CN1690199A - Three urokinase mutants - Google Patents
Three urokinase mutants Download PDFInfo
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- CN1690199A CN1690199A CN 200410033936 CN200410033936A CN1690199A CN 1690199 A CN1690199 A CN 1690199A CN 200410033936 CN200410033936 CN 200410033936 CN 200410033936 A CN200410033936 A CN 200410033936A CN 1690199 A CN1690199 A CN 1690199A
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Abstract
For eliminating application point of the thrombase, weakening bond of plaminogen activator inhabitor-1 of profibrinolytic original and urokinase original, advancing resistance of urokinase original tomutant to thrombase and PAI-1, and thus prolonging the half life of urokinase original in plasm and reducing the clinical dosage of urokinase,the invention introduces mutating natural urokinase original gene with PCP mutagenesis method of fixed points, mutating para-arginine156(Arg156) to histidine (His156), para-Arg178,179,181 to Lys178,Lys179 and His181,and Arg156,178,179,181 to His156,Lys178,Lys179 and His181, and obtaining three kinds of urokinase original tomutant. After sequential analysis, linking to animal gene expression carrier, shifting urokinase original tomutant into Chinese rat's ovary cell with liposome medium to express, then the analyze the characteristic. It can also apply to the expression of bacteria, byeast and animal ioreactor.
Description
Technical field
The novel uPA that the present invention relates in thrombolytic treatment, use (prourokinase, pro-UK) mutant (pro-Ukm), pro-Ukm with to zymoplasm or/and inhibitors of plasminogen activator inhibitor-1 has resistibility is characteristics.
Background information
Pro-UK is that a kind of single chain serine protease is former, forms molecular mass 54kDa by 411 amino-acid residues.The prominent feature of pro-UK is that it has higher intrinsic activity (intrinsic activity), its intrinsic activity is higher more than 10000 times than other serine stretch protein proenzyme (as trypsinogen and chymotrypsinogen), shows as the activation to the substrate and the plasminogen of synthetic.
Pro-UK activates at Lys through kallikrein (kallikrein) or plasmin (plasmin)
158-Ile
159Disconnect and form double chain urokinase (urokinase).Pro-UK and urokinase be energy activation fiber protein dissolution proenzyme (plasminogen all, be called for short Profibrinolysin), make it to become fibrinoclase (plasmin), fibrinoclase can be with the scleroproein at thrombus place and the degraded of other plasma proteinss, thereby pro-UK and urokinase all can be used for the treatment of thrombus infraction.
In addition, zymoplasm (thrombin) but the hydrolysis of peptide bond between the Arg156-Phe157 of catalysis pro-UK, produce the urokinase two strands of zymoplasm breaking type, this two strands only has the activity of faint plasminogen activation, and can effectively resist the activation of fibrinoclase to it.Concentration of thrombin is higher in the new thrombus that forms, and makes the active reduction rapidly of pro-UK, thereby has influenced the effect of pro-UK treatment thrombus.Discover that zymoplasm has the action site specificity, peptide bond (Chang J Y.Thrombin specificity between can only hydrolysis Arg/Lys-Xaa, requirement for apolar amino acids adjacent to thethrombin cleavage site of polypeptide substrate.Eur J Biochem, 1985,151:217～224.).
The catalytic activity of pro-UK is subjected to the regulation and control of four kinds of serpins at least, is respectively pro-UK activator inhibitor (plaminogen activator inhibitor, PAI) 1,2,3 and nexin-1 (nexin 1).Wherein PAI-1 is (the plaminogen activator of plasminogen activator in the blood plasma, PA) main inhibitor, it can form stable PA-PAI-1 mixture with PA, this mixture can be caught by hepatic parenchymal cells and degrade very soon, the major cause of Here it is PA transformation period in thrombolytic treatment.Discover, many positively charged amino-acid residue (R178RHRGGS184) in the variable loop of pro-UK surface can form " salt bridge " with the electronegative amino-acid residue in the PAI-1 molecule, this salt bridge (Adams DS that in PA and PAI-1 cohesive process, plays a decisive role, Griffin LA, Nachajko WR, Reddy VB, Wei CM.A synthetic DNA encoding a modified human urokinase resistant to inhibition by serumplasminogen activator inhibitor.J Biol Chem.1991; 266 (13): 8476-82.).Handle uPA with phenylglyoxal, can cause the Arg residue to change, result of study shows that changing four Arg residues can make derivative the most stable in blood plasma, and has higher thrombolysis activity, infer 3 variable loops (178RRHRGGS184) that are arranged in the uPA surface are arranged in these four Arg residues, an Arg residue in addition may be exactly Arg156 (Moukhametova LI, Aisina RB, YuLG, et al.Properties of the urokinase-type plasminogen activator modified with phenylglyoxal.Russian Journal of Bioorganic Chemistry, 2002,28 (4): 278-283.).
Urokinase does not have selectivity to scleroproein, can activate the Profibrinolysin in the circulation of blood non-specificly, produces the free plasmin of higher concentration, thereby produces systematic fibrinolytic state, causes bleeding or side effect such as apoplexy.Compare with urokinase, the notable feature of pro-UK is to have more selectivity when low dosage, and it has activation to the Profibrinolysin that is combined in the scleroproein surface and be very weak to the activation of free Profibrinolysin when lower concentration.But, when the clinical treatment thrombus, use higher dosage, the specificity when pro-UK has just lost its low dosage in order to guarantee the thrombolysis effect.
To the objective of the invention is in order overcoming the above problems the pro-UK gene to be transformed, to obtain to have the pro-UKm of good thrombus effect, it has the aminoacid sequence of natural pro-UK, the arginine (Arg to the 156th
156) sport Methionin (Lys), be called urokinase zymogen mutant 1 (pro-Ukm1); To the 178th, 179,181 arginine (Arg
178 179,181) sport 178Lys, 179Lys, 181His is called urokinase zymogen mutant 2 (pro-Ukm2); Also with the 156th and the 178th, 179,181 arginine (Arg
156,178,181) site mutation become 156Lys, 178Lys, 179Lys, 181His, this mutant is called urokinase zymogen mutant 3 (pro-Ukm3).These mutant can make its resistibility to zymoplasm and PAI-1 improve, thus the clinical usage quantity that prolongs the survival time of pro-UK in blood plasma and reduction pro-UK.
Summary of the invention
The present invention is a goal gene with the pro-UK gene, adopts PCR site-directed mutagenesis method that specific site is suddenlyd change, and changes the pro-UK mutant over to eukaryotic cell and expresses by liposome-mediated.
Technology contents of the present invention comprises:
Pass through rite-directed mutagenesis, make up two kinds of pro-UK mutator genes that zymoplasm and/or PAI-1 had resistibility, that is: with the 156th Arg and the 178th of pro-UK, 179,181 arginine becomes Methionin or Histidine and combination mutant respectively, to reduce zymoplasm combining the hydrolysis of pro-UK and PAI-1 and pro-UK.The present invention is according to above-mentioned design philosophy, adopts PCR site-directed mutagenesis method to obtain mutator gene, is connected into pGEM-3ZF, transduces to competent escherichia coli cell, selects positive colony and the pro-UK gene order in its plasmid DNA is measured.
The structure of expression vector, that is: reclaim pro-UKm from the cloning vector pGEM-3ZF that imports three kinds of pro-UKm, be connected into pcDNA3.1, transduce to competent escherichia coli cell, select positive colony and the pro-UK gene order in its plasmid DNA is measured.
The expression of pro-UKm in Chinese hamster ovary (CHO) cell that is: with the expression vector linearizing, by the liposome transfection Chinese hamster ovary celI, obtains positive colony by the G418 resistance screening.With the positive colony enlarged culturing, collect supernatant liquor.
The property analysis of pro-UKm, promptly agarose scleroproein solusphere method is measured activity, and SDS-PAGE measures the double-stranded ratio of list of expressing protein, and expressing protein is to zymoplasm resistibility and stable in blood plasma.
Description of drawings
156 Arg of Fig. 1 pro-UKm1 sport the synoptic diagram of His.
178 of Fig. 2 pro-UKm2 and 179 s' Arg sports the synoptic diagram that Lys, 181 Arg sport His.
The Arg that 156 Arg of Fig. 3 pro-UKm3 sport His, 178 and 179 sports the synoptic diagram that Lys, 181 Arg sport His.
Embodiment
1. the structure that zymoplasm or PAI-1 is had the pro-Ukm gene of resistibility
1.1 zymoplasm is had the structure of the pro-Ukm1 gene of resistibility
According to the requirement of the specificity Arg/Lys-Xaa in thrombin action site, the present invention adopts will the encode base CGC of Arg156 of PCR site-directed mutagenesis method to sport CAC (coding His), to avoid zymoplasm to its active influence.
Designing four primers is:
A1 5’CTC?GGT?ACC?ATG?AGA?GCC?CTG?CTG?GCG?CGC?3’·
A2 5’CCC?AAT?AAT?CTT?AAA?GTG?GGG?CCT?CAG?AGT?CT?3’
A3 5’AG?ACT?CTG?AGG?CCC?CAC?TTT?AAG?ATT?ATT?GGG3’
A4 5’TCT?GAT?ATC?AGA?GGG?CCA?GGC?CAT?TCT?CTT?CC?3’
Respectively with A1 and A2, A3 and A4 are that the upstream and downstream primer adopts high-fidelity DNA polymerase pcr amplification pro-UK gene (see figure 1), and condition is 94 ℃, 5min → (94 ℃, 30sec → 65 ℃, 30 sec → 72 ℃, 45 sec) * 30 → 72 ℃, 6min.1% agarose electrophoresis reclaims the fragment that length is respectively 0.54kb and 0.78kb, is template with these two fragments, is primer with A1 and A4, and pcr amplification obtains mutator gene, and amplification condition as above.Mutator gene connects into the pGEM-3ZF of same double digestion after the KpnI+EcoRV enzyme is cut, transformed into escherichia coli competence DH5 α, selecting positive colony is seeded in 5ml LB/Amp (the 100 μ g/ml) nutrient solution, 37 ℃ of shaking culture 10 hours, use the alkaline lysis method of extracting recombinant plasmid, with KpnI+EcoRV (3.2kb+1.3kb), Bgl I (2.7kb+1.6kb+0.2kb), PstI (3.6kb+0.9kb) carries out after enzyme cuts evaluation, enzyme is cut correct recombinant plasmid transformed intestinal bacteria competence DH5 α, 37 ℃ of shaking culture 10 hours, utilize test kit to extract recombinant plasmid, enzyme is cut evaluation, and enzyme is cut correct plasmid the uPA gene order in its plasmid DNA is measured.
1.2 PAI-1 is had the structure of the pro-Ukm2 gene of resistibility
The present invention Arg178 that will encode, Arg179, the base AGG of Arg181, AGG, CGG sports AAG respectively, AAG, CAT (Lys that encodes respectively, Lys, His), reduce the positive charge of scu-PA surface variable loop, reducing itself and the combining of PAI-1, thus the mutant of acquisition stability and high efficiency.
Designing two primers is:
C2 5’GT?GAC?AGA?GCC?CCC?ATG?GTG?CTT?CTT?GTA?GAT?3’
C3 5’ATC?TAC?AAG?AAG?CAC?CAT?GGG?GGC?TCT?GTC?AC?3’
Respectively with A1 and C2, C3 and A4 are that the upstream and downstream primer adopts pcr amplification pro-UK gene (see figure 2), and condition is 94 ℃, 5min → (94 ℃, 30sec → 65 ℃, 30 sec → 72 ℃, 45 sec) * 30 → 72 ℃, 6min.1% agarose electrophoresis reclaims the fragment of length for difference 0.62kb and 0.70kb, is template with these two fragments, is primer with A1 and A4, and pcr amplification obtains mutator gene, and amplification condition as above.Mutator gene inserts the pGEM-3ZF of same double digestion after the KpnI+EcoRV enzyme is cut, transformed into escherichia coli competence DH5 α, selecting positive colony is seeded in 5ml LB/Amp (the 100 μ g/ml) nutrient solution, 37 ℃ of shaking culture 10 hours, use the alkaline lysis method of extracting recombinant plasmid, with KpnI+EcoRV (3.2kb+1.3kb), BglI (2.7kb+1.6kb+0.2kb), PstI (3.6kb+0.9kb) carries out after enzyme cuts evaluation, enzyme is cut correct recombinant plasmid transformed intestinal bacteria competence DH5 α, utilize test kit to extract recombinant plasmid, enzyme is cut evaluation, and enzyme is cut correct plasmid the pro-UK gene order in its plasmid DNA is measured.
1.3 structure to zymoplasm and the resistive pro-Ukm3 gene of PAI-1
Being template to the resistive Pro-UKm gene of PAI-1, respectively with A1 and A2, A3 and A4 are the upstream and downstream primer, adopt the concrete grammar (see figure 3) that makes up the resistive pro-UKm gene of zymoplasm, acquisition is carried out the structure of expression vector to zymoplasm and all resistive pro-UKm gene of PAI-1 after the order-checking.
2. the structure that contains the expression vector of urokinase zymogen mutant
The KpnI+EcoRV enzyme is cut reorganization pGEM-3ZF, utilize test kit to reclaim the 1.3kb fragment, connect into pcDNA3.1 through same double digestion, transformed into escherichia coli competence DH5 α, obtain positive colony through the amicillin resistance screening, be seeded in 5ml LB/Amp (the 200 μ g/ml) nutrient solution, 37 ℃ of shaking culture 10 hours, use the alkaline lysis method of extracting recombinant plasmid, use KpnI+EcoRV, PstI, the NcoI enzyme is cut reorganization pcDNA3.1, and the fragment that obtains is followed successively by 5.4kb+1.3kb, 2.3kb+4.4kb, (3.3kb+2.1kb+0.7kb+0.6kb the reorganization pcDNA3.1 that contains Pro-UKm2 and Pro-Ukm3 gene is cut to 3.3kb+1.7kb+0.7kb+0.6kb+0.3kb through the NcoI enzyme).
3. the expression of mutant in Chinese hamster ovary (CHO) cell
Enzyme is cut correct plasmid transformation escherichia coli DH5 α, be seeded in 5ml LB/Amp (the 100 μ g/ml) nutrient solution, 37 ℃ of shaking culture 10 hours, extract plasmid with test kit, with MfeI enzyme tangent line shape recombinant plasmid pcDNA3.1, test kit reclaims linearizing fragment (size is about 6.7kb), cuts with the Bst1107I enzyme again, reclaims the 4.33kb fragment.The recover Chinese hamster ovary celI of freezing preservation, 37 ℃, 5%CO
2Carry out cell cultures under the condition, added 1% non-essential amino acid, 1% Sodium.alpha.-ketopropionate and 1% couple of anti-(Qing Meisu ﹠amp in the cell culture fluid (1/3CHO-S-SFMII+2/3DMEM); Streptomycin sulphate), when treating that it grows to 70%-90% and converges, adopt liposome 4.33kb fragment transfection CHO cell.In 96 orifice plates and 90mm culture dish, screen resistance clone respectively with Geneticin, the concentration of Geneticin is 1200 μ g/mL, changed liquid every 3 days, change behind the liquid 3 times resistance clone with gained and go to 24 orifice plates after with 0.25% tryptic digestion, all collect supernatant liquor every day, it is active to adopt scleroproein solusphere method to measure, and picks out and expresses the active high cell strain of foreign protein, behind 0.25% tryptic digestion it is gone to the cell cultures flask culture.
4.pro-UKm property analysis
Cell conditioned medium liquid in the collecting cell culturing bottle carries out property analysis through behind the preliminary purification.
4.1 agarose scleroproein solusphere method is measured active
1% sepharose is heated to boiling, draw the 10ml hot solution to 50ml fine taper bottle, be cooled to about 40 ℃, add 1ml ox blood fibrinogen solution (10mg/ml), mix rapidly, add thrombin solution (1.68U/ml) 1ml again, mix rapidly, pour the 90mm Tissue Culture Dish immediately into, gently inclining makes it uniform distribution, bubble is abandoned in suction, placing 30min under the room temperature, is the punch tool punching of 2mm with internal diameter, adds sample 8 μ l in every hole, culture dish is placed in the wet box, place 10h for 37 ℃.Compare with the urokinase standard substance, measure the solusphere size of recombinant protein, with the guestimate thrombolysis activity.
4.2.SDS-PAGE measure the double-stranded ratio of list of expressing protein
The target protein of preliminary purification is carried out polyacrylamide gel reduction electrophoresis, compare with the pro-UK standard substance, used resolving gel concentration is 12%, and voltage is 100V.Behind the usefulness of the gel behind electrophoresis coomassie brilliant blue staining 4h, with the methyl alcohol that does not contain dyestuff-acetic acid solution decolouring, observation 50kb left and right sides protein band calculates its proportion.
4.3. expressing protein to the zymoplasm resistibility and in blood plasma Study on Stability
The pro-UKm of preliminary purification and 1ml human plasma are mixed and place 37 ℃ of water temperature to bathe, and every 1 hour, take out 20 μ l and utilize scleroproein solusphere method to measure thrombolysis activity.
4.4. be used for the expression study of bacterium, yeast and animal bioreactor
The gene DNA of three kinds of pro-Ukm is connected respectively to bacterial expression vector, Yeast expression carrier and animal's mammary gland expression vector carries out expression study.
Urokinase zymogen mutant 1 nucleotide sequence
SEQUENCE?LISTING
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉three kinds of novel urokinase zymogen mutants
＜130〉urokinase zymogen mutant 1 nucleotide sequence
<141>2004-04-20
<160>1
<170>PatentIn?version?3.2
<210>1
<211>1296
<212>DNA
<213>human
<400>1
atgagagccc?tgctggcgcg?cctgcttctc?tgcgtcctgg?tcgtgagcga?ctccaaaggc 60
agcaatgaac?ttcatcaagt?tccatcgaac?tgtgactgtc?taaatggagg?aacatgtgtg 120
tccaacaagt?acttctccaa?cattcactgg?tgcaactgcc?caaagaaatt?cggagggcag 180
cactgtgaaa?tagataagtc?aaaaacctgc?tatgagggga?atggtcactt?ttaccgagga 240
aaggccagca?ctgacaccat?gggccggccc?tgcctgccct?ggaactctgc?cactgtcctt 300
cagcaaacgt?accatgccca?cagatctgat?gctcttcagc?tgggcctggg?gaaacataat 360
tactgcagga?acccagacaa?ccggaggcga?ccctggtgct?atgtgcaggt?gggcctaaag 420
ctgcttgtcc?aagagtgcat?ggtgcatgac?tgcgcagatg?gaaaaaagcc?ctcctctcct 480
ccagaagaat?taaaatttca?gtgtggccaa?aagactctga?ggccccactt?taagattatt 540
gggggagaat?tcaccaccat?cgagaaccag?ccctggtttg?cggccatcta?caggaggcac 600
cgggggggct?ctgtcaccta?cgtgtgtgga?ggcagcctca?tcagcccttg?ctgggtgatc 660
agcgccacac?actgcttcat?tgattaccca?aagaaggagg?actacatcgt?ctacctgggt 720
cgctcaaggc?ttaactccaa?cacgcaaggg?gagatgaagt?ttgaggtgga?aaacctcatc 780
ctacacaagg?actacagcgc?tgacacgctt?gctcaccaca?acgacattgc?cttgctgaag 840
atccgttcca?aggagggcag?gtgtgcgcag?ccatcccgga?ctatacagac?catctgcctg 900
ccctcgatgt?ataacgatcc?ccagtttggc?acaagctgtg?agatcactgg?ctttggaaaa 960
gagaattcta?ccgactatct?ctatccggag?cagctgaaaa?tgactgttgt?gaagctgatt 1020
tcccaccggg?agtgtcagca?gccccactac?tacggctctg?aagtcaccac?caaaatgctg 1080
tgtgctgctg?acccacagtg?gaaaacagat?tcctgccagg?gagactcagg?gggacccctc 1140
gtctgttccc?tccaaggccg?catgactttg?actggaattg?tgagctgggg?ccgtggatgt 1200
gccctgaagg?acaagccagg?cgtctacacg?agagtctcac?acttcttacc?ctggatccgc 1260
agtcacacca?aggaagagaa?tggcctggcc?ctctga 1296
The aminoacid sequence of human pro-urokinase's mutant 1
SEQUENCE?LISTING
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉three kinds of novel urokinase zymogen mutants
＜130〉aminoacid sequence of human pro-urokinase's mutant 1
<141>2004-04-20
<160>1
<170>PatentIn?version?3.2
<210>1
<211>431
<212>PRT
<213>human
<400>1
Met?Arg?Ala?Leu?Leu?Ala?Arg?Leu?Leu?Leu?Cys?Val?Leu?Val?Val?Ser
1 5 10 15
Asp?Ser?Lys?Gly?Ser?Asn?Glu?Leu?His?Gln?Val?Pro?Ser?Asn?Cys?Asp
20 25 30
Cys?Leu?Asn?Gly?Gly?Thr?Cys?Val?Ser?Asn?Lys?Tyr?Phe?Ser?Asn?Ile
35 40 45
His?Trp?Cys?Asn?Cys?Pro?Lys?Lys?Phe?Gly?Gly?Gln?His?Cys?Glu?Ile
50 55 60
Asp?Lys?Ser?Lys?Thr?Cys?Tyr?Glu?Gly?Asn?Gly?His?Phe?Tyr?Arg?Gly
65 70 75 80
Lys?Ala?Ser?Thr?Asp?Thr?Met?Gly?Arg?Pro?Cys?Leu?Pro?Trp?Asn?Ser
85 90 95
Ala?Thr?Val?Leu?Gln?Gln?Thr?Tyr?His?Ala?His?Arg?Ser?Asp?Ala?Leu
100 105 110
Gln?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Asn?Arg
115 120 125
Arg?Arg?Pro?Trp?Cys?Tyr?Val?Gln?Val?Gly?Leu?Lys?Pro?Leu?Val?Gln
130 135 140
Glu?Cys?Met?Val?His?Asp?Trp?Ala?Asp?Gly?Lys?Lys?Pro?Ser?Ser?Pro
145 150 155 160
Pro?Glu?Glu?Leu?Lys?Phe?Gln?Cys?Gly?Gln?Lys?Thr?Leu?Arg?Pro?His
165 170 175
Phe?Lys?Ile?Ile?Gly?Gly?Glu?Phe?Thr?Thr?Ile?Glu?Asn?Gln?Pro?Trp
180 185 190
Phe?Ala?Ala?Ile?Tyr?Arg?Arg?His?Arg?Gly?Gly?Ser?Val?Thr?Tyr?Val
195 200 205
Cys?Gly?Gly?Ser?Leu?Ile?Ser?Pro?Cys?Trp?Val?Ile?Ser?Ala?Thr?His
210 215 220
Cys?Phe?Ile?Asp?Tyr?Pro?Lys?Lys?Glu?Asp?Tyr?Ile?Val?Tyr?Leu?Gly
225 230 235 240
Arg?Ser?Arg?Leu?Asn?Ser?Asn?Thr?Gln?Gly?Glu?Met?Lys?Phe?Glu?Val
245 250 255
Glu?Asn?Leu?Ile?Leu?His?Lys?Asp?Tyr?Ser?Ala?Asp?Thr?Leu?Ala?His
260 265 270
His?Asn?Asp?Ile?Ala?Leu?Leu?Lys?Ile?Arg?Ser?Lys?Glu?Gly?Arg?Cys
275 280 285
Ala?Gln?Pro?Ser?Arg?Thr?Ile?Gln?Thr?Ile?Cys?Leu?Pro?Ser?Met?Tyr
290 295 300
Asn?Asp?Pro?Gln?Phe?Gly?Thr?Ser?Cys?Glu?Ile?Thr?Gly?Phe?Gly?Lys
305 310 315 320
Glu?Asn?Ser?Thr?Asp?Tyr?Leu?Tyr?Pro?Glu?Gln?Leu?Lys?Met?Thr?Val
325 330 335
Val?Lys?Leu?Ile?Ser?His?Arg?Glu?Cys?Gln?Gln?Pro?His?Tyr?Tyr?Gly
340 345 350
Ser?Glu?Val?Thr?Thr?Lys?Met?Leu?Cys?Ala?Ala?Asp?Pro?Gln?Trp?Lys
355 360 365
Thr?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Ser?Leu
370 375 380
Gln?Cys?Arg?Met?Thr?Leu?Thr?Gly?Ile?Val?Ser?Trp?Gly?Arg?Gly?Cys
385 390 395 400
Ala?Leu?Lys?Asp?Lys?Pro?Gly?Val?Tyr?Thr?Arg?Val?Ser?His?Phe?Leu
405 410 415
Pro?Trp?Ile?Arg?Ser?His?Thr?Lys?Glu?Glu?Asn?Gly?Leu?Val?Leu
420 425 430
Urokinase zymogen mutant 2 nucleotide sequences
SEQUENCE?LISTING
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉three kinds of novel urokinase zymogen mutants
＜130〉urokinase zymogen mutant 2 nucleotide sequences
<141>2004-04-20
<160>1
<170>PatentIn?version?3.2
<210>1
<211>1296
<212>DNA
<213>human
<400>1
atgagagccc?tgctggcgcg?cctgcttctc?tgcgtcctgg?tcgtgagcga?ctccaaaggc 60
agcaatgaac?ttcatcaagt?tccatcgaac?tgtgactgtc?taaatggagg?aacatgtgtg 120
tccaacaagt?acttctccaa?cattcactgg?tgcaactgcc?caaagaaatt?cggagggcag 180
cactgtgaaa?tagataagtc?aaaaacctgc?tatgagggga?atggtcactt?ttaccgagga 240
aaggccagca?ctgacaccat?gggccggccc?tgcctgccct?ggaactctgc?cactgtcctt 300
cagcaaacgt?accatgccca?cagatctgat?gctcttcagc?tgggcctggg?gaaacataat 360
tactgcagga?acccagacaa?ccggaggcga?ccctggtgct?atgtgcaggt?gggcctaaag 420
ctgcttgtcc?aagagtgcat?ggtgcatgac?tgcgcagatg?gaaaaaagcc?ctcctctcct 480
ccagaagaat?taaaatttca?gtgtggccaa?aagactctga?ggccccgctt?taagattatt 540
gggggagaat?tcaccaccat?cgagaaccag?ccctggtttg?cggccatcta?caagaagcac 600
catgggggct?ctgtcaccta?cgtgtgtgga?ggcagcctca?tcagcccttg?ctgggtgatc 660
agcgccacac?actgcttcat?tgattaccca?aagaaggagg?actacatcgt?ctacctgggt 720
cgctcaaggc?ttaactccaa?cacgcaaggg?gagatgaagt?ttgaggtgga?aaacctcatc 780
ctacacaagg?actacagcgc?tgacacgctt?gctcaccaca?acgacattgc?cttgctgaag 840
atccgttcca?aggagggcag?gtgtgcgcag?ccatcccgga?ctatacagac?catctgcctg 900
ccctcgatgt?ataacgatcc?ccagtttggc?acaagctgtg?agatcactgg?ctttggaaaa 960
gagaattcta?ccgactatct?ctatccggag?cagctgaaaa?tgactgttgt?gaagctgatt 1020
tcccaccggg?agtgtcagca?gccccactac?tacggctctg?aagtcaccac?caaaatgctg 1080
tgtgctgctg?acccacagtg?gaaaacagat?tcctgccagg?gagactcagg?gggacccctc 1140
gtctgttccc?tccaaggccg?catgactttg?actggaattg?tgagctgggg?ccgtggatgt 1200
gccctgaagg?acaagccagg?cgtctacacg?agagtctcac?acttcttacc?ctggatccgc 1260
agtcacacca?aggaagagaa?tggcctggcc?ctctga 1296
The aminoacid sequence of human pro-urokinase's mutant 2
SEQUENCE?LISTING
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉three kinds of novel urokinase zymogen mutants
＜130〉aminoacid sequence of human pro-urokinase's mutant 2
<141>2004-04-20
<160>1
<170>PatentIn?version?3.2
<210>1
<211>431
<212>PRT
<213>human
<400>1
Met?Arg?Ala?Leu?Leu?Ala?Arg?Leu?Leu?Leu?Cys?Val?Leu?Val?Val?Ser
1 5 10 15
Asp?Ser?Lys?Gly?Ser?Asn?Glu?Leu?His?Gln?Val?Pro?Ser?Asn?Cys?Asp
20 25 30
Cys?Leu?Asn?Gly?Gly?Thr?Cys?Val?Ser?Asn?Lys?Tyr?Phe?Ser?Asn?Ile
35 40 45
His?Trp?Cys?Asn?Cys?Pro?Lys?Lys?Phe?Gly?Gly?Gln?His?Cys?Glu?Ile
50 55 60
Asp?Lys?Ser?Lys?Thr?Cys?Tyr?Glu?Gly?Asn?Gly?His?Phe?Tyr?Arg?Gly
65 70 75 80
Lys?Ala?Ser?Thr?Asp?Thr?Met?Gly?Arg?Pro?Cys?Leu?Pro?Trp?Asn?Ser
85 90 95
Ala?Thr?Val?Leu?Gln?Gln?Thr?Tyr?His?Ala?His?Arg?Ser?Asp?Ala?Leu
100 105 110
Gln?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Asn?Arg
115 120 125
Arg?Arg?Pro?Trp?Cys?Tyr?Val?Gln?Val?Gly?Leu?Lys?Pro?Leu?Val?Gln
130 135 140
Glu?Cys?Met?Val?His?Asp?Trp?Ala?Asp?Gly?Lys?Lys?Pro?Ser?Ser?Pro
145 150 155 160
Pro?Glu?Glu?Leu?Lys?Phe?Gln?Cys?Gly?Gln?Lys?Thr?Leu?Arg?Pro?Arg
165 170 175
Phe?Lys?Ile?Ile?Gly?Gly?Glu?Phe?Thr?Thr?Ile?Glu?Asn?Gln?Pro?Trp
180 185 190
Phe?Ala?Ala?Ile?Tyr?Lys?Lys?His?His?Gly?Gly?Ser?Val?Thr?Tyr?Val
195 200 205
Cys?Gly?Gly?Ser?Leu?Ile?Ser?Pro?Cys?Trp?Val?Ile?Ser?Ala?Thr?His
210 215 220
Cys?Phe?Ile?Asp?Tyr?Pro?Lys?Lys?Glu?Asp?Tyr?Ile?Val?Tyr?Leu?Gly
225 230 235 240
Arg?Ser?Arg?Leu?Asn?Ser?Asn?Thr?Gln?Gly?Glu?Met?Lys?Phe?Glu?Val
245 250 255
Glu?Asn?Leu?Ile?Leu?His?Lys?Asp?Tyr?Ser?Ala?Asp?Thr?Leu?Ala?His
260 265 270
His?Asn?Asp?Ile?Ala?Leu?Leu?Lys?Ile?Arg?Ser?Lys?Glu?Gly?Arg?Cys
275 280 285
Ala?Gln?Pro?Ser?Arg?Thr?Ile?Gln?Thr?Ile?Cys?Leu?Pro?Ser?Met?Tyr
290 295 300
Asn?Asp?Pro?Gln?Phe?Gly?Thr?Ser?Cys?Glu?Ile?Thr?Gly?Phe?Gly?Lys
305 310 315 320
Glu?Asn?Ser?Thr?Asp?Tyr?Leu?Tyr?Pro?Glu?Gln?Leu?Lys?Met?Thr?Val
325 330 335
Val?Lys?Leu?Ile?Ser?His?Arg?Glu?Cys?Gln?Gln?Pro?His?Tyr?Tyr?Gly
340 345 350
Ser?Glu?Val?Thr?Thr?Lys?Met?Leu?Cys?Ala?Ala?Asp?Pro?Gln?Trp?Lys
355 360 365
Thr?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Ser?Leu
370 375 380
Gln?Cys?Arg?Met?Thr?Leu?Thr?Gly?Ile?Val?Ser?Trp?Gly?Arg?Gly?Cys
385 390 395 400
Ala?Leu?Lys?Asp?Lys?Pro?Gly?Val?Tyr?Thr?Arg?Val?Ser?His?Phe?Leu
405 410 415
Pro?Trp?Ile?Arg?Ser?His?Thr?Lys?Glu?Glu?Asn?Gly?Leu?Val?Leu
420 425 430
SEQUENCE?LISTING
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉three kinds of novel urokinase zymogen mutants
＜130〉urokinase zymogen mutant 3 nucleotide sequences
<141>2004-04-20
<160>1
<170>PatentIn?version?3.2
<210>1
<211>1296
<212>DNA
<213>human
<400>1
atgagagccc?tgctggcgcg?cctgcttctc?tgcgtcctgg?tcgtgagcga?ctccaaaggc 60
agcaatgaac?ttcatcaagt?tccatcgaac?tgtgactgtc?taaatggagg?aacatgtgtg 120
tccaacaagt?acttctccaa?cattcactgg?tgcaactgcc?caaagaaatt?cggagggcag 180
cactgtgaaa?tagataagtc?aaaaacctgc?tatgagggga?atggtcactt?ttaccgagga 240
aaggccagca?ctgacaccat?gggccggccc?tgcctgccct?ggaactctgc?cactgtcctt 300
cagcaaacgt?accatgccca?cagatctgat?gctcttcagc?tgggcctggg?gaaacataat 360
tactgcagga?acccagacaa?ccggaggcga?ccctggtgct?atgtgcaggt?gggcctaaag 420
ctgcttgtcc?aagagtgcat?ggtgcatgac?tgcgcagatg?gaaaaaagcc?ctcctctcct 480
ccagaagaat?taaaatttca?gtgtggccaa?aagactctga?ggccccactt?taagattatt 540
gggggagaat?tcaccaccat?cgagaaccag?ccctggtttg?cggccatcta?caagaagcac 600
catgggggct?ctgtcaccta?cgtgtgtgga?ggcagcctca?tcagcccttg?ctgggtgatc 660
agcgccacac?actgcttcat?tgattaccca?aagaaggagg?actacatcgt?ctacctgggt 720
cgctcaaggc?ttaactccaa?cacgcaaggg?gagatgaagt?ttgaggtgga?aaacctcatc 780
ctacacaagg?actacagcgc?tgacacgctt?gctcaccaca?acgacattgc?cttgctgaag 840
atccgttcca?aggagggcag?gtgtgcgcag?ccatcccgga?ctatacagac?catctgcctg 900
ccctcgatgt?ataacgatcc?ccagtttggc?acaagctgtg?agatcactgg?ctttggaaaa 960
gagaattcta?ccgactatct?ctatccggag?cagctgaaaa?tgactgttgt?gaagctgatt 1020
tcccaccggg?agtgtcagca?gccccactac?tacggctctg?aagtcaccac?caaaatgctg 1080
tgtgctgctg?acccacagtg?gaaaacagat?tcctgccagg?gagactcagg?gggacccctc 1140
gtctgttccc?tccaaggccg?catgactttg?actggaattg?tgagctgggg?ccgtggatgt 1200
gccctgaagg?acaagccagg?cgtctacacg?agagtctcac?acttcttacc?ctggatccgc 1260
agtcacacca?aggaagagaa?tggcctggcc?ctctga 1296
The aminoacid sequence of human pro-urokinase's mutant 3
SEQUENCE?LISTING
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉three kinds of novel urokinase zymogen mutants
＜130〉aminoacid sequence of human pro-urokinase's mutant 3
<141>2004-04-20
<160>1
<170>PatentIn?version?3.2
<210>1
<211>431
<212>PRT
<213>human
<400>1
Met?Arg?Ala?Leu?Leu?Ala?Arg?Leu?Leu?Leu?Cys?Val?Leu?Val?Val?Ser
1 5 10 15
Asp?Ser?Lys?Gly?Ser?Asn?Glu?Leu?His?Gln?Val?Pro?Ser?Asn?Cys?Asp
20 25 30
Cys?Leu?Asn?Gly?Gly?Thr?Cys?Val?Ser?Asn?Lys?Tyr?Phe?Ser?Asn?Ile
35 40 45
His?Trp?Cys?Asn?Cys?Pro?Lys?Lys?Phe?Gly?Gly?Gln?His?Cys?Glu?Ile
50 55 60
Asp?Lys?Ser?Lys?Thr?Cys?Tyr?Glu?Gly?Asn?Gly?His?Phe?Tyr?Arg?Gly
65 70 75 80
Lys?Ala?Ser?Thr?Asp?Thr?Met?Gly?Arg?Pro?Cys?Leu?Pro?Trp?Asn?Ser
85 90 95
Ala?Thr?Val?Leu?Gln?Gln?Thr?Tyr?His?Ala?His?Arg?Ser?Asp?Ala?Leu
100 105 110
Gln?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Asn?Arg
115 120 125
Arg?Arg?Pro?Trp?Cys?Tyr?Val?Gln?Val?Gly?Leu?Lys?Pro?Leu?Val?Gln
130 135 140
Glu?Cys?Met?Val?His?Asp?Trp?Ala?Asp?Gly?Lys?Lys?Pro?Ser?Ser?Pro
145 150 155 160
Pro?Glu?Glu?Leu?Lys?Phe?Gln?Cys?Gly?Gln?Lys?Thr?Leu?Arg?Pro?His
165 170 175
Phe?Lys?Ile?Ile?Gly?Gly?Glu?Phe?Thr?Thr?Ile?Glu?Asn?Gln?Pro?Trp
180 185 190
Phe?Ala?Ala?Ile?Tyr?Lys?Lys?His?His?Gly?Gly?Ser?Val?Thr?Tyr?Val
195 200 205
Cys?Gly?Gly?Ser?Leu?Ile?Ser?Pro?Cys?Trp?Val?Ile?Ser?Ala?Thr?His
210 215 220
Cys?Phe?Ile?Asp?Tyr?Pro?Lys?Lys?Glu?Asp?Tyr?Ile?Val?Tyr?Leu?Gly
225 230 235 240
Arg?Ser?Arg?Leu?Asn?Ser?Asn?Thr?Gln?Gly?Glu?Met?Lys?Phe?Glu?Val
245 250 255
Glu?Asn?Leu?Ile?Leu?His?Lys?Asp?Tyr?Ser?Ala?Asp?Thr?Leu?Ala?His
260 265 270
His?Asn?Asp?Ile?Ala?Leu?Leu?Lys?Ile?Arg?Ser?Lys?Glu?Gly?Arg?Cys
275 280 285
Ala?Gln?Pro?Ser?Arg?Thr?Ile?Gln?Thr?Ile?Cys?Leu?Pro?Ser?Met?Tyr
290 295 300
Asn?Asp?Pro?Gln?Phe?Gly?Thr?Ser?Cys?Glu?Ile?Thr?Gly?Phe?Gly?Lys
305 310 315 320
Glu?Asn?Ser?Thr?Asp?Tyr?Leu?Tyr?Pro?Glu?Gln?Leu?Lys?Met?Thr?Val
325 330 335
Val?Lys?Leu?Ile?Ser?His?Arg?Glu?Cys?Gln?Gln?Pro?His?Tyr?Tyr?Gly
340 345 350
Ser?Glu?Val?Thr?Thr?Lys?Met?Leu?Cys?Ala?Ala?Asp?Pro?Gln?Trp?Lys
355 360 365
Thr?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Ser?Leu
370 375 380
Gln?Cys?Arg?Met?Thr?Leu?Thr?Gly?Ile?Val?Ser?Trp?Gly?Arg?Gly?Cys
385 390 395 400
Ala?Leu?Lys?Asp?Lys?Pro?Gly?Val?Tyr?Thr?Arg?Val?Ser?His?Phe?Leu
405 410 415
Pro?Trp?Ile?Arg?Ser?His?Thr?Lys?Glu?Glu?Asn?Gly?Leu?Val?Leu
420 425 430
Claims (8)
1. three kinds of uPA (pro-UK) mutant (pro-UKm) with thrombolysis activity, through artificial orientation's sudden change, except that the sequence of sudden change place, other parts are constant on the aminoacid sequence basis of natural pro-UK for they.Three kinds of mutant have the thrombolysis ability same or higher with natural pro-UK, than natural pro-UK better stability and longer transformation period are arranged.
2. according to claims 1 described pro-UKm, it is characterized in that resisting-1 pair of active influence of uPA of zymoplasm and/or inhibitors of plasminogen activator inhibitor, thereby improve the thrombolysis ability and reduce dosage.
3. according to claims 2 described pro-UKm, wherein in the aminoacid sequence of natural pro-UK the 156th, 178,179,181 arginine (Arg
156,178,179,181) replaced by other amino acid.
4. according to claims 3 described pro-UKm, it is characterized in that the 156th, 178,179,181 arginine (Arg
156,178,179,181), or replace with Methionin, or replace and multiple combinatorial mutagenesis with Histidine.
5. protein molecular of claims 2,3 and 4 described pro-UKm of encoding.
6. with claims 5 described encoding histone dna molecular cells transfected and yeast.
7. the bacterium of using claims 5 described encoding histone dna moleculars to transform.
8. use the animal bioreactor of claims 5 described encoding histone dna moleculars preparations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410033936 CN1690199A (en) | 2004-04-21 | 2004-04-21 | Three urokinase mutants |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410033936 CN1690199A (en) | 2004-04-21 | 2004-04-21 | Three urokinase mutants |
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| Publication Number | Publication Date |
|---|---|
| CN1690199A true CN1690199A (en) | 2005-11-02 |
Family
ID=35345957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410033936 Pending CN1690199A (en) | 2004-04-21 | 2004-04-21 | Three urokinase mutants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1690199A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441683C (en) * | 2006-03-24 | 2008-12-10 | 中国人民解放军军事医学科学院生物工程研究所 | Low molecular-weight urokinase mutant and its expression vector |
| CN102559720A (en) * | 2010-12-16 | 2012-07-11 | 中国科学院福建物质结构研究所 | Expression, purification and crystallization of human coagulation factor XI catalytic structural domain |
-
2004
- 2004-04-21 CN CN 200410033936 patent/CN1690199A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441683C (en) * | 2006-03-24 | 2008-12-10 | 中国人民解放军军事医学科学院生物工程研究所 | Low molecular-weight urokinase mutant and its expression vector |
| CN102559720A (en) * | 2010-12-16 | 2012-07-11 | 中国科学院福建物质结构研究所 | Expression, purification and crystallization of human coagulation factor XI catalytic structural domain |
| CN102559720B (en) * | 2010-12-16 | 2015-05-13 | 中国科学院福建物质结构研究所 | Expression, purification and crystallization of human coagulation factor XI catalytic structural domain |
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