactive site")
8, Woman & D, Co11en:Th
eJ-ournal of Biological
Chemistry, 254°9291-92
97 (1979)], and the second is the plasmin binding site on the carboxy-terminal side [3, Wiman & D.
Co11en;European Journal
l ofBiochemistry, 84. 5
73-578 (1978)], and the third is the fibrin binding site on the amine terminal side [Y, 5akat
a et al; Thrombosis
Re5search, 16. 279-282 (19
79) Reference.
ヒトα2−プラスミンインヒビターにおけるこれら3種
類の活性部位のうち、フィブリン結合部位はヒトα2−
プラスミンインヒビターのアミノ末端より2番目のアミ
ノ酸であるQlnであることが確められティる[T、T
amaki & N。Among these three active sites in human α2-plasmin inhibitor, the fibrin binding site is the human α2-plasmin inhibitor.
It has been confirmed that Qln is the second amino acid from the amino terminus of plasmin inhibitor [T,T
amaki & n.
A oki ; T he J ournal
OF B iologicalChemistry、
257. 14767−14772 (19
82) 参照]。また、プラスミン結合部位を含む2
6アミノ酸から成るペプチド断片も知られており、この
ペプチド断片は、ヒトα2−プラスミンインヒビターの
カルボキシ末端近くに存在する事も報告されティる[T
、 5asaki 、 et al、 ; TMJ
ournal of 3iochemistry
、 99.1699−1705(1986)参照]が、
その位置は明らかではない。Aoki; The Journal
OF B iological Chemistry,
257. 14767-14772 (19
82) Reference]. In addition, 2 containing the plasmin binding site
A peptide fragment consisting of 6 amino acids is also known, and it has been reported that this peptide fragment exists near the carboxy terminus of human α2-plasmin inhibitor [T
, 5asaki, et al; TMJ
internal of 3iochemistry
, 99.1699-1705 (1986)], but
Its location is not clear.
またヒトα2−プラスミンインヒビターのりアクティブ
サイトに関しては、1eu−1yjetであるという報
告[B、 Wtman Chemistry、 2
54.9291−9297 (1979)参照]或はA
r(+−M atであるという記載[HoR,L t
jnen et at、 ; Thrombosi
sResearch 、 39. 625−630(1
985) 参照]はあるもののその位置は明らかとはな
っていない。これらプラスミン結合部位、及びリアクテ
ィブサイトの構造、その周辺の構造0位置が明らかとな
ればヒトα2−プラスミンインヒビターとの競争剤(C
OmpetitOr )としての利用及び開発等に関し
、非常に有益であり、興味のあることである。Regarding the active site of human α2-plasmin inhibitor, it has been reported that it is 1eu-1yjet [B, Wtman Chemistry, 2
54.9291-9297 (1979)] or A
r(+-M at [HoR, L t
Thrombosi
sResearch, 39. 625-630 (1
985)], but its location is not clear. If the structures of these plasmin-binding sites and reactive sites, as well as the surrounding structure 0 position, are clarified, it will be possible to identify the competitive agent (C) with human α2-plasmin inhibitor.
It is very useful and interesting for its use and development as OmpetitOr).
一方、ヒトα2−プラスミンインヒビターの一部あるい
は全部をコードする遺伝子(断片)は取られていない。On the other hand, the gene (fragment) encoding part or all of human α2-plasmin inhibitor has not been obtained.
この遺伝子もしくはその断片が得られれば、ヒ1〜α2
−プラスミンインヒビターの一部、あるいは全部を頂伝
子操作手法により生産することも可能となる。また、こ
の遺伝子もしくはその断片を利用して、ヒトα2−プラ
スミンインヒビター欠損症の診断も可能となると思われ
る。If this gene or its fragment is obtained, human 1-α2
- It becomes possible to produce part or all of the plasmin inhibitor by apical gene manipulation techniques. Furthermore, it is thought that it will be possible to diagnose human α2-plasmin inhibitor deficiency using this gene or a fragment thereof.
C6本発明の構成
本発明者らは、ヒトα2−プラスミンインヒビター遺伝
子について研究をおこなった結果、ヒトα2−プラスミ
ンインヒビターのカルボキシ末端より403個のアミノ
酸配列をコードする1209個のヌクレオチドを含むヒ
トα2−プラスミンインヒビターの相補DNA (以下
CDNAと略記する)を単離し、その塩基配列の解析に
よってヒトα2−プラスミンインヒビターのカルボキシ
端側の詳細な構造が明らかとなり本発明に到達した。C6 Structure of the Present Invention As a result of research on the human α2-plasmin inhibitor gene, the present inventors found that the human α2-plasmin inhibitor gene contains 1209 nucleotides encoding a 403 amino acid sequence from the carboxy terminus of human α2-plasmin inhibitor. Complementary DNA (hereinafter abbreviated as CDNA) of plasmin inhibitor was isolated, and the detailed structure of the carboxy-terminal side of human α2-plasmin inhibitor was clarified through analysis of its base sequence, leading to the present invention.
すなわち、本発明は添付第1図における第1番目(ph
e)から第403番目(LVS)までのアミノ酸部列で
示されたヒトα2−プラスミンインヒビター断片をコー
ドした遺伝子断片であり、また該遺伝子断片が添付第1
図における第1番目(T)から第1209番目(G)ま
での塩基配列で示されたものである遺伝子断片であり、
また添付第1図における第1番目(Phe)から第40
3番目(Lys)までのアミノ酸配列で示されたヒトα
2−プラスミンインヒビターのアミノ酸配列であり、ま
た添付第1図のアミノ酸配列における第314番目(S
er) 、第315番目(Aro)、第316番目(M
et)または第317番目(Ser)のいずれかのアミ
ノ酸をアミノ酸末端とし、第403番目(Lys)まで
のアミノ酸をカルボキシ末端とするアミノ酸配列である
。 前記本発明の403個のアミノ酸配列中にはヒトα
2−プラスミンインヒビターのプラスミン結合部位及び
リアクティブサイトが含まれる。That is, the present invention is directed to the first (ph) in FIG.
This is a gene fragment encoding the human α2-plasmin inhibitor fragment shown by the amino acid sequence from e) to the 403rd amino acid sequence (LVS), and the gene fragment is attached in attached No. 1.
It is a gene fragment shown by the base sequence from the 1st (T) to the 1209th (G) in the figure,
Also, from number 1 (Phe) to number 40 in attached Figure 1.
Human α shown by the amino acid sequence up to the third (Lys)
This is the amino acid sequence of 2-plasmin inhibitor, and is the 314th (S) amino acid sequence in the attached Figure 1.
er), 315th (Aro), 316th (M
et) or the 317th amino acid (Ser) as the amino acid terminus, and the amino acids up to the 403rd position (Lys) as the carboxy terminus. The 403 amino acid sequence of the present invention includes human α
2-Contains the plasmin binding site and reactive site of the plasmin inhibitor.
特にプラスミン結合部位を含む26アミノ酸(添付第1
歯にアンダーラインで示す)は、ヒトα2−プラスミン
インヒビターの最もカルボキシ端側に見出された。また
、リアクティブサイトは、カルボキシ端より91番目よ
り87番目まで′のM et −A r(]−Met−
8erの中に見出される。また本発明は、プラスミンと
ヒトα2−プラスミンインヒビターが反応してプラスミ
ン−ヒトα2−プラスミンインヒビター複合体を形成し
た時に遊離される低分子ペプチド(分子量約io、oo
o>である。すなわち、この低分子ペプチドのアミノ末
端は第1図に示されたアミノ酸配列の314番目の3
er、或は315番目のAra、或は316番目のMe
t或は317番目のSepのいずれかをアミノ末端とし
、403番目のLVSをカルボキシ末端とする90〜8
7アミノ酸より成るペプチドである。In particular, the 26 amino acids containing the plasmin binding site (attached
(indicated by underlined teeth) was found at the most carboxy-terminal side of the human α2-plasmin inhibitor. In addition, the reactive site is Met-A r(]-Met-
Found in the 8er. The present invention also provides a low-molecular-weight peptide (with a molecular weight of about io, oo
o>. That is, the amino terminus of this low-molecular-weight peptide is located at position 314 of the amino acid sequence shown in Figure 1.
er, or the 315th Ara, or the 316th Me
90-8 with either Sep at position 317 as the amino terminus and LVS at position 403 as the carboxy terminus
It is a peptide consisting of 7 amino acids.
以下実施例を掲げてヒト肝細胞よりメッセンジt−RN
A(以下”mRNA”と記す)の単離。The following is an example of message t-RN from human hepatocytes.
Isolation of A (hereinafter referred to as "mRNA").
CD N Aライブラリーの作製、ヒトα2−プラスミ
ンインヒビターのスクリーニング、ヒトα2−プラスミ
ンインヒビターCD N Aの制限エンドヌクレアーゼ
(以下“制限酵素″と記す)切断地図の作製、ヒトα2
−プラスミンインヒビターC[)NAのりクローニング
、ヒトα2−プラスミンインヒビターCD N Aの塩
基配列の決定について詳細に説明する。Preparation of CD N A library, screening of human α2-plasmin inhibitor, preparation of restriction endonuclease (hereinafter referred to as “restriction enzyme”) cleavage map of human α2-plasmin inhibitor CD N A, human α2
- Plasmin inhibitor C [)NA paste cloning and determination of the base sequence of human α2-plasmin inhibitor CD N A will be explained in detail.
なお、本明細書及び図面において、アミノ酸。Note that in this specification and drawings, amino acids are referred to as amino acids.
ポリペプチドはfUPAc−IUB生化学委員会(CB
N)で採用された方法により略記するものとし、たとえ
ば下記の略号を用いる。The polypeptide is fUPAc-IUB Biochemistry Committee (CB
N) shall be abbreviated according to the method adopted, for example, the following abbreviations will be used.
Alal−−アラニン
Ar!IL−アルギニン
AsnL−アスパラギン
Asp L−アスパラギン酸
Cys L−システィン
Gln L−グルタミン
GIIJL−グルタミン酸
Gly グリシン
H+SL−ヒスチジン
■tel−−イソロイシン
1−eul−一ロイシン
LySL−リジン
Met L−メチオニン
phel−−フェニルアラニン
一7=
prol−プロリン
5erl−−セリン
Thrl−−スレオニン
TrpL−トリプトファン
TVr L−チロシン
Vat L−バリン
また、DNAの配列はそれを構成する各デオキシリボヌ
クレオチドに含まれる塩基の種類で略記するものとし、
たとえば下記の略号を用いる。Alal--alanine Ar! IL-arginine AsnL-asparagine Asp L-aspartic acid Cys L-cysteine Gln L-glutamine GIIJL-glutamic acid Gly Glycine H+SL-histidine tel--isoleucine 1-eul-monoleucine LySL-lysine Met L-methionine phel--phenylalanine 1 7=prol-Proline 5erl--Serine Thrl--Threonine TrpL-Tryptophan TVr L-Tyrosine Vat L-valine Furthermore, the sequence of DNA shall be abbreviated by the type of base contained in each deoxyribonucleotide constituting it,
For example, use the following abbreviations.
△ アデニン(デオキシアデニル酸を示す。)Cシトシ
ン(デオキシシチジル酸を示す。)G グアニン(デオ
キシグアニル酸を示す。)T チミン (デオキシチミ
ジル酸を示す。)実施例1 ヒト肝細胞よりmRN A
の単離ヒト肝細胞よりmRNAの単離は、グアニジンチ
オシアネート法[J、 M、 Chirowin e
t al。△ Adenine (indicates deoxyadenylic acid) C Cytosine (indicates deoxycytidylic acid) G Guanine (indicates deoxyguanylic acid) T Thymine (indicates deoxythymidylic acid) Example 1 mRNA from human hepatocytes
Isolation of mRNA from human hepatocytes was performed using the guanidine thiocyanate method [J. M. Chirowin et al.
tal.
; B iochemistry 、 18.5294
−5299 (1979)参照]に従った。ヒト肝細胞
2 X 108個に5dのGTC溶液(6Mグアニジン
イソチオシアネー1〜,5111Mクエン酸ナトリウム
、0,1M2−メルカプトエタノール、0.5%N−ラ
ウロイルザルコシン酸ナトリウム)を加え、ホモゲナイ
ズした。3.8dの5.7M Cs Cj、 0.
IM EDTA水溶液の上に重層し、これをRPS−
40Tローター(HITACHI)を用いて、35.O
OOrpmで15時間、25℃で超遠心した。超遠心後
注意深く溶液を取り除いた後、エタノール約1戒で3回
リンスし、1.4mの水に溶解後エタノール沈澱させた
。この沈澱を0.5M Na (J、 10 mM
Tris −HCj (pH7,5) 、 1 m
M EDTA、 0.05%5DS(7)組成の洗
浄液0.5−に溶解し、0.5−の○Iig。;Biochemistry, 18.5294
-5299 (1979)]. 5d GTC solution (6M guanidine isothiocyanate 1~, 5111M sodium citrate, 0.1M 2-mercaptoethanol, 0.5% sodium N-lauroyl sarcosinate) was added to 2 x 108 human hepatocytes and homogenized. . 3.8d 5.7M Cs Cj, 0.
IM was layered on top of the EDTA aqueous solution, and this was then subjected to RPS-
Using a 40T rotor (HITACHI), 35. O
Ultracentrifuged at 25° C. for 15 hours at OOrpm. After ultracentrifugation, the solution was carefully removed, rinsed three times with approximately one drop of ethanol, dissolved in 1.4 m of water, and precipitated with ethanol. This precipitate was diluted with 0.5M Na (J, 10mM
Tris-HCj (pH 7,5), 1 m
M EDTA, 0.05% 5DS (7) Dissolved in 0.5- composition cleaning solution, 0.5-○Iig.
(dT)セルロースカラムを通した。このカラムを上記
洗浄液で洗った後、10mM Tris −HCj(
pH7,5) 、 1 mM EDTA、 0.
05%SDSの組成の溶出液で溶出し、約31μ3のp
olyA”mRNAを得た。(dT) was passed through a cellulose column. After washing this column with the above washing solution, 10mM Tris-HCj (
pH 7.5), 1 mM EDTA, 0.
Eluted with an eluate with a composition of 0.05% SDS, and a p
olyA” mRNA was obtained.
実施例2 CDNAライブラリーの作製ヒト肝細胞
由来polyA ” mRN AよりCD N Aの
合成は、Gublerと@ offmanの方法[U。Example 2 Preparation of CDNA Library CDNA was synthesized from human hepatocyte-derived polyA'' mRNA using the method of Gubler and @offman [U.
Gubler & B、J、Hoffman:
Gen)、25゜263−269 (1983)参照]
に従い、アマージャム製CD N A合成キットを用い
ておこなった。Gubler & B. J. Hoffman:
Gen), 25°263-269 (1983)]
It was carried out using a CD DNA synthesis kit manufactured by Amerjam in accordance with the following.
5μy(7)polyA” mRNA (に50ユニ
ツトのヒト胎盤由来RN ase阻害酵素(HPRI>
の存在下5μグのOligo (dT ) 12−18
を加え100ユニツトの逆転写酵素を42℃で1.5時
間働かせて約30%の収率で1本鎖 c DNAを合成
した。この反応液に4ユニツトの大腸菌リボヌクレアー
ゼHと115ユニツトの大腸菌DNAポリメラーゼ■を
加え12℃で1時間、22°Cで1時間反応させた後7
0℃で10分間放置して酵素を失活させた。その後10
ユニツトのT4DNAポリメラーゼを加え37°Cで1
0分間反応させて、約95%の収率で2重鎖cD N
Aを得た。この2重鎖cDNAを20ユニツトのEC0
RIメチラーゼを37℃で1時間反応させた。これに1
6ユニツトのEcoRT(宝酒造製)を結合させた。こ
れに16ユニツトのEcoRI(宝酒造製)を加え37
℃で2時間反応させた後、3 epharose C
L−4Bカラムを通し、純化し約0.8μグのC[)N
Aを得た。次にこのCDNA0.4μ3とλ(ltlO
アーム1.0μg(ベクタークローニングシステムズ製
)との連結をおこない、ヒト肝細胞由来のCD N A
が挿入されたハイブリッドDNAを得た。5μy(7)polyA” mRNA (with 50 units of human placenta-derived RNase inhibitory enzyme (HPRI)
5 μg of Oligo (dT) 12-18 in the presence of
was added and 100 units of reverse transcriptase was operated at 42°C for 1.5 hours to synthesize single-stranded cDNA with a yield of about 30%. To this reaction solution, 4 units of E. coli ribonuclease H and 115 units of E. coli DNA polymerase ■ were added and reacted at 12°C for 1 hour and at 22°C for 1 hour.
The enzyme was inactivated by standing at 0°C for 10 minutes. then 10
Add Unit T4 DNA polymerase and incubate at 37°C.
The double-stranded cDN was reacted for 0 min with a yield of about 95%.
I got an A. This double-stranded cDNA was converted into EC0 of 20 units.
RI methylase was reacted at 37°C for 1 hour. 1 for this
6 units of EcoRT (manufactured by Takara Shuzo) were combined. Add 16 units of EcoRI (manufactured by Takara Shuzo) to 37
After reacting at ℃ for 2 hours, 3 epharose C
Passed through an L-4B column to purify approximately 0.8 μg of C[)N.
I got an A. Next, this CDNA0.4μ3 and λ(ltlO
After ligation with 1.0 μg of Arm (manufactured by Vector Cloning Systems), human hepatocyte-derived CD N A
A hybrid DNA into which was inserted was obtained.
得られたハイブリッドDNAについてin Vitr
Oパッケージング[A、[3ecker & M、
Qold :Proc 、 Natl 、 Acad
、 Sci、USA、 72゜581 (1975)
参照]をおこない、ヒト肝細胞由来CD N Aライブ
ラリーを得た。About the obtained hybrid DNA in Vitr
O packaging [A, [3ecker & M,
Qold: Proc, Natl, Acad
, Sci, USA, 72°581 (1975)
Reference] was carried out to obtain a human hepatocyte-derived CD DNA library.
前記実施例2で得られたヒト肝細胞由来cDNAライブ
ラリーを大腸菌C600MM−株に感染させ、プラーク
を形成させた。ヒトα2−プラスミンインヒビター遺伝
子を含むクローンは、[32Pコで標識した合成りNA
P−1及びP−2をプローブとし用い3 ento
nとQaViSのプラークハイブリダイゼーション法[
W、 D、 Benton & R。E. coli C600MM- strain was infected with the human hepatocyte-derived cDNA library obtained in Example 2 to form plaques. A clone containing the human α2-plasmin inhibitor gene was created using a synthetic RNA labeled with [32P].
Using P-1 and P-2 as probes, 3 ento
Plaque hybridization method of n and QaViS [
W, D, Benton & R.
W、 Davis; 5cienc)、 196.
180−(1977)参照]に従って選別した。プロー
ブとして用いた合成りNAはC01lenらによって報
告されていたヒトα2−プラスミンインヒビターの部分
アミノ酸配列[D、 Co11en et at、
;Thrombosisand l−1aemo
s1−1a)、 48.311−314(1982)参
照]と対応するDNA配列をDNAシンセサイザー(ア
プライド・バイオシステムズ製)を用いて合成した。W, Davis; 5cienc), 196.
180-(1977)]. The synthetic RNA used as a probe was the partial amino acid sequence of human α2-plasmin inhibitor reported by Colen et al.
; Thrombosis and l-1aemo
s1-1a), 48.311-314 (1982)] was synthesized using a DNA synthesizer (manufactured by Applied Biosystems).
(P−1) Met Glu Glu Asp Ty
r Pr。(P-1) Met Glu Glu Asp Ty
r Pr.
3’ TACCTT CTT CTG ATG GG
5’CCA A
(P−2) Vat Glu Met Met Gl
n Ala3’ CAG TCT TACTACGT
T CG 5’ACC
ヒトα2−プラスミンインヒヒダター遺伝子を含むλ0
t10ファージからのDNAの調製は、Thomasと
□avts方法[M、 Thomas & R。3' TACCTT CTT CTG ATG GG
5'CCA A (P-2) Vat Glu Met Met Gl
n Ala3' CAG TCT TACTACGT
T CG 5'ACC λ0 containing human α2-plasmin inhibitor gene
Preparation of DNA from t10 phage was performed using the Thomas and □avts method [M, Thomas & R.
W 、 D avts : J ournal of
M olecular3 iology、鮭、
315 (1974)参照]によりおこなった。W, Davts: Journal of
Molecular3 iology, salmon,
315 (1974)].
実施例3で得られたヒトα2−プラスミンインヒビター
cDNAを含むλ0t10D N AをEC0RIで消
化し、挿入されていたヒトα2−プラスミンインヒビタ
ーCD N Aを切り出し、0.8%アガロースゲル電
気泳動によって単離した。このヒトα2−プラスミンイ
ンヒビターcD N A断片0.1μグを制限81%’
消化用バッファー[EcoRI消化では100 mM
Na Cf、 50 mM Tris −HCj
(1)87.5) 、 10 mM M(I C92
,1mMジチオスレイトール水溶液を、BamHI 、
PstI 。The λ0t10 DNA containing the human α2-plasmin inhibitor cDNA obtained in Example 3 was digested with EC0RI, and the inserted human α2-plasmin inhibitor cDNA was cut out and isolated by 0.8% agarose gel electrophoresis. did. This human α2-plasmin inhibitor cDNA fragment of 0.1 μg was restricted to 81%'
Digestion buffer [100 mM for EcoRI digestion
NaCf, 50mM Tris-HCj
(1)87.5), 10mM M(IC92
, 1mM dithiothreitol aqueous solution, BamHI,
PstI.
HindI[[消化では50 mM Na CL 1
0 mM’rris −HCj (p+ 7.5
)、 10 mM MOC12。HindI [[50 mM Na Cl 1 for digestion
0 mM'rris -HCj (p+ 7.5
), 10 mM MOC12.
1 111Mジチオスレイトール水溶液を、3 ma
i消化では20 mM K(J、 10 mM T
ris−HCR(pH8,0) 10 mM M(]
CR2,1mMジチオスレイトール水溶液をそれぞれ
用いた。]10μ文に溶解させ、制限酵素(宝酒造製)
2ユニツトを添加して37℃で1時間消化した。なお、
二種類の制限酵素による消化をおこなう場合には、まず
低温濃度で作用づる制限酵素で処理し、次に所定濃度ま
で塩濃度を上げてから、より高塩濃度で作用する制限酵
素で処理した。制限酵素による消化後、1μ文の0.2
5%ブロモフェノールブルー、 50%グリセO−ル水
溶液を加え1μg/dのエチジウムブロマイドを含む0
.8%〜1.2%アガロースでゲル電気泳動をおこなっ
た。この電気泳動の際、DNA断片の分子量マーカーと
してλファージのDNAを)−1indllIで消化し
たものを用いた。電気泳動後このゲルに対して紫外線を
照射して消化パターンを観察した。各種制限酵素単独に
よる消化、及び二種の制限酵素の組わせによる消化での
消化パターンを解析して後述した。かくして得られた約
2.2Kbのヒトα2−プラスミンインヒビターcD
N Aの制限酵素地図を第2図に示した。1 111M dithiothreitol aqueous solution was added to 3 ma
i digestion, 20 mM K(J, 10 mM T
ris-HCR (pH 8,0) 10 mM M(]
CR2 and a 1mM dithiothreitol aqueous solution were used, respectively. ] Dissolve in 10 μg of restriction enzyme (manufactured by Takara Shuzo)
2 units were added and digested at 37°C for 1 hour. In addition,
When performing digestion with two types of restriction enzymes, the sample is first treated with a restriction enzyme that acts at a low concentration, then the salt concentration is raised to a predetermined concentration, and then treated with a restriction enzyme that acts at a higher salt concentration. After digestion with restriction enzymes, 0.2 of 1μ statement
Add 5% bromophenol blue and 50% glycerinol aqueous solution, and add 0.5% bromophenol blue containing 1 μg/d of ethidium bromide.
.. Gel electrophoresis was performed in 8% to 1.2% agarose. During this electrophoresis, λ phage DNA digested with )-1indllI was used as a molecular weight marker for the DNA fragment. After electrophoresis, the gel was irradiated with ultraviolet light and the digestion pattern was observed. The digestion patterns of digestion using various restriction enzymes alone and digestion using a combination of two restriction enzymes were analyzed and described later. The approximately 2.2 Kb human α2-plasmin inhibitor cD thus obtained
The restriction enzyme map of NA is shown in FIG.
大腸菌用プラスミド1)tJc8 2μりを実施例4に
準じて制限酵素E(IORIで消化したものに対してア
ルカリ性ホスファターゼ(E、 coli C75)
(宝酒造製)を1.0ユニット加えて58℃で2時間反
応させた。反応終了後、反応液中のアルカリ性フォスフ
ァターゼを失活・除去するためにフェノール抽出を3回
繰返した。このようにして得られたptJC8のEco
RI/アルカリ性フォスファターゼ処理液に、実施例4
で得たヒトα2−プラスミンインヒビターECORI消
化cD N A断片を加え、2ユニツトのT4−DNA
リガーゼを12℃で16時間反応させて連結させた。Plasmid for E. coli 1) 2 μl of tJc8 was digested with restriction enzyme E (IORI) according to Example 4, and then treated with alkaline phosphatase (E, coli C75).
(manufactured by Takara Shuzo) was added and reacted at 58° C. for 2 hours. After the reaction was completed, phenol extraction was repeated three times to inactivate and remove alkaline phosphatase in the reaction solution. Eco of ptJC8 thus obtained
Example 4 was added to the RI/alkaline phosphatase treatment solution.
Add the human α2-plasmin inhibitor ECORI-digested cDNA fragment obtained from 2 units of T4-DNA.
Ligation was performed by reacting ligase at 12° C. for 16 hours.
大腸菌LE392株の形質転換は、通常のCaCR2法
[M、 V、 Norgard et al、; Ge
ne 。Transformation of E. coli strain LE392 was performed using the standard CaCR2 method [M, V, Norgard et al.;
ne.
ユ、 297(178)参照コに従い調製し、前記p
UC8にヒトα2−プラスミンインヒビターEcoRI
消化CD N A断片を連結させたハイブリットDNA
を形質転換した。形質転換した大腸菌LE392を50
μg/fnflの濃度のアンピシリンを含んだL−プロ
スプレートに接種した。このプレートを37℃で1晩培
養して形質転換株を生育させた。得られたコロニーより
公知の方法を用いてDNAを調製し、アガロースゲル電
気泳動により目的のハイブリッドDNAを確認し、この
1つをpPI41と命名した。Yu, 297 (178), and prepared according to the above p.
Human α2-plasmin inhibitor EcoRI in UC8
Hybrid DNA ligated with digested CD DNA fragments
was transformed. 50 transformed E. coli LE392
L-pros plates containing ampicillin at a concentration of μg/fnfl were inoculated. This plate was cultured overnight at 37°C to grow the transformed strain. DNA was prepared from the obtained colonies using a known method, and the desired hybrid DNA was confirmed by agarose gel electrophoresis, and one of these was named pPI41.
ヒトα2−プラスミンインヒビター〇DNA塩基配列は
S anoerのジデオキシシーケンス法CF。Human α2-plasmin inhibitor DNA base sequence was Sanoer's dideoxy sequencing method CF.
5anaer et al、 : Proc 、
Natl 、 Acad 。5anaer et al.: Proc.
Natl, Acad.
S ci、 U S A 74.5463−5467
(1977)参照]により決定した。Sci, USA 74.5463-5467
(1977)].
たとえばヒトα2−プラスミンインヒビターcDNA+
7)EcoRI消化断片を公知の方法に従ってM13m
p18又はM13111 p19ベクターのEOOR
Iサイトに挿入し、大腸菌JM105株に形質転換させ
る。形質転換させたJM105株によって生産される一
重鎖ハイブリットDNAを公知の方法に従って調製した
。この−重鎖DNAを用いて、M13シーケンスキット
(アマージャム製)を用いてシーケンス反応をおこない
、7M尿素を含む6%ポリアクリルアミドゲル電気泳動
で分離した。ゲルを乾燥後−80℃で一晩オートラジオ
グラフィーをおこなった後、分離パターンの解析をおこ
ない、ヒトα2−プラスミンインヒビターの塩基配列決
定のための資料とした。For example, human α2-plasmin inhibitor cDNA+
7) The EcoRI-digested fragment was converted into M13m according to a known method.
EOOR of p18 or M13111 p19 vector
It is inserted into the I site and transformed into E. coli JM105 strain. Single-stranded hybrid DNA produced by the transformed JM105 strain was prepared according to a known method. Using this heavy chain DNA, a sequence reaction was performed using an M13 sequence kit (manufactured by Amerjam), and the DNA was separated by electrophoresis on a 6% polyacrylamide gel containing 7M urea. After drying the gel and performing autoradiography at -80°C overnight, the separation pattern was analyzed and used as data for determining the base sequence of human α2-plasmin inhibitor.
かくして第1図に示されたヒトα2−プラスミンインヒ
ビターをコードする塩基配列が決定された。Thus, the nucleotide sequence encoding the human α2-plasmin inhibitor shown in FIG. 1 was determined.
添付第1図は、本発明によるヒトα2−プラスミンイン
ヒビターのアミノ酸配列及び遺伝子断片を締ものであり
、第2図はヒトα2−プラスミンインヒビターのcDN
△の制限酵素地図を示したものであり、同第2図におい
て白い部分は本発明のアミノ酸配列のコード領域であり
、黒い部分は非コード領域である。Attached Figure 1 shows the amino acid sequence and gene fragment of human α2-plasmin inhibitor according to the present invention, and Figure 2 shows the cDNA of human α2-plasmin inhibitor.
A restriction enzyme map of Δ is shown, and in FIG. 2, the white part is the coding region of the amino acid sequence of the present invention, and the black part is the non-coding region.
Claims (1)
3番目(Lys)までのアミノ酸配列で示されたヒトα
_2プラスミンインヒビター断片をコードした遺伝子断
片。 2、該遺伝子断片が添付第1図における第1番目(T)
から第1209番目(G)までの塩基配列で示されたも
のである第1項記載の遺伝子断片。 3、添付第1図における第1番目(Phe)から第40
3番目(Lvs)までのアミノ酸配列で示されたヒトα
_2−プラスミンインヒビターのアミノ酸配列。 4、添付第1図のアミノ酸配列における第314番目(
Ser)、第315番目(Arg)、第316番目(M
et)または第317番目(Ser)のいずれかのアミ
ノ酸をアミノ末端とし、第403番目(Lys)までの
アミノ酸をカルボキシル末端とするアミノ酸配列。[Claims] 1. 1st (Phe) to 40th in attached Figure 1
Human α shown by the amino acid sequence up to the third (Lys)
A gene fragment encoding a _2 plasmin inhibitor fragment. 2. The gene fragment is number 1 (T) in the attached Figure 1.
2. The gene fragment according to item 1, which is represented by the base sequence from position 1209 to position (G). 3. 1st (Phe) to 40th in attached Figure 1
Human α shown in the amino acid sequence up to the third (Lvs)
_2-Amino acid sequence of plasmin inhibitor. 4. 314th position (in the amino acid sequence shown in attached Figure 1)
Ser), 315th (Arg), 316th (M
et) or the 317th amino acid (Ser) as the amino terminal, and the amino acids up to the 403rd (Lys) as the carboxyl terminal.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP30175386A JPS63157984A (en) | 1986-12-19 | 1986-12-19 | Amino acid sequence and gene fragment of human alpha2-plasmin inhibitor |
DE8787118686T DE3777359D1 (en) | 1986-12-19 | 1987-12-16 | AMINO ACID SEQUENCE OF THE HUMAN ALPHA 2 PLASMIN INHIBITOR AND CDNS, AND GENOIC DNA CODING THIS AMINO ACID SEQUENCE. |
EP19870118686 EP0272609B1 (en) | 1986-12-19 | 1987-12-16 | Amino acid sequence of human alpha2-plasmin inhibitor and cdna and genomic dna encoding said amino acid sequence |
US07/803,896 US5221738A (en) | 1986-12-19 | 1991-12-09 | cDNA and genomic DNA encoding the amino acid sequence of human α2 -plasmin inhibition |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP30175386A JPS63157984A (en) | 1986-12-19 | 1986-12-19 | Amino acid sequence and gene fragment of human alpha2-plasmin inhibitor |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS63157984A true JPS63157984A (en) | 1988-06-30 |
Family
ID=17900750
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP30175386A Pending JPS63157984A (en) | 1986-12-19 | 1986-12-19 | Amino acid sequence and gene fragment of human alpha2-plasmin inhibitor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS63157984A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0286778A (en) * | 1988-09-21 | 1990-03-27 | Teijin Ltd | Gene capable of coding human alpha2-plasmin inhibitor protein |
JPH02227091A (en) * | 1988-09-05 | 1990-09-10 | Teijin Ltd | New plasmin-inhibiting protein |
-
1986
- 1986-12-19 JP JP30175386A patent/JPS63157984A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH02227091A (en) * | 1988-09-05 | 1990-09-10 | Teijin Ltd | New plasmin-inhibiting protein |
JPH0286778A (en) * | 1988-09-21 | 1990-03-27 | Teijin Ltd | Gene capable of coding human alpha2-plasmin inhibitor protein |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ueyama et al. | Structure of a human smooth muscle actin gene (aortic type) with a unique intron site | |
VOORDOUW et al. | Cloning and sequencing of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) | |
Anderson et al. | Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe. | |
Huang et al. | A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60 | |
MAURER‐FOGY et al. | Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+‐dependent phospholipid‐binding protein | |
Jenne et al. | Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the" pexin" family and a model for their evolution | |
Ferretti et al. | Total synthesis of a gene for bovine rhodopsin. | |
Ernst et al. | Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA. | |
Gitt et al. | Evidence that a human soluble beta-galactoside-binding lectin is encoded by a family of genes. | |
Rixon et al. | Nucleotide sequence of the gene for the. gamma. chain of human fibrinogen | |
DK174927B1 (en) | DNA encoding human factor VIII: C, vector comprising this DNA, transformed host comprising this DNA, and method for producing human factor VIII: C | |
US4994559A (en) | Human basic fibroblast growth factor | |
Horiuchi et al. | Core sequence of two separable terminus sites of the R6K plasmid that exhibit polar inhibition of replication is a 20 bp inverted repeat | |
JPH0811073B2 (en) | Hirudin's genetic engineering method | |
US5089406A (en) | Method of producing a gene cassette coding for polypeptides with repeating amino acid sequences | |
Chung et al. | Characterization of a cDNA clone coding for the beta chain of bovine fibrinogen. | |
JPH0866194A (en) | Gene carrying code for human tumor necrosis factor mutein | |
JPS6228A (en) | Cdna coding against human von willebrand's factor, plasminogen or phage having cdna or fragment, microbial, animal or human cell having same, manufacture of protein by host cultivation, protein obtained and bioactive drug composition | |
EP0209568B1 (en) | Dna sequences, recombinant dna molecules and processes for producing human lipocortin-like polypeptides | |
Case et al. | Repeated nucleotide sequence arrays in Balbiani ring 1 of Chironomus tentans contain internally nonrepeating and subrepeating elements. | |
JPS63157984A (en) | Amino acid sequence and gene fragment of human alpha2-plasmin inhibitor | |
EP0226181B1 (en) | Human placenta angiogenic factor capable of stimulating capillary endothelial cell protease synthesis, DNA synthesis and migration | |
US5081019A (en) | DNA sequences, recombinant DNA molecules and processes for producing lipocortin-like polypeptides | |
US4950646A (en) | DNA sequences, recombinant DNA molecules and processes for producing human lipocortin-like polypeptides | |
Domdey et al. | Sequence analysis of the cloned mRNA coding for glyceraldehyde‐3‐phosphate dehydrogenase from chicken heart muscle |