CN1924013A - Prourokinase modifier, preparation method, pharmaceutical composition, use, encoding gene, carrier containing gene and transformation cell - Google Patents
Prourokinase modifier, preparation method, pharmaceutical composition, use, encoding gene, carrier containing gene and transformation cell Download PDFInfo
- Publication number
- CN1924013A CN1924013A CNA2005100149619A CN200510014961A CN1924013A CN 1924013 A CN1924013 A CN 1924013A CN A2005100149619 A CNA2005100149619 A CN A2005100149619A CN 200510014961 A CN200510014961 A CN 200510014961A CN 1924013 A CN1924013 A CN 1924013A
- Authority
- CN
- China
- Prior art keywords
- modifier
- prourokinase
- district
- pro
- cell
- 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
- 239000003607 modifier Substances 0.000 title claims abstract description 74
- 108010073863 saruplase Proteins 0.000 title claims description 39
- 108090000623 proteins and genes Proteins 0.000 title claims description 20
- 238000002360 preparation method Methods 0.000 title claims description 8
- 239000008194 pharmaceutical composition Substances 0.000 title description 4
- 230000009466 transformation Effects 0.000 title description 3
- -1 preparation method Substances 0.000 title description 2
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 claims abstract description 43
- NNRFRJQMBSBXGO-CIUDSAMLSA-N (3s)-3-[[2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NNRFRJQMBSBXGO-CIUDSAMLSA-N 0.000 claims abstract description 16
- 101000829980 Homo sapiens Ral guanine nucleotide dissociation stimulator Proteins 0.000 claims abstract description 15
- 102100023320 Ral guanine nucleotide dissociation stimulator Human genes 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims description 28
- 230000002537 thrombolytic effect Effects 0.000 claims description 21
- 208000007536 Thrombosis Diseases 0.000 claims description 19
- 230000014509 gene expression Effects 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 12
- 235000001014 amino acid Nutrition 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 230000006870 function Effects 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 238000005304 joining Methods 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 claims description 4
- 241000699802 Cricetulus griseus Species 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 210000001672 ovary Anatomy 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 108020004511 Recombinant DNA Proteins 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 238000001890 transfection Methods 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims 3
- 102000039446 nucleic acids Human genes 0.000 claims 3
- 239000000969 carrier Substances 0.000 claims 1
- 125000004395 glucoside group Chemical group 0.000 claims 1
- 238000012856 packing Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 abstract description 42
- 206010050661 Platelet aggregation inhibition Diseases 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 229960005356 urokinase Drugs 0.000 description 40
- 230000000694 effects Effects 0.000 description 25
- 102000010911 Enzyme Precursors Human genes 0.000 description 23
- 108010062466 Enzyme Precursors Proteins 0.000 description 23
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 19
- 108091008146 restriction endonucleases Proteins 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 102000013566 Plasminogen Human genes 0.000 description 12
- 108010051456 Plasminogen Proteins 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 101710195328 Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex Proteins 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 102100033571 Tissue-type plasminogen activator Human genes 0.000 description 10
- 108050006955 Tissue-type plasminogen activator Proteins 0.000 description 10
- 230000029087 digestion Effects 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 229940012957 plasmin Drugs 0.000 description 9
- 208000005189 Embolism Diseases 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 238000010369 molecular cloning Methods 0.000 description 8
- 230000013595 glycosylation Effects 0.000 description 7
- 238000006206 glycosylation reaction Methods 0.000 description 7
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 7
- 238000011144 upstream manufacturing Methods 0.000 description 7
- 230000002490 cerebral effect Effects 0.000 description 6
- 210000004351 coronary vessel Anatomy 0.000 description 6
- 239000003527 fibrinolytic agent Substances 0.000 description 6
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 5
- 208000006193 Pulmonary infarction Diseases 0.000 description 5
- 208000001435 Thromboembolism Diseases 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 230000003480 fibrinolytic effect Effects 0.000 description 5
- 230000007575 pulmonary infarction Effects 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 206010008190 Cerebrovascular accident Diseases 0.000 description 4
- 101150074155 DHFR gene Proteins 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 208000032843 Hemorrhage Diseases 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 208000006011 Stroke Diseases 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000001772 blood platelet Anatomy 0.000 description 4
- 230000003073 embolic effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 206010061216 Infarction Diseases 0.000 description 3
- 108010038807 Oligopeptides Proteins 0.000 description 3
- 102000015636 Oligopeptides Human genes 0.000 description 3
- 102000001938 Plasminogen Activators Human genes 0.000 description 3
- 108010001014 Plasminogen Activators Proteins 0.000 description 3
- 102100033237 Pro-epidermal growth factor Human genes 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000007574 infarction Effects 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 229940127126 plasminogen activator Drugs 0.000 description 3
- 230000002797 proteolythic effect Effects 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102220023258 rs387907548 Human genes 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 102000053602 DNA 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
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 108010035030 Platelet Membrane Glycoprotein IIb Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000002429 anti-coagulating effect Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 229940023064 escherichia coli Drugs 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 229940116978 human epidermal growth factor Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 244000309715 mini pig Species 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 102220023257 rs387907546 Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229960000103 thrombolytic agent Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000003151 transfection method Methods 0.000 description 2
- 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 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010072454 CTGCAG-specific type II deoxyribonucleases Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- 101800001224 Disintegrin Proteins 0.000 description 1
- 101800000620 Disintegrin-like Proteins 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000801481 Homo sapiens Tissue-type plasminogen activator Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 101150046652 M2 gene Proteins 0.000 description 1
- 241001563100 Mixcoatlus barbouri Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000271477 Sistrurus Species 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010064129 Thrombogen Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 102220369447 c.1352G>A Human genes 0.000 description 1
- 102220369445 c.668T>C Human genes 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000004004 carotid artery internal Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 231100000284 endotoxic Toxicity 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- CTCHDPAJHVDPRN-UHFFFAOYSA-N integrin Chemical compound C=1C(OC)=CC(O)=C(C(C=2CC=C(C)C)=O)C=1OC=2C1=CC=C(O)C=C1O CTCHDPAJHVDPRN-UHFFFAOYSA-N 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000007794 irritation Effects 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
- 210000004731 jugular vein Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- YDTFRJLNMPSCFM-YDALLXLXSA-M levothyroxine sodium anhydrous Chemical compound [Na+].IC1=CC(C[C@H](N)C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 YDTFRJLNMPSCFM-YDALLXLXSA-M 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000033885 plasminogen activation Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108010003233 preprourokinase Proteins 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001210 retinal vessel Anatomy 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 230000008470 skin growth Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention discloses a new abbokinase modifier in the genetic engineering domain, which consists of KGD and RGDS, wherein the tPA K2 segment and abbokinase P area are induced to the N end of molecular structure, which is compatible with platelet aggregation inhibition.
Description
Technical field
The invention belongs to the genetically engineered field, specifically, relate to uPA (prourokinase, abbreviation pro-UK) modifier, the preparation method of this modifier contains the pharmaceutical composition and the purposes of this modifier and this modifier encoding gene, the carrier that contains its gene contains the transformant of this carrier.
Background technology
1979, Husain etc. were purified into uPA (US Patent No.4381346,1979) first from urine.UPA claims strand urine type plasminogen activator (single chain urine-type plasminogen activator again, be called for short scu-PA) form by 411 amino acid, be that a kind of molecular weight is the single chain protein matter molecule of 50～54Kda (its difference may be different with cell and the purification process produced, thereby cause due to the level of glycosylation difference).Natural pro-UK is an alkaline glycoprotein, contains 12 pairs of disulfide linkage, and its precursor molecule contains the hydrophobic signal peptide of being made up of 20 amino-acid residues.The pro-UK iso-electric point is 8.9-9.05, and specific activity is commonly considered as 1-1.3 * 10
5IU/mg, measuring method mainly contain dissolving circle method (Xu Kangsen etc.. the research of urokinase quality standard. work of drug test communication, 1979; 9:241; Gao Lihuas etc., the improvement of uPA measuring method reaches the observation to CL-11G cell expressing uPA horizontal stability. biotechnology communication, 1997; 8:94) with S2444 substrate colorimetry (Winkler M.E.﹠amp; Blaber M. (1986) Purification and characterization of recombinant single-chain urokinase producedin Escherichea coli.Biochemistry25 (5): 4041-14)).Pro-UK has identical antigenic determinant with urokinase, can with the urokinase antibody response.
Sophisticated proUK is divided into three protein structure domains by its function: Urogastron structural domain (EGF domain, be called for short the E district), handle structural domain (Ktingle domain is called for short the K district) and proteolytic ferment structural domain (protease domain is called for short the P district).E district: form by 5-49 amino acids residue, its sequence and human epidermal growth factor's height homology, be rich in halfcystine, amino-acid residue 11 and 19,13 and 31,33 and 42 a pair of disulfide linkage is arranged respectively, wherein the amino acid of 12-32 position is responsible for combining with the cell surface specific receptors, particularly Asn
22, Asn
27, His
29, Trp
30Residue has irreplaceable effect.K district: form by the 50-136 amino acids, gain the name because of its three-dimensional structure is folded into a handle shape structure.3 pairs of disulfide linkage are arranged, and its position is respectively between 50 and 130,71 and 113,102 and 126 residues.Also there is the Kringle district among Profibrinolysin (plasminogen is called for short plg), tissue-type plasminogen activator's (being called for short t-PA), thrombogen and the lipoprotein α.The function in this district main with scleroproein in conjunction with relevant, by K domain structure and function in the analysis blood coagulation component, infer that this district may participate in the interaction between albumen and the albumen, but concrete function it be unclear that.P district: form by the 159-411 amino acids, be positioned at pro-UK shuttle cardinal extremity, have 5 pairs of disulfide linkage in this district, constitute a serine protease catalytic center.His wherein
204, Asp
255, Ser
356Constitute the active centre of this enzyme, Asn
302Be glycosylation site.Mutating experiment shows: Ile
159Existence not only determine the activity of double-stranded UK derivative, and the activity of decision strand pro-UK.Lys
300High catalytic activity to pro-UK has vital role.Be to comprise activating site Lys between handle structural domain and the proteolytic ferment structural domain
158-Ile
159At interior joining peptide, Lys under the effect of pro-UK at plasmin
158-Ile
159After the peptide splitting of chain of key becomes double-stranded UK, by Cys
148-Cys
279Between disulfide linkage be connected to form stable secondary structure.
The Pro-UK full length gene is 6387bp, be positioned on No. 10 karyomit(e), contain 10 introns, 11 exons, the exon II proUK signal peptide sequence of mainly encoding, exon IV coding schedule skin growth factor-class formation territory, exon V and VI coding kringle district, exon VII-XI encoding serine proteolytic enzyme structural domain.The Pro-UK gene can be transcribed into the ripe mRNA of 2.4kb, and initial translation product is preceding pro-UK, contains the signal peptide sequence of being made up of 20 amino-acid residues, during emiocytosis signal peptide is removed and produces ripe pro-UK.
Natural Pro-UK molecule is a glycoprotein, and its glycosylation site is at Asn
302On.Pro-UK is mediated by the specific receptors on the liver plasma membrane mainly in vivo, and is degraded in the liver cell lysosome, about 8 minutes of the transformation period in the blood.Studies show that nonglycosylated pro-UK is strong by plasmin (plasmin) activatory energy force rate glycosylation pro-UK, its catalytic activity is also strong than glycosylation pro-UK, but non-glycosylated more unstable than glycosylation in blood plasma, easily degraded.Think that at present the reason that glycosylation influences enzymic activity may be Asn
302Site (the Lys that present position and pro-UK are activated
158-Ile
159) and enzyme active center more approaching.
Pro-UK belongs to the serine stretch protein enzyme family, is a kind of of plasminogen activator, and its thrombolysis mechanism is not have active Profibrinolysin to be transformed into activated plasmin, and plasmin has the ability of crosslinked fibrin in the degraded clot, with thrombus.The process of Pro-UK plasminogen activation can be summarized as three-step reaction: a small amount of Profibrinolysin of the inherent enzymic activity catalysis of pro-UK becomes plasmin; The a spot of plasmin catalysis pro-UK that produces changes UK into; 3) UK catalysis Profibrinolysin produces a large amount of plasmins.
Pro-UK has special thrombus dissolving effect, and it only acts on the Profibrinolysin that is adsorbed by scleroproein and does not act on free Profibrinolysin in the blood circulation.Therefore be difficult for causing whole body Profibrinolysin and fibrinogen degradation and exhaustion during the Pro-UK thrombolysis, avoid that systematicness is hemorrhage to wait side effect to take place.Pro-UK itself is very weak to fibrinous affinity, does not also have consensus at present for its optionally fibrinolytic mechanism.
UPA makes [Homes et al, 1985 by gene recombination technology; Winkler et al, 1986; Nolli etal, 1989; Nelles et al, 1987; Li Fengzhi etc., 1991; Satoh et al, 1993; Satoh et al, 1996; Xu Yang etc., 1993; Weaver et al, 1994], show fibrinolytic effect in vivo and in vitro.Since (Collen D. such as Collen in 1986, etal.Coronary thrombolysis in patients with acute myocardial infarction by intravenous infusion ofsynergic thrombolytic agents.Am Heart is (5) J.112: 1083-4.) adopted first since 6 heart stalk patients of pro-UK thromboembolism treatment, existing a large amount of clinical trials confirm that it can be used for the treatment of thrombotic diseases clinically.
T-PA also has special thrombus dissolving effect.T-PA is made up of 527 amino acid, molecular weight 70,000th, and a kind of glycoprotein belongs to the serine stretch protein enzyme.The t-PA of strand can be cut at the R275-I276 place by plasmin becomes double-stranded t-PA.The N-end is called heavy chain (or A chain) for one section, and the C end is called light chain (or B chain) for one section.The A chain is made up of four structural domains: (1) refers to type district (F district): comprise residue 1-43, with the finger type district homology of Fibronectin; (2) epidermal growth factor subarea (EGF district or E district): comprise residue 50-84, with the human epidermal growth factor homology; (3) handle district (Kringle) 1: comprise residue 92-172, with handle district homology in the Profibrinolysin; (4) handle district (Kringle) 2: comprise residue 180-265.B chain and serine protease homology claim proteolytic ferment district (P district) again, and its active centre is by H322, D371, and S478 forms.T-PA has strong affinity to scleroproein, when scleroproein does not exist, t-PA is very weak activator just, but scleroproein can significantly increase the active rate of t-PA to Profibrinolysin, increase the stability of t-PA and Profibrinolysin mixture, plasmin is only produced on the scleroproein surface, thus the scleroproein in the special thrombus.
RGDS sequence (arginine-glycine-aspartic acid-Serine) and KGD (LYS-GLY-aspartic acid) sequence, it is the oligopeptides sequence that is present on the class crude substance (disintegrin), by this specificity site can be specifically with suppress GP IIb/IIIa receptors bind on the thrombocyte, GP IIb/IIIa is the glycoprotein on the activatory platelet membrane, is the core element in the thrombosis that causes of mediation thrombocyte.Therefore, RGDS and KGDW oligopeptides can suppress platelet function, suppress thrombosis.Experimental results show that the natural and synthetic oligopeptides that contains RGDS and KGDW sequence can significantly suppress hematoblastic aggreation.(Ruoslahiti?E，et?al.New?perspective?in?cell?adhesion，RGD?and?integrins.Science，1987，154：367-382；Scarborough?RM，et?al.Barbouri：a?GP?IIb-IIIa-specific?integrin?antagonistfrom?the?venom?of?Sistrurus?m.Barbouri.J.Biol.Chem.1991，266，15，9359-9362.)
Although uPA and t-PA have the narrow spectrum thrombolytics of scleroproein, the thrombolysis effect better and hemorrhage side effect is less, but in medicine production and clinical treatment, development active higher, security is bigger, target better, longer thrombolytic drug of transformation period in lower, the body of stalk rate again, will be long-term and persistent problem.
Summary of the invention
Technical problem to be solved
The present invention aims to provide a kind of new, high reactivity, high thrombolysis specificity and has the modifier of the uPA of anticoagulant effect concurrently.
Summary of the invention
The invention provides a kind of new prourokinase modifier, its molecular structure is for to introduce the K2 district fragment of tPA and the P district of uPA in proper order at the N end, this molecule also comprises KGD and RGDS small peptide, KGD and RGDS small peptide can be held at N, can hold at C, also can be between the P district of the K2 district fragment of tPA and uPA, can also be in the P district of the K2 district of tPA or uPA, KGD also can not link to each other (the part-structure synoptic diagram is seen Fig. 1) that can link to each other with the RGDS small peptide.At each structural domain of this molecule, promptly can also comprise the joining region between the K2 district of KGD, RGDS, tPA and the uPA P district, this joining region length is preferably between 10 to 20 amino acid.The present invention also comprises the derivative of above-mentioned prourokinase modifier, this derivative is in the aminoacid sequence of above-mentioned prourokinase modifier, process is replaced, is lacked or increases one or several amino acid derived protein, and has identical functions with above-mentioned prourokinase modifier.Derivative polypeptide-NH2 end can have an additional methionine residue, maybe can have one or several glycosyl side chain.This purified prourokinase modifier or its derivative are compared with the uPA of prototype, and thrombolytic activity is higher, and specificity is stronger, has the anticoagulant effect simultaneously concurrently.
The present invention also further comprises the medicinal compositions that contains above-mentioned this prourokinase modifier or modifier derivative, this medicinal compositions by above-mentioned this prourokinase modifier or modifier derivative be suitable for medicinal carrier and mix mutually.This medicinal compositions can be used to dissolve the thrombus of humans and animals, is used for the treatment of thrombotic diseases clinically, as myocardium infarct, cerebral infarction, retinal vessel embolism, lower limbs vein infarction, pulmonary infarction etc.
The present invention comprises a kind of dna molecular in addition again, for example, a kind of recombinant DNA molecules, prourokinase modifier that this molecule encoding is above-mentioned or modifier derivative, perhaps at the preference codon of different expression systems, the recombinant DNA molecules that designs according to the aminoacid sequence of this prourokinase modifier or modifier derivative.The present invention also comprises the recombinant vectors that contains above-mentioned dna molecular, and with above-mentioned carrier cell transformed.This carrier and host cell can be any general and commercial expression vector and host cell, it both can be prokaryotic expression system, as escherichia expression system, the expression vector and the host cell of Bacillus subtilus expression system etc., it also can be eukaryotic expression system, as yeast expression system, the expression vector of insect baculovirus expression system and mammalian cell expression system etc. and host cell (Sa nurse Bruce (Sambrool, work such as J.), Huang Peitang etc. translate, molecular cloning experiment guide (the 3rd edition), Beijing, Science Press, 2002,8:1605).
The present invention also comprises the prourokinase modifier that a kind of preparation is described or the method for modifier derivative herein, this method comprise with the coding a kind of dna molecular that changes the configuration uPA transform a kind of cell, cultivate this cell, and express this modifier, and separate this modifier (albumen).
Beneficial effect
The compound that these recruits form found at occurring in nature never in the past that this was a purpose of the present invention.These prourokinase modifiers have special thrombolytic effect, can substitute uPA effectively.Test shows that they compare with uPA, has higher fibrinolytic, and better thrombolysis specificity in addition, also has the effect of platelet aggregation-against concurrently.Therefore, when clinical use, can reduce dosage, reduce hemorrhage and again side effect such as infraction take place.
Description of drawings
The structural representation of prourokinase modifier M2
Embodiment
Specifically the present invention is specifically described below in conjunction with embodiment.
Embodiment 1
Prourokinase modifier M
2The structure of gene (Nucleotide and aminoacid sequence are seen sequence table SEQ ID NO.1 and SEQID NO.2 respectively)
1.t-PA K
2The district makes up
According to document (Pennica, D., et al.Cloning and expression of human tissue-type plasminogenactivator cDNA in E.coli.Nature 301 (5897), 214-221 (1983) .), K
2District's total order is classified as:
GTGACTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAG
TCGGGTGCCTCCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACAC
AGCACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGG
AATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGA
CGTGGGAGTACTGTGATGTGCCCTCCTGCT
Divide three sections synthetic K
2Template:
First section:
AAGTGGATATCATGTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCAC
AGCCTCACCGAGTCGGGTGCCTCCTGCCTGCCG?TGGAATTCCATGAT
Upstream primer is: 5 '-GGATATCATGTGCTACTTTGGGAA-3 ' restriction enzyme site is EcoRV
Downstream primer is: 5 '-AAGAATTCCACGGGAGGCAGGA-3 ' restriction enzyme site is EcoRI
Second section:
TGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAAC
CGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCATGCATCCAC
Upstream primer is: 5 '-GGAATTCAATGATCCTGATAG-3 ' restriction enzyme site is EcoRI
Downstream primer is: 5 '-TCCGGCAGTAGTTGTGTTTG-3 ' restriction enzyme site is HpaII
The 3rd section:
TGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGC
CCAGGCACTGGGCCTGGGCAAACACAACTACTGCCGGAAT
Upstream primer is: 5 '-TGCCGGAATCCTGATGGGGAT-3 ' restriction enzyme site is HpaII
Downstream primer is: 5 '-AATGCATGAGGGCACATCACAGT-3 ' restriction enzyme site is NsiI
The above-mentioned three sections sequences of difference pcr amplification, amplified production carries out enzyme with corresponding enzyme and cuts after reclaiming purifying, and enzyme is cut and is connected after product reclaims purifying.Connect product after deactivation, be that template is that primer carries out pcr amplification with article one upstream primer and the 3rd downstream primer, amplified production is t-PA K
2The district.Be connected Transformed E .coliDH5 α with the pGEM-Teasy carrier after reclaiming purifying.
2.ProUK-P the district makes up
According to bibliographical information (Jacobs, P., et al.Molecular cloning, sequencing, and expression in Escherichiacoli of human preprourokinase cDNA.DNA 4 (2), 139-146 (1985) .), ProUK-P region sequence (upstream and downstream respectively contains the non-P of part district encoding sequence) is:
GCATGACTGCGCAGATGGAAAAAAGCCCTCCTCTCCTCCAGAAGAATTAAAATTTC
AGTGTGGCCAAAAGACTCTGAGGCCCCGCTTTAAGATTATTGGGGGAGAATTCACC
ACCATCGAGAACCAGCCCTGGTTTGCGGCCATCTACAGGAGGCACCGGGGGGGCTC
TGTCACCTACGTGTGTGGAGGCAGCCTCATCAGCCCTTGCTGGGTGATCAGCGCCA
CACACTGCTTCATTGATTACCCAAAGAAGGAGGACTACATCGTCTACCTGGGTCGCT
CAAGGCTTAACTCCAACACGCAAGGGGAGATGAAGTTTGAGGTGGAAAACCTCATC
CTACACAAGGACTACAGCGCTGACACGCTTGCTCACCACAACGACATTGCCTTGCT
GAAGATCCGTTCCAAGGAGGGCAGGTGTGCGCAGCCATCCCGGACTATACAGACCA
TCTGCCTGCCCTCGATGTATAACGATCCCCAGTTTGGCACAAGCTGTGAGATCACTG
GCTTTGGAAAAGAGAATTCTACCGACTATCTCTATCCGGAGCAGCTGAAAATGACTG
TTGTGAAGCTGATTTCCCACCGGGAGTGTCAGCAGCCCCACTACTACGGCTCTGAA
GTCACCACCAAAATGCTATGTGCTGCTGACCCCCAATGGAAAACAGATTCCTGCCA
GGGAGACTCAGGGGGACCCCTCGTCTGTTCCCTCCAAGGCCGCATGACTTTGACTG
GAATTGTGAGCTGGGGCCGTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACG
AGAGTCTCACACTTCTTACCCTGGATCCGCAGTCACACCAAGGAAGAGAATGGCCT
GGCCCTCTGAGGGTCCCCAGGGAGGAAACGGGCACCACCCGCTTTCTTGCTGGTTG
TCATTTTTGCAGTAGAGTCATCTCCATCAGCTGTAAGAAGAGACTGGGAAGATAGGC
TCTGCACAGATGGATTTGCCTGTGGCACCACCAGGGTGAACGACAATAGC
Chemosynthesis ProUK-P district encoding sequence, and its 5 ' end add that restriction enzyme site is NsiI, 3 ' end add that restriction enzyme site is XbaI.NsiI/XbaI double digestion, enzyme are cut and are connected Transformed E .coli DH5 α with the pGEM-Teasy carrier after product reclaims purifying.
3.KGD+RGDS lead the structure of peptide
The synthetic double-stranded DNA segment that contains the KGD+RGDS sequence of gene, as leading the peptide template:
GAAGCTTCGCGGCGACAGCAAGGGTGACTGGGATATCC
Upstream, downstream restriction enzyme site be HindIII and EcoRV respectively.
4.M2 structure
Obtain the KGD+RGDS district with HimdIII and EcoRV double digestion Teasy-KGD+RGDS, with EcoRV and NsiI double digestion Teasy-K
2Obtain K
2Distinguish, obtain the P district with NsiI and XbaI double digestion Teasy-P, with HindIII and XbaI double digestion expression vector pcDNA3.1 (+), reclaim purifying, enzyme is cut the product connection and is spent the night, and connects product Transformed E .coli DH5 α, modifier M
2Structure is finished.
Embodiment 2
Contain prourokinase modifier M
2Expression in Chinese hamster ovary cell
1.t-PA K
2The district makes up: the method by embodiment 1 is carried out.
2.ProUK-P the district makes up: the method by embodiment 1 is carried out.
3.Signal+KGD+RGDS lead the structure in peptide district
The synthetic single stranded DNA segment that contains the Signal+KGD+RGDS sequence is as leading the peptide template
GAAGCTTATGAGAGCCCTGCTGGCGCGCCTGCTTCTCTGCGTCCTGGTCCTGAGCG
ACTCCAAAGGCCGCGGCGACAGCAAGGGTGACTGGGATATCC
Upstream primer is: 5 '-GAAGCTTATGAGAGCCCTGCTG-3 ' restriction enzyme site is HindIII
Downstream primer is: 5 '-AGATATCCCAGTCACCCTTGCT-3 ' restriction enzyme site is EcoRV
Pcr amplification product is Signal+KGD+RGDS and leads peptide.Amplified production is connected Transformed E .coli DH5 α after reclaiming purifying with the pGEM-Teasy carrier.
4.pcDNA-M
2The structure of plasmid
Obtain the Signal+KGD+RGDS district with HindIII and EcoRV double digestion Teasy-Signal+KGD+RGDS, with EcoRV and NsiI double digestion Teasy-K
2Obtain K
2Distinguish, obtain the P district with NsiI and XbaI double digestion Teasy-P, with HindIII and XbaI double digestion expression vector pcDNA3.1 (+), reclaim purifying, enzyme is cut the product connection and is spent the night, and connects product Transformed E .coliDH5 α, obtains pcDNA-M
2Plasmid.
5.pTSL-M
2Structure
Synthetic dhfr gene complete sequence
According to GeneBank sequence number: NM_010049, check in dhfr gene total order and classify as:
ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAG
AACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGA
CCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGG
TTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGA
GAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTT
AAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAG
GCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTCAGACTCTTTGTGACA
AGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATAT
AAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCA
AGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAA
This sequence of synthetic, and add the EcoRV restriction enzyme site at 5 ' end of sequence, 3 ' end adds the PstI restriction enzyme site.
With pGL3-enhancer is template, design primer, synthetic promoter and polyA sequence respectively
Design of primers is as follows:
Promoter primer: 5 '-GGAGCTCTGTCAGTTAGGGTGTGGAAA-3 ' restriction enzyme site is SacI
5 '-GGATATCGCCTCCTCACTACTTCTGGA-3 ' restriction enzyme site is EcoRV
The polyA aligning primer: 5 '-GCTGCAGCGTGTAATTCTAGAGTCGG-3 ' restriction enzyme site is PstI
5 '-GAGATCTTACCACATTTGTAGAGGTTTT-3 ' restriction enzyme site is BglII
With pEGFP-1 is template, design primer, synthetic SV40 enhanser
The enhanser primer is: 5 '-GAGATCTGCTGTGGAATGTGTGTCAG-3 ' restriction enzyme site is BglIl
5 '-GGAGCTCTACAAATGTGGTAAAATCGA-3 ' restriction enzyme site is SacI
Synthetic enhanser, promotor and polyA sequence of pcr amplification respectively, purified pcr product, respectively with corresponding enzyme double digestion, enzyme is cloned into the Teasy carrier after cutting product recovery purifying, conforms to expection through determined dna sequence with this three segment DNA and dhfr gene order.The Teasy carrier of this reorganization is cut with the BglII enzyme, and enzyme is cut product and is reclaimed back and the linearizing pcDNA-M of BglII
2Plasmid connects jointly, connects product Transformed E .coli DH5 α, so far, and modifier M
2Expression vector--pTSL-M
2Structure is finished.
6.CHO-dhfr
-Cell expressing modifier M
2
Utilize the big extraction reagent kit of endo-free plasmid, extract high purity in a large number and do not have endotoxic pTSL-M
2Plasmid is with calcium phosphate precipitation infection protocol stable transfection CHO-dhfr-cell, with containing 2 * 10
-8The selection substratum of the methotrexate of M (MTX) carries out dhfr and the dual screening of MTX, obtains the stabilized cell clone, according to the activity detection of target protein, the Chinese hamster ovary celI that screening obtains efficiently expressing from cell clone.Identify that through the SDS-PAGE electrophoresis molecular weight of the prourokinase modifier M2 of this cell expressing is about 48kd, its expression level can reach 25pg/cell/d.Rolling bottle is carried out in this Chinese hamster ovary celI strain cultivate, the fermented liquid of results is behind ion-exchange chromatography, gel chromatography and affinity chromatography purifying, and low-temperature freeze drying becomes protein powder, 4 ℃ of preservations.This is prourokinase modifier M
2Pure product.
The big extraction reagent kit of endo-free plasmid is available from German QIAGEN company, and the plasmid extracting method carries out according to this test kit operation instruction.The calcium phosphate precipitation transfection method carries out (R.I. Fu Leixieni work, Zhang Jingbo are deposited slowly to fasten etc. and translated animal cell culture---basic fundamental guide (the 4th edition), Beijing, Science Press, 2004,9) with reference to the method for " animal cell culture---basic fundamental guide ".
Embodiment 3
Utilize insect baculovirus expression system to express modifier M
2
With modifier M
2Gene clone is gone among the baculovirus expression carrier pFast-BacHTa, through liposome Superfect mediation transfection Sf 9 insect cell, results substratum supernatant, after the affinity column affinity chromatography by coupling antiurokinase original antibody, desalination and concentration, freeze-drying obtains the prourokinase modifier M of purifying
2Protein powder.Identify that through the SDS-PAGE electrophoresis molecular weight of the prourokinase modifier M2 of Sf9 insect cell expression conforms to expection.
The affinity column of coupling antiurokinase original antibody is provided by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, transfection method is with reference to " molecular cloning experiment guide " (Sa nurse Bruce (Sambrool, work such as J.), Huang Peitang etc. translate, molecular cloning experiment guide (the 3rd edition), Beijing, Science Press, 2002,8).
Embodiment 4
Prourokinase modifier M
2The structure, acquisition, expression, purifying and the evaluation (protokaryon) of host strain of expression vector
The M2 gene order is cloned in the prokaryotic expression carrier pKK233-2 plasmid, after being converted into intestinal bacteria JA221 respectively with Calcium Chloride Method, induce with IPTG, express prourokinase modifier on a small scale, the collecting cell extract therefrom screens the high bacterial strain of expression amount as engineering bacteria through SDS-PAGE electrophoresis rough determination expression amount.Identify the prourokinase modifier M that this project bacterium is expressed through the SDS-PAGE electrophoresis
2Proteic molecular weight is approximately 46kd.Because there is reference literature (Winkler ME ﹠amp in target protein inclusion body form with non-activity in thalline; Blaber M, Purification and characterization ofrecombinant single-chain urokinase produced in Escherichia coli.1986, Biochemistry15; 25 (14): the method that 4041-4045.) provides gives external change renaturation, can obtain the active prourokinase modifier M of 300000 units from 1 liter of substratum
2
((Sambrool J.) waits work to Sa nurse Bruce to methods such as the structure of expression vector, the acquisition of host strain and protein expression, and Huang Peitang etc. translate with reference to " molecular cloning experiment guide ", molecular cloning experiment guide (the 3rd edition), Beijing, Science Press, 2002,8).
Embodiment 5
Prourokinase modifier M
2Thrombolysis activity and the Comparative Examples of the thrombolysis activity of uPA
The fibrin plate method:
With reference to 2005 editions Chinese Pharmacopoeia urokinase activity measuring methods, with 0.1mol/LPBS (pH7.4) preparation 1.4% agarose, water-bath is boiled molten earlier, get the bovine fibrinogen (2mg/ml) and 0.45ml (10u/ml) of 11ml and 11ml37 ℃ preheating, the thrombin of beef of 37 ℃ of preheatings mixes, and pours 24 orifice plates into and covers, and coagulates the back punching, aperture 3mm, add 10 μ l/ aperture urokinase standard substance (5U, 10U, 20U respectively, 40U is totally four holes) and testing sample, 37 ℃ of 6-18h.According to the typical curve of drawing, calculate the activity of testing sample.
Bubble method is measured with reference to 2005 editions Chinese Pharmacopoeia urokinase activity measuring methods.
Recording the proteic external fibrinolytic of prourokinase modifier M2 according to fiber flat band method and bubble method is 2000IU/10
6Cell, and the external fibrinolytic of simultaneously treated uPA is 1000IU/10
6Cell.
Embodiment 6
Prourokinase modifier M
2The anticoagulant experiment
Concrete test operation is according to document (Jia LG, et al., Function of disintegrin-like/cysteine-rich domains ofatrolysin A.Inhibition of platelet aggregation by recombinant protein and peptide antagonists.JBiol Chem.1997,16; 272 (20): 13094-102) carry out, the concentration of inductor ADP is 5mmol/L, and sample is to inhibiting rate=(solvent control MA-sample MA)/solvent MA * 100% of platelet aggregation.The platelet aggregation-against test shows prourokinase modifier M
2What albumen suppressed 50% platelet aggregation partly suppresses constant IC
50Be 11.2 μ mol/L, almost do not have a rejection capability of platelet aggregation and natural urokinase is former.As seen prourokinase modifier M
2Albumen is compared with natural urokinase is former, has tangible rejection capability of platelet aggregation.
Embodiment 7
Contain prourokinase modifier M
2Pharmaceutical composition and the treatment thrombotic diseases purposes
The prourokinase modifier M that the Chinese hamster ovary celI strain that obtains among the embodiment 2 is expressed
2Through ion-exchange chromatography, gel chromatography and affinity chromatography purifying, be lyophilized into protein powder after purified product added protective material, vehicle, make powder injection, 4 ℃ of cryopreservation.
1. recombinant human urokinase zymogen modifier M
2Solvency action to thrombus in the miniature pig coronary artery
Miniature pig is tested by China directly cause the endarterium damage, form thrombus in the coronary artery gradually with the electricity irritation coronary artery.With various dose recombinant human urokinase zymogen modifier M
2Carry out thromboembolism treatment, analyze the thrombolysis effect, measure epicardial electrogram, serum biochemistry creatine kinase, hemorrhage and index observing thrombolytic effects such as clotting time, Profibrinolysin with the section of coronary artery pathology.Found that recombinant human urokinase zymogen modifier M
240000IU/kg, 80000IU/kg, three dosage groups of 160000IU/kg are compared with control group all and can obviously be dwindled the coronary artery thrombus cross-sectional area, alleviate degree of myocardial ischemia and scope, dwindle infarct.Compare with tr-PA and recombinant human urokinase zymogen simultaneously, the result shows: three kinds of medicines of same dose (80000IU/kg) to the solvency action of coronary artery thrombus, to myocardial ischemia and myocardial infarction and related biochemical indicator to improve effect similar, but recombinant human urokinase zymogen modifier M
2Hemorrhage side effect be starkly lower than tr-PA, and onset is faster, simultaneously recombinant human urokinase zymogen modifier M
2Effect strengthen with dosage.
2. recombinant human urokinase zymogen modifier M
2To Golden Hamster pulmonary infarction and venothrombotic solvency action
In Golden Hamster pulmonary infarction animal model, animal subject is divided into 7 groups, 10 every group.10min begins administration form pulmonary infarction in embolus injects body after, adopts 10% (volume 0.1ml injects time 1min) of first intravenous injection total dose, remains 90% vein constant speed gasing injection (volume 0.9ml, the infusion time is 1h).Control group gives physiological saline, is subjected to 3 dosage groups of reagent to give injection recombinant human urokinase zymogen modifier M
2, dosage is respectively 1,3,8 * 10
4IU/kg, 3 positive drug control group give urokinase injection (UK), injection recombinant human urokinase zymogen, rt-PA respectively, and corresponding dosage is respectively 8 * 10
4, 8 * 10
4IU/kg and 1mg/kg.Above-mentionedly respectively organize every animals administer cumulative volume and be 1ml.120min puts to death animal.Core after the off-test, lung measures residual blood grumeleuse radioactivity, is calculated as follows thrombolysis percentage ratio:
And get its radioactivity of tissue test such as Tiroidina, liver, kidney, urine, blood, muscle, calculate recovery rate simultaneously.Judge that as the effective standard of thromboembolism treatment each dosage group is efficient above 50% with dissolution rate.
In Golden Hamster phlebothrombosis model, animal is divided into 7 groups immediately by body weight, 10 every group.Unclamp ligature 30min (being thrombosis 60min), begin administration, adopt 10% (volume 0.1ml injects time 1min) of first intravenous injection total dose, remain 90% vein constant speed gasing injection (volume 0.9ml, the infusion time is 2h) through the offside external jugular vein.Control group gives physiological saline, is subjected to 3 dosage groups of reagent to give recombinant human urokinase zymogen modifier M
2, dosage is respectively 1,3,8 * 10
4IU/kg, 3 positive drug control group give urokinase injection (UK), injection recombinant human urokinase zymogen, rt-PA respectively, and corresponding dosage is respectively 8 * 10
4, 8 * 10
4IU/kg and 1mg/kg.Above-mentionedly respectively organize every animals administer cumulative volume and be 1ml.180min puts to death animal.The method of calculation of thrombolysis percentage ratio are with the pulmonary infarction model.
Found that the thrombolytic effect of recombinant human urokinase zymogen modifier and uPA, tPA are suitable, but thrombolysis speed is fast than uPA and tPA.
3. recombinant human urokinase zymogen modifier M
2Therapeutic action to the embolic cerebral apoplexy
Adopt the internal carotid artery injection to form rabbit and rat embolic cerebral apoplexy model from body whole blood grumeleuse, the application of radiation isotropic substance (
99mTc) external dynamic tracer technique and nucleus magnetic resonance video method find that vein gives injection recombinant human urokinase zymogen modifier M on rabbit embolic cerebral apoplexy model
22.0,4.0,8.0 * 10
4IU/kg can produce gradual significant thrombolytic effect, its ED
50Be 3.2 * 10
4IU/kg.Recombinant human urokinase zymogen modifier M
2(8 * 10
4IU/kg) thrombolysis speed is fast than urokinase, rt-PA (1mg/kg), and thrombolytic efficacy is also stronger, and is suitable with the recombinant human urokinase zymogen effect.Recombinant human urokinase zymogen modifier M
2In the thromboembolism treatment process, hematencephalon degree and scope are light than urokinase, rt-PA, recombinant human urokinase zymogen.
On rat embolic cerebral apoplexy model, vein gives injection recombinant human urokinase zymogen modifier M
24,8,16 * 10
4IU/kg, the animal behavior obstacle obviously improves, and magnetic resonance detection shows that cerebral lesion volume and scope obviously dwindle, and TTC dyeing shows that cerebral infarction volume and scope also obviously dwindle, the conventional organization pathological examination finds that the brain tissue impairment severity makes moderate progress, and does not see obvious hematencephalon.Recombinant human urokinase zymogen modifier M
2(8 * 10
4IU/kg) the provide protection effectiveness to cerebral tissue is suitable with isodose urokinase, recombinant human urokinase zymogen and rt-PA (2mg/kg) behind the thromboembolism treatment, but the hematencephalon side reaction is few than urokinase and rt-PA.
4. recombinant human urokinase zymogen modifier M
2Solvency action to rat retina central artery thrombus
Adopt photochemical method to cause that arter centralis retinae forms thrombus, use doppler's microcirculation blood flow instrument and measure the retina relative discharge, find 10% and 1 hour subsequently venoclysis surplus 90% of intravenous injection total dose, recombinant human urokinase zymogen modifier (2.0,4.0,8.0 * 10
4IU/kg) the obvious thrombus of energy makes the arter centralis retinae of embolism occur leading to again, and recanalization rate is respectively 60%, 75%, 90%, and thrombolysis speed is than uPA (8.0 * 10
4IU/kg), urokinase (8.0 * 10
4IU/kg) fast with rt-PA (2mg/kg).
Above-mentioned template and primer adopt the dna synthesizer by specification to carry out, and sequencing adopts automatic determining nucleic acid sequence instrument by specification to carry out.PGEM-Teasy carrier, pGL3-enhancer plasmid, pKK233-2 plasmid, bacterial strain E.coli DH5 α and JA221 are available from Promega company, used enzyme and reagent are available from Promega company, the pEGFP-1 plasmid is available from Clonetech company, and liposome Superfect is available from invitrogen company.Enzyme is cut with ligation condition by specification and is carried out.The PCR product reclaims the QIAEX II Gel Extraction Kit test kit of purifying employing available from QIAGEN company.PCR reaction, electrophoresis, competent cell preparation, transform method that the equimolecular biological method adopts " molecular cloning experiment guide " and carry out that ((Sambrool J.) waits work to Sa nurse Bruce, and Huang Peitang etc. translate, molecular cloning experiment guide (the 3rd edition), Beijing, Science Press, 2002,8).Plasmid extracts, and adopts Shanghai to give birth to worker's test kit K192, carries out according to operation instruction.
Nucleotide and aminoacid sequence table
SEQ?ID?NO.1
cgcggcgaca?gcaagggtga?ctgggatatc?atgtgctact?ttgggaatgg?gtcagcctac 60
cgtggcacgc?acagcctcac?cgagtcgggt?gcctcctgcc?tcccgtggaa?ttcaatgatc 120
ctgataggca?aggtttacac?agcacagaac?cccagtgccc?aggcactggg?cctgggcaaa 180
cacaactact?gccggaatcc?tgatggggat?gccaagccct?ggtgccacgt?gctgaagaac 240
cgcaggctga?cgtgggagta?ctgtgatgtg?ccctcatgca?tgcatgactg?cgcagatgga 300
aaaaagccct?cctctcctcc?agaagaatta?aaatttcagt?gtggccaaaa?gactctgagg 360
ccccgcttta?agattattgg?gggagaattc?accaccatcg?agaaccagcc?ctggtttgcg 420
gccatctaca?ggaggcaccg?ggggggctct?gtcacctacg?tgtgtggagg?cagcctcatc 480
agcccttgct?gggtgatcag?cgccacacac?tgcttcattg?attacccaaa?gaaggaggac 540
tacatcgtct?acctgggtcg?ctcaaggctt?aactccaaca?cgcaagggga?gatgaagttt 600
gaggtggaaa?acctcatcct?acacaaggac?tacagcgctg?acacgcttgc?tcaccacaac 660
gacattgcct?tgctgaagat?ccgttccaag?gagggcaggt?gtgcgcagcc?atcccggact 720
atacagacca?tctgcctgcc?ctcgatgtat?aacgatcccc?agtttggcac?aagctgtgag 780
atcactggct?ttgg?aaaga?gaattctacc?gactatctct?atccggagca?gctgaaaatg 840
actgttgtga?agctgatttc?ccaccgggag?tgtcagcagc?cccactacta?cggctctgaa 900
gtcaccacca?aaatgctgtg?tgctgctgac?ccacagtgga?aaacagattc?ctgccaggga 960
gactcagggg?gacccctcgt?ctgttccctc?caaggccgca?tgactttgac?tggaattgtg 1020
agctggggcc?gtggatgtgc?cctgaaggac?aagccaggcg?tctacacgag?agtctcacac 1080
ttcttaccct?ggatccgcag?tcacaccaag?gaagagaatg?gcctggccct?ctga 1134
SEQ?ID?NO.2
Arg?Gly?Asp?Ser?Lys?Gly?Asp?Trp?Asp?Ile?Met?Cys?Tyr?Phe?Gly?Asn
1 5 10 15
Gly?Ser?Ala?Tyr?Arg?Gly?Thr?His?Ser?Leu?Thr?Glu?Ser?Gly?Ala?Ser
20 25 30
Cys?Leu?Pro?Trp?Asn?Ser?Met?Ile?Leu?Ile?Gly?Lys?Val?Tyr?Thr?Ala
35 40 45
Gln?Asn?Pro?Ser?Ala?Gln?Ala?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys
50 55 60
Arg?Asn?Pro?Asp?Gly?Asp?Ala?Lys?Pro?Trp?Cys?His?Val?Leu?Lys?Asn
65 70 75 80
Arg?Arg?Leu?Thr?Trp?Glu?Tyr?Cys?Asp?Val?Pro?Ser?Cys?Met?His?Asp
85 90 95
Cys?Ala?Asp?Gly?Lys?Lys?Pro?Ser?Ser?Pro?Pro?Glu?Glu?Leu?Lys?Phe
100 105 110
Gln?Cys?Gly?Gln?Lys?Thr?Leu?Arg?Pro?Arg?Phe?Lys?Ile?Ile?Gly?Gly
115 120 125
Glu?Phe?Thr?Thr?Ile?Glu?Asn?Gln?Pro?Trp?Phe?Ala?Ala?Ile?Tyr?Arg
130 135 140
Arg?His?Arg?Gly?Gly?Ser?Val?Thr?Tyr?Val?Cys?Gly?Gly?Ser?Leu?Ile
145 150 155 160
Ser?Pro?Cys?Trp?Val?Ile?Ser?Ala?Thr?His?Cys?Phe?Ile?Asp?Tyr?Pro
165 170 175
Lys?Lys?Glu?Asp?Tyr?Ile?Val?Tyr?Leu?Gly?Arg?Ser?Arg?Leu?Asn?Ser
180 185 190
Asn?Thr?Gln?Gly?Glu?Met?Lys?Phe?Glu?Val?Glu?Asn?Leu?Ile?Leu?His
195 200 205
Lys?Asp?Tyr?Ser?Ala?Asp?Thr?Leu?Ala?His?His?Asn?Asp?Ile?Ala?Leu
210 215 220
Leu?Lys?Ile?Arg?Ser?Lys?Glu?Gly?Arg?Cys?Ala?Gln?Pro?Ser?Arg?Thr
225 230 235 240
Ile?Gln?Thr?Ile?Cys?Leu?Pro?Ser?Met?Tyr?Asn?Asp?Pro?Gln?Phe?Gly
245 250 255
Thr?Ser?Cys?Glu?Ile?Thr?Gly?Phe?Gly?Lys?Glu?Asn?Ser?Thr?Asp?Tyr
260 265 270
Leu?Tyr?Pro?Glu?Gln?Leu?Lys?Met?Thr?Val?Val?Lys?Leu?Ile?Ser?His
275 280 285
Arg?Glu?Cys?Gln?Gln?Pro?His?Tyr?Tyr?Gly?Ser?Glu?Val?Thr?Thr?Lys
290 295 300
Met?Leu?Cys?Ala?Ala?Asp?Pro?Gln?Trp?Lys?Thr?Asp?Ser?Cys?Gln?Gly
305 310 315 320
Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Ser?Leu?Gln?Gly?Arg?Met?Thr?Leu
325 330 335
Thr?Gly?Ile?Val?Ser?Trp?Gly?Arg?Gly?Cys?Ala?Leu?Lys?Asp?Lys?Pro
340 345 350
Gly?Val?Tyr?Thr?Arg?Val?Ser?His?Phe?Leu?Pro?Trp?Ile?Arg?Ser?His
355 360 365
Thr?Lys?Glu?Glu?Asn?Gly?Leu?Ala?Leu
370 375
Claims (10)
1. prourokinase modifier, its molecular structure is
(i) introduce the K2 district fragment of tPA and the P district of uPA in proper order at the N end, this molecule also comprises KGD and RGDS small peptide;
(ii) in the aminoacid sequence of (i), one or several amino acid derived protein is replaced, lacks or increased to process, and have identical functions with above-mentioned prourokinase modifier, perhaps derivative has an additional methionine residue at the N end, or has one or several glucoside side chain.
2. as claim 1 prourokinase modifier, also comprise the joining region between each structural domain of this molecule, this joining region length is between 10 to 20 amino acid.
3. a medicinal compositions that is used for thrombolysis is made up of with suitable medicinal carrier claim 1 or 2 described prourokinase modifiers.
4. the application of the composition of claim 3 in the medicine of preparation treatment thrombotic diseases.
5. the nucleic acid molecule of the prourokinase modifier of the claim 1 of encoding, its sequence is:
(i) nucleic acid molecule of the described aminoacid sequence of coding claim 1;
(ii) at the preference codon of different expression systems, the recombinant DNA molecules that designs according to the aminoacid sequence of this prourokinase modifier.
6. the expression vector that contains the nucleic acid molecule of claim 5.
7. expression vector as claimed in claim 6, wherein said carrier are carrier for expression of eukaryon.
8. the cell strain that forms of the carrier transfection host cell of claim 7.
9. cell strain as claimed in claim 8, wherein said host cell is a Chinese hamster ovary celI.
10. preparation method who prepares the prourokinase modifier of claim 1, this method comprise that a kind of appropriate host cell of transfection is cultivated this cell, expresses and this modifier of separation and purification with the dna molecular of claim 5 appropriate carriers of packing into.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005100149619A CN1924013A (en) | 2005-09-01 | 2005-09-01 | Prourokinase modifier, preparation method, pharmaceutical composition, use, encoding gene, carrier containing gene and transformation cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005100149619A CN1924013A (en) | 2005-09-01 | 2005-09-01 | Prourokinase modifier, preparation method, pharmaceutical composition, use, encoding gene, carrier containing gene and transformation cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1924013A true CN1924013A (en) | 2007-03-07 |
Family
ID=37816838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2005100149619A Pending CN1924013A (en) | 2005-09-01 | 2005-09-01 | Prourokinase modifier, preparation method, pharmaceutical composition, use, encoding gene, carrier containing gene and transformation cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1924013A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139578B (en) * | 2007-08-24 | 2010-09-15 | 河北师范大学 | Double-functional staphylokinase mutant having platelet aggregation resistance and fibrinolytic activity, application and preparation method thereof |
-
2005
- 2005-09-01 CN CNA2005100149619A patent/CN1924013A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139578B (en) * | 2007-08-24 | 2010-09-15 | 河北师范大学 | Double-functional staphylokinase mutant having platelet aggregation resistance and fibrinolytic activity, application and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1103815C (en) | Thrombin mutants | |
| JPH07507448A (en) | Tissue plasminogen activator glycosylation variants with improved therapeutic properties | |
| GB2119804A (en) | Human tissue plasminogen activator pharmaceutical compositions containing it processes for making it and dna and transformed cell intermediates therefor | |
| JPH07147984A (en) | Gene coding for polykringleplasminogen activator and vector containing the same | |
| AU2012315516B2 (en) | Compositions and methods for modulating hemostasis | |
| CN1402788A (en) | Fibrinolytically active polypeptide | |
| IE62832B1 (en) | Methode and materials for expression of human plasminogen variant | |
| CN1125450A (en) | Factor VII-derived peptides | |
| CN1102438A (en) | Bifunctional variants of urokinase with improved fibrinolytic properties and thrombin-inhibiting activity | |
| JP2003532375A (en) | DNA encoding human serine protease T | |
| CN1924013A (en) | Prourokinase modifier, preparation method, pharmaceutical composition, use, encoding gene, carrier containing gene and transformation cell | |
| CN1786175A (en) | Plasmid for expressing recombination human tPA and its construction method | |
| CN1563387A (en) | Gene of earthworm plasmin and genetic engineerng strain, construction and application | |
| WO2000049871A1 (en) | An anti-angiogenic kringle protein and its mutants | |
| CN1429909A (en) | Production method of tissue plasminogen activator mutant | |
| CN1244894A (en) | Tissue type plasminogen activator (t-PA) variants: compositions and methods of use | |
| CN1226926A (en) | Plasminogen Activator capable of being activated by thrombin | |
| EP2494040A1 (en) | Method of producing recombinant vitamin k dependent proteins | |
| CN1380408A (en) | A polypeptide-Glu/Leu/Phe/Val dehydrogenase-11.55 and polynucleotide for coding this polypeptide | |
| CN1380319A (en) | A polypeptide-haematoglobin-9.24 and polynucleotide for coding this polypeptide | |
| CN1393549A (en) | Polypeptide-trypsinogen-26.51 and polynucleotide for coding it | |
| CN1341641A (en) | A novel polypeptide-human protein 11.33 with cysteine proteinase active site and polynucleotide for coding said polypeptide | |
| CN1324931A (en) | New polypeptide polycopper oxidase 12 and polynucleotides for encoding same | |
| CN1345942A (en) | Novel polypeptide-human protein 10.34 containing pyridine nucleotide disulfide bond oxido-reductase I grade active site and polynucleotide for encoding said polypeptide | |
| CN1807603A (en) | Low pyrogen staphylokinase and its preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |