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 |
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