CN1096326A - A kind of method for preparing recombined streptokinase - Google Patents
A kind of method for preparing recombined streptokinase Download PDFInfo
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- CN1096326A CN1096326A CN 94112106 CN94112106A CN1096326A CN 1096326 A CN1096326 A CN 1096326A CN 94112106 CN94112106 CN 94112106 CN 94112106 A CN94112106 A CN 94112106A CN 1096326 A CN1096326 A CN 1096326A
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Abstract
A kind of manufacture method of recombined streptokinase.The gene constructed process of prior art is loaded down with trivial details, and the expression in escherichia coli level is low, the purification scheme complexity.The inventive method pcr amplification streptokinase genes is assembled into expression plasmid PSTE-SK-1 with prokaryotic expression carrier such as PLY-4, behind the transformed into escherichia coli with temperature-induced r-SK genetic expression.Engineering bacteria is through fermenting and amplifying, broken bacterium, and centrifugal collection inclusion body, renaturation is after two step method purifying r-SK.The output of present method products obtained therefrom and purity are all very high, and cost is low.
Description
The invention belongs to biotechnological means.
The special efficacy thrombolytic drug has TISSURE-TYPE PLASMINOGEN ACTIVATOR (t-PA), streptokinase (SK), benzene methoxy acyl plasminogen activator streptokinase mixture (APSAC) etc. at present.In September, 1993 U.S. Brigharm and Women's hospital, professors Ridker etc. have summed up the thromboembolism treatment of 100,000 routine Acute Myocardial Infarctions, its result shows that the drug effect of above-mentioned all kinds of thrombolytic drugs and side reaction do not have significant difference.Rt-PA costs an arm and a leg, and 2300 dollars every dose, the SK price is lower 10 times than rt-PA, and the embolism recurrence rate is low, uses easier.
At present external each company does to produce bacterial strain with pathogenic Hemolytic streptococcus, need take environment and safety of workers into account, removes to cause ypotension, the toxin of side reactions such as heating, and the thrombus dissolving suis SK reasons such as being difficult for purifying that yields poorly make the Processes and apparatus complexity, the cost height, price is expensive.The beginning of the eighties, Ferrtti J.J. etc. was separated to the SK gene from the Hemolytic streptococcus phage, and expressed in intestinal bacteria, but expression level is very low, has only hundreds of units/ml bacterium liquid, does not form gene engineering product.The SK gene isolation is identical with Ferrtti J.J. in the same patents with the U.S. in 1981 and Japan in 1986, but expression vector is different with culture condition, expression level in large intestine sense bacterium is 60～274ug/ml bacterium liquid, i.e. 6420～29300 units/ml bacterium liquid, do not have data such as pure product yield, do not see the formation gene engineering product yet.Cuba Herrena L. had developed recombined streptokinase in 1992, but its gene constructed process is loaded down with trivial details, and the expression level in intestinal bacteria is low, the purification process complexity, and prices of raw and semifnished materials costliness such as Profibrinolysin affinity adsorbent, its product purity and yield are low, the cost height.
Purpose of the present invention is to overcome the shortcoming dangerous in the prior art, that cost is high, invents a kind ofly to prepare the method for recombined streptokinase with biological high-technology, makes the yield of safe preparation process, product and purity is all very high and cost is low.
The present invention adopts following technical proposals:
(1) separate Hemolytic streptococcus from human oral cavity, the streptokinase of purifying from nutrient solution is measured the reference that 5 amino-acid sequences of N-terminal and C-terminal are made the design primer.DNA makes template with the Hemolytic streptococcus macromole, by the PCR streptokinase genes that directly increases, streptokinase genes for example contains PL with prokaryotic expression carrier after the nucleotide sequence analysis confirms, the PR promotor, the PLY-4 plasmid of controlling elements such as CIT857 gene and 5SRNA termination signal is assembled into efficient expression plasmid pSTE-SK-1;
(2) pSTE-SK-1 transformed into escherichia coli, with temperature-induced r-SK expression of gene, SDS-PAGE confirms that expression product r-SK molecular weight is 43000, forms with inclusion body to be present in the engineering bacteria, accounts for more than 65% of tropina, engineering bacteria name PSTE-SK-1;
(3) by fermentation technique amplification engineering bacteria, make basic culture solution, fluid infusion material such as temperature-induced front and back LB, glucose, rare elements solution with M9CAA, fermentation parameter is temperature 30-42 ℃, the molten amount of oxygen is 50-80%, PH6-8, and the broken bacterium of high pressure is used in the fermentation back, centrifugal collection inclusion body, inclusion body washs through damping fluid, after the renaturation of guanidine hydrochloride dissolution and r-SK, use gel-filtration again, ion-exchange two step method purified product.
Use the inventive method, every liter of fermented liquid must wet about bacterium 20 grams, and the r-SK product purity reaches 97-99%, specific activity 100,000 IU/mg, and every liter of fermented liquid of pure product output gets 600 milligrams.
Add human serum albumin in the pure product r-SK solution, behind the stablizers such as Sodium Glutamate and phosphoric acid salt, use the filtering with microporous membrane degerming, lyophilize becomes white powder shape finished product.Use water for injection dissolving back, is main application with thrombotic diseases such as treatment Acute Myocardial Infarctions.
Treat experimental dog myocardial infarction, all thrombolysises behind 30-60min such as (0.3mg/kg) of rabbit femoral artery thrombosis, revascularization.In the treatment lagophthalmos during hematocele (10-20 μ/eye), hematocele disappearance after 24 hours.During treatment lagophthalmos internal hemorrhage, exudate disappears after a few hours.
The present invention uses recombinant DNA technology, in the non-virulent intestinal bacteria, efficiently express r-SK, method safety, bring any harm for environment and operator, because it efficiently expresses with purification process simple, make the purity and the output of product all very high, therefore greatly reduce production cost and the price thereof of r-SK, following table is the contrast of Cuba Herrena L and the inventive method.
Cuba Herrena L the present invention
Expression level accounts for coli somatic protein 25 % more than 65%
The affine ion-exchange of purification process Profibrinolysin-Sepharose, gel-filtration
Chromatography, ion-exchange, gel-filtration etc.
Purity specific activity 5.2 ten thousand IU/mg 100,000 IU/mg
SDS-PAGE shows that r-SK accounts for 95% 98～99%
Pure product yield r-SK 50mg/L fermented liquid 500～600mg/L fermented liquid
Embodiment (one):
1, uses the pcr amplification streptokinase genes.
Collect Hemolytic streptococcus 60 strains from the hospital laboratory, through screening, the maximum bacterium (NO.8) of a large amount of cultivation secretion SK, and from training liquid purification SK, then with amino-acid sequence analyser and manual N and 5 amino orders of C-terminal measured respectively.Press the order of each 5 amino-acid sequence design PCR nucleotide primer of streptokinase N end C end,, be used for the pcr amplification streptokinase genes behind the purifying with synthetic these two primers of solid phase phosphotriester method mat dna synthesizer.5 of two PCR primers ' end is restricted property restriction endonuclease recognition sequence respectively.
Prepare the streptococcic macromole DNA of NO.8 by Frederick M. Ausubel et al method, make A
260/ A
280Ratio＞2.0.
PCR reaction system and condition are as follows:
Reaction system: 5 * PCR damping fluid 20ul
ddH
2O 64ul
Template DNA 3ul(1ug)
Primer I 1ul(50 pmoles)
Primer II 1ul(50 pmoles)
dNTP 10ul（2mM）
Tag archaeal dna polymerase 1ul(2.5U)
90 ℃ of denaturation temperatures 60 seconds, 72 ℃ of elongating temperatures 120 seconds, anneal 52 ℃ 60 seconds, add the 2.5UTag enzyme again after 15 circulations, proceed 15 circulations.Get reactant 2ul, identify (Fig. 1), observe the single band of 1.3Kb, meet the length of complete streptokinase genes with agarose gel electrophoresis.Reactant is through the restriction endonuclease hydrolysis, and agarose gel electrophoresis is identified, shows distinctive restriction enzyme site in the streptokinase genes.
2, streptokinase genes clone's structure and DNA sequence analysis:
Each adds T4 dna ligase reaction system above-mentioned PCR reaction product and pUC19 plasmid after identical two restriction endonuclease complete hydrolysis, room temperature was placed 1 hour, and transformed into escherichia coli JM83 is with penbritin-LB flat board screening and culturing in 37 ℃ of incubators.
Get single bacterium colony and prepare plasmid, use several restriction endonuclease hydrolysis plasmids respectively, agarose electrophoresis is identified, is presented SK gene expression characteristics band person and name plasmid (pST-1).
PST-1 plasmid nucleotide sequence is analyzed: because of containing Hind III point of contact in the SK gene, so at first make up 770bp and two subclones of 570bp fragment, carry out the nucleotide sequence analysis then, certainly the nucleotide sequence and the reading frame of streptokinase genes.
3, streptokinase genes is cloned in the expression in the intestinal bacteria:
The structure of expression plasmid pSTE:
With restriction enzyme enzymic hydrolysis pST-1 plasmid, separate through agarose gel electrophoresis, reclaim 1.3Kb SK gene, recombinate with prokaryotic expression matter carrier (containing PL, PR promotor, controlling elements such as CIt857 repressor gene and 5s RNA t1t2 termination signal) then.The ligation thing is with calcium phosphate method transformed into escherichia coli K802, through the penbritin screening and culturing.Select single bacterium colony, the preparation plasmid is identified with the restriction enzyme fragment length, contains SK gene expression characteristics fragment person, is called the pSTE-1 expression plasmid, and the bacterium that contains the pSTE-1 expression plasmid is called engineering bacteria PSTE-1.
4, the expression of PSTE-1 in intestinal bacteria and the evaluation of expression product:
The single bacterium colony of picking pSTE-1,30 ℃ of shaking culture are spent the night in the LB liquid nutrient medium, and after the dilution in 1: 50 of training liquid, 30 ℃ are cultured to A in shaking bottle
600Be warming up to 42 ℃ in～0.6,3 minutes, continue cultivated 3-4 hour, centrifugal collections bacterium is with PBS(pH 7) wash once, be stored in the evaluation and the purifying of-20 ℃ or the product of do expression at once.
5, the evaluation of expression product:
SPS-PAGE-
With the molecular weight and the expression level of sulfuric acid ten diester sodium-polyacrylamide gel electrophoresises (SDS-PAGE) evaluation expression product, and fibrinolytic.
Sample dissolution liquid: contain 0.0625M Tris-HCl(pH 6.7), 2% SDS, 10% glycerine, 5% mercaptoethanol, 0.001% bromjophenol blue.
Sample preparation and point sample: bacterium liquid centrifuged deposit is suspended from 100ul sample dissolution liquid behind the 1ml inducing culture, puts in the boiling water bath 5 minutes, makes resolution of precipitate.The sample equal-volume of chromatographic peak, 2 times of concentration sample dissolution liquid mixing back reheat are handled, and the point sample amount respectively is 20ug, contrasts bacterium or induces the sample of preceding bacterium to use with the quadrat method cultivation, handles sample with similarity condition.
Gel-colored: as to examine the bright orchid of Ma Shi.
Expression level: proteinic dyeing band is done blackness scanning with Tianjin, island CS-910 dual-wavelength scanner in the gel, and Computer Processing is printed the shared per-cent in r-SK peak.
Point sample is 42 ℃ and induces bacterium SDS lysate to be equivalent to 43000 places at molecular weight the band that concentrates is very much arranged as a result, point sample be bacterium before inducing lysate almost lose band in same area.Scanning result shows that r-SK accounts for more than 65% of bacterial protein.
After the evaluation of expression product fibrinolytic-gel slab is used Triton X-100 and physiological saline flush away SDS, be covered on the gel of fibre-bearing albumen, Profibrinolysin and agarose, 37 ℃ the insulation a few hours after, have only temperature-induced bacterium lysate very clear thorough radiolucent zone to be arranged at 43000 molecular weight places, be that this position scleroproein dissolves, show that 43000 molecular weight position protein have fibrinolytic.
Expression product exists state-thalline ultrasonic wave and high-pressure homogenization pump rupture of membranes in thalline.After centrifugal, the other point sample of last cleer and peaceful precipitation that will contain same protein content, carry out SDS-PAGE, the result shows, point sample is in the row of resolution of precipitate thing, be that 43000 places dyeing band is very dark at molecular weight, and point sample is the inherent same position of row of supernatant liquor, almost do not see dyeing, show that the r-SK expression product has formed inclusion body.
Specific activity mensuration-usefulness fibrin gel plate method, chromophoric substrate method or fibrin clot dissolution method are measured the r-SK activity, survey protein content with the Lowry colorimetry, protein content (mg) in r-SK activity unit in specific activity=unit volume sample/unit volume sample.
6, the fermentation of engineering bacteria:
The single colony inoculation of shake flask fermentation-PSTE-1 is trained liquid in LB-Amp, 30 ℃ of overnight incubation, and the dilution in 1: 50 of inferior daily improvement M9 nutrient solution, 30 ℃ of shake wells are cultured to A600～0.6, are warming up to inducing temperature in several minutes, continue to cultivate 3 hours.Bacterium liquid is with the centrifuge tube of known bare weight, and 5000 r.p.m., 4 ℃ of centrifugal 10min abandon supernatant, the bacterium piece is with PBS(pH 7.4) washing once, weigh, calculate bacterium piece weight in wet base, be stored in-20 ℃.
The single bacterium colony of NBS BIO FIII jar fermentation-PSTE-1 is 30 ℃ of overnight incubation in 100ml LB-Amp training liquid, survey A
600,, use for fermentation as seed liquor.
(120 ℃ of Bio FIII jar water vapors, 15 pounds) sterilized 30 minutes, after the cooling, be seated to operator's console, 30 ℃ of design temperatures, pH6-7, dissolved oxygen amount 50-80%, stirring velocity 225-800 time/minute (stirring velocity is speed change with oxygen level) is after parameters is stable, by 1: 50 volume ratio inoculation PSTE-1 bacterium liquid (A
600=1-2).
Keeping the condition bottom fermentation of above-mentioned parameter, treat A
600When reaching 0.6 left and right sides, be warming up to inducing temperature in several minutes and continue fermentation 3 hours, regulate automatically about pH6-8, make great efforts to make oxygen level to be not less than 50-80%.
After the fermentation ends, second order fermentation usefulness is preserved or done to bacterium liquid after centrifugal.
7, Rotofor isoelectrofocusing-gel-filtration method purification r-SK: be applicable to a small amount of preparation r-SK:
1, gets engineering bacteria 4gm and be suspended from hypotonic buffer liquid (0.05M pH7.8 Tris-HCl, 0.02M EDTA, N,O-Diacetylmuramidase 0.5mg/ml) 25ml, stirring at room 60 minutes, add Triton X-100 to 2%, NaCl to 0.5M is behind the placement certain hour, with the broken bacterium of homogenizer, 8000r.p.m.10 ℃ centrifugal 60 minutes precipitations are with 20ml damping fluid (0.05M pH7.8 Tris-HCl, 0.02M EDTA) washing three times, collecting precipitation is suspended from 20ml H
2O, and to H
2The O dialysed overnight adds urea to final concentration and reaches 4M in the dialysate, make solubilization of inclusion bodies.
2, the Rotofor isoelectrofocusing separates the renaturation of r-SK and r-SK:
Get above-mentioned solubilization of inclusion bodies liquid 49ml, add Ampholine(pH 3-10) 1ml, after focusing on 4 hours with 4 ℃ of Rotofor isoelectrofocusing instrument 12W, make SDS-PAGE from the sampling of each section solution and analyze, merge and contain each section of SK solution, 0.05M pH7.4 PBS, 0.02M urea is removed in the EDTA dialysis, again with amino acid-hydrotropy damping fluid dilution, 4 ℃, renaturation 72 hours.Centrifugal through 8000r.p.m., collect supernatant and carry out ultrafiltration and concentration.
3, gel-filtration and sterilising filtration:
Get above-mentioned concentrated solution application of sample through ultrafiltration in Sephacryl S-200(with the abundant balance of hydrotropy damping fluid) ion exchange column, flow velocity 1ml/min, substep is collected, detect protein peak with ECONO low pressure liquid chromatography instrument, with SDS-PAGE, γ-SK activity and determining the protein quantity, analysis and collection contain the r-SK part, after 0.2 μ micro-pore-film filtration sterilization back packing and freeze-drying.
The protein content, r-SK activity, specific activity, purification multiple and the rate of recovery that respectively go on foot product in the purge process are summarized in:
The active number that reclaims of protein content r-SK gross activity specific activity purifying multiple
(mg) (×10
6) (IU/mg) (%)
Wet bacterium--7.8 540--100
Inclusion body 101 5.6 5,500 10.1 72
Rotofor purifying 46 4.4 95,000 175 57
Sephadex G-100 32 3.2 100000 185 41
Embodiment (two):
1, the preservation of engineering bacteria
Set up seed bank and seed lot, be stored in-70 ℃ of special-purpose profound hypothermia refrigerators.
Whether make regular check on the inscribe zymogram of plasmid and the expression level of r-SAK maintains more than 65%.
2, engineering bacteria is cultivated
Substratum
The LB+Amp flat board:
Veast Extract 5G Tryptone 10G
NaCI 5G Agar 10G
H
2O 1000ml
High pressure steam sterilization 1Kg/cm
220 minutes, add penbritin 100 μ g/ml LB training liquid after the cooling: except that not adding Agar and Amp, all the other were the same.
From-70 ℃ of profound hypothermia refrigerators, take out kind of a daughter bacteria, draw LB flat board (containing penbritin 100mg/ml), 30 ℃ of overnight incubation, picking list bacterium colony from the flat board is inoculated in the LB nutrient solution, and 30 ℃ of joltings are spent the night, this is a primary seed solution, primary seed solution was inoculated in the LB substratum with 1: 10 again, cultivated 3-4 hour for 30 ℃, this is a secondary seed solution.
3, fermentation
After fermentor tank (5L or the 10L) sterilization, emit the H in the jar
2O changes to nutrient solution, proofreaies and correct and be provided with various parameters.Take out the secondary seed bacterium, be inoculated in the fermentor tank with 1: 10, bubbling air ferments.
Keep following condition in the fermenting process: temperature: 30 ℃, PH:6-8, DO:50-80%, stirring velocity: with DO is main control automatically, per hour takes out 5ml bacterium liquid and detects.
Bacterial density (OD
600) and Glucose concentration
Regulate the fermented liquid potential of hydrogen with ammoniacal liquor, keep PH6-8, foam adds defrother for a long time.
Induce the front and back of heating up to replenish casein hydrolyzate, glucose and various trace elements.
After inducing culture 3-4 hour, OD
600Reach 15 when above, stop fermentation, emit bacterium liquid from fermentor tank, the centrifugation bacterium is also weighed, and is many that bacterium break bacterium immediately, the separation inclusion body, or bacterium is frozen in-70 ℃.
4, bacteria breaking
Broken bacterium: wet bacterium is suspended in the damping fluid, joins in the high-pressure homogenization pump, with suitable pressure explosion bacterium.
Quality control index: after the microscopy bacteria breaking, inspection method: with the bacterial suspension before the homogenate, even picture after suitably diluting, gramstaining, the bacterial count (A) in 4 visuals field of microscopy is beaten the suspension after the homogenate, after the same dilution, evenly picture is observed 4 not broken bacterial counts in the visual field (B).
Bacteria breaking rate %=(A-B)/A * 100
After the bacteria breaking rate reaches more than 95%, centrifugal collecting precipitation, promptly rough inclusion body, r-SK accounts for more than the bacterial protein 75-85% in the thick inclusion body.
5, inclusion body purification
Thick bag is contained body and function washing method purifying, makes r-SK account for about 90% of bacterial protein.
6, the dissolving of r-SK and renaturation
Inclusion body behind purifying 6M guanidine hydrochloride dissolution, centrifugal 30 minutes of 10000rmp leaves and takes supernatant.To PB dialysis repeatedly, centrifugal 30 minutes of 20000rmp stays supernatant, and this moment, r-SK accounted for tropina about 95%, specific activity more than 6.5 ten thousand IU/mg, r-SK yield about 65%.
7, purifying
Ion exchange chromatography: detect with Waters650 or Econo chromatographic system.Anion-exchange column PB balance, the r-SK solution after the renaturation is crossed post, flow velocity: 1.0ml/min, wash post with PB then, with the PB wash-out r-SK of linear hydrochloric acid gradient and linear PH gradient, flow velocity: 4.0ml/min collects first elution peak (r-SK).
SDS-PAGE analyzes, and r-sk purity should reach 94%.Specific activity: more than the 8.0IU/mg.The r-SK yield:＞55%, protein concentration 7mg/ml.
Gel-filtration: gel, with no thermal source, aseptic, deionized water swelling, autoclave sterilization, dress post, PB balance, sample is gone up sample through the filter filtration back of 0.22U, uses the PB wash-out, flow velocity: 5.0ml/min.The fibrin gel dissolution method is measured the r-SK activity, merges each pipe of active peak.About product specific activity 100,000 IU/mg, every L fermented liquid gets about pure product 600mg, purity 97-99% at this moment.R-SK yield: 40-50%.
8, sterile filtration, packing, freeze-drying:
According to protein content and activity, add auxiliary material and stablizer in following ratio
r-SK 500，000IU
Phosphoric acid salt Na2HPO4 1.33mg, NaII2PO4 0.31mg 1,500,000 IU/ bottles
Sodium Glutamate (injection) 30mg/ bottle
Human serum albumin 13mg/ bottle
Above raw material must meet the requirement of injecting drug use
Sterile filtration: reach at cleanliness factor under 100 grades the condition, filter with 0.22 μ sterilizing filter.
Use the portioning machine packing, every bottle contains 500,000 or 1,500,000 IU.
Divide the sample that installs, insert the interior freeze-drying of Freeze Drying Equipment and in Freeze Drying Equipment, add bottle stopper automatically, add aluminium lid then, label vanning.
Be stored in 8-12 ℃ of refrigerator.
With vasography proof r-SK treatment dog femoral artery thrombus and dog coronary artery thrombosis significant curative effect is arranged.
Description of drawings:
Figure one is a streptokinase genes expression plasmid design of graphics
Figure two is nucleic acid sequence of streptokinase genes
Claims (2)
1, a kind of preparation method of recombined streptokinase comprises the structure of streptokinase genes plasmid, and the expression in intestinal bacteria and the purifying of expression product thereof and the technology that manufactures a product on a large scale is characterized in that:
(1) separate Hemolytic streptococcus from human oral cavity, the streptokinase of purifying in the nutrient solution is measured the reference that N-terminal and C-terminal amino-acid sequence are made design PCR primer.DNA makes template with the Hemolytic streptococcus macromole, by the PCR streptokinase genes that directly increases, streptokinase genes is after the nucleotide sequence analysis confirms and prokaryotic expression carrier, for example contain PL, the PR promotor, the PLY-4 plasmid of controlling elements such as CIT857 gene and 5SRNA termination signal is assembled into efficient expression plasmid pSTE-SK-1;
(2) pSTE-SK-1 transformed into escherichia coli with temperature-induced r-SK expression of gene, is present in the engineering bacteria with inclusion body formation;
(3) by fermentation technique amplification engineering bacteria, make basic culture solution with M9CAA, the several fluid infusion is carried out with LB, glucose, rare elements solution etc. in temperature-induced front and back, and fermentation parameter is temperature 30-42 ℃, and the molten amount of oxygen is 50-80%, PH6-8;
(4) the broken bacterium of high pressure is used in the fermentation back, centrifugal collection inclusion body, and inclusion body washs through damping fluid, after the renaturation of guanidine hydrochloride dissolution and r-SK, uses gel-filtration again, ion-exchange two step method purified product.
2, behind the stablizers such as r-SK adding serum albumin according to claim 1 preparation, sterile filtration divides the bottle of packing into, forms finished product after the freeze-drying.Be stored in 8-12 ℃ of refrigerator.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94112106 CN1035193C (en) | 1994-04-04 | 1994-04-04 | Preparing method for recomposing streptokinase |
| RU96121788/13A RU2127758C1 (en) | 1994-04-04 | 1995-04-03 | Method of recombinant streptokinase preparing |
| PCT/CN1995/000024 WO1995027050A1 (en) | 1994-04-04 | 1995-04-03 | A method of preparing recombinant streptokinase |
| CH03481/95A CH688653A5 (en) | 1994-04-04 | 1995-04-03 | Prepn. of streptokinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94112106 CN1035193C (en) | 1994-04-04 | 1994-04-04 | Preparing method for recomposing streptokinase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1096326A true CN1096326A (en) | 1994-12-14 |
| CN1035193C CN1035193C (en) | 1997-06-18 |
Family
ID=5035911
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 94112106 Expired - Fee Related CN1035193C (en) | 1994-04-04 | 1994-04-04 | Preparing method for recomposing streptokinase |
Country Status (4)
| Country | Link |
|---|---|
| CN (1) | CN1035193C (en) |
| CH (1) | CH688653A5 (en) |
| RU (1) | RU2127758C1 (en) |
| WO (1) | WO1995027050A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064406C (en) * | 1998-05-11 | 2001-04-11 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for producing glucokinase by gene engrg. technique |
| CN102936603A (en) * | 2012-10-31 | 2013-02-20 | 上海昊海生物科技股份有限公司 | Expression of uridine diphosphate-glucose dehydrogenase and measurement of enzymatic activity |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR071169A1 (en) * | 2008-03-31 | 2010-06-02 | Council Scient Ind Res | VARIANTS OF STREPTOQUINASE CONTAINING CISTEINE AND ITS COVALENTLY MODIFIED FORMS |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU561372B2 (en) * | 1983-10-10 | 1987-05-07 | Board Of Regents Of The University Of Oklahoma, The | Streptokinase-coding recombinant vectors |
| US5066589A (en) * | 1984-03-02 | 1991-11-19 | Board Of Regents Of The University Of Okla. | Streptokinase-coding recombinant vectors |
| US5240845A (en) * | 1989-07-11 | 1993-08-31 | Otsuka Pharmaceutical Factory, Ltd. | Mutated streptokinase proteins |
| CU22277A1 (en) * | 1990-05-23 | 1995-01-31 | Cigb | Procedure for the isolation and expression of a codifying gene for streptokinase, the nucleotidic sequence obtained, dna recombiners and the transformed micro-organisms. |
-
1994
- 1994-04-04 CN CN 94112106 patent/CN1035193C/en not_active Expired - Fee Related
-
1995
- 1995-04-03 RU RU96121788/13A patent/RU2127758C1/en not_active IP Right Cessation
- 1995-04-03 WO PCT/CN1995/000024 patent/WO1995027050A1/en active Search and Examination
- 1995-04-03 CH CH03481/95A patent/CH688653A5/en not_active IP Right Cessation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064406C (en) * | 1998-05-11 | 2001-04-11 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for producing glucokinase by gene engrg. technique |
| CN102936603A (en) * | 2012-10-31 | 2013-02-20 | 上海昊海生物科技股份有限公司 | Expression of uridine diphosphate-glucose dehydrogenase and measurement of enzymatic activity |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2127758C1 (en) | 1999-03-20 |
| CH688653A5 (en) | 1997-12-31 |
| WO1995027050A1 (en) | 1995-10-12 |
| CN1035193C (en) | 1997-06-18 |
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