CN101928704A - Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof - Google Patents
Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof Download PDFInfo
- Publication number
- CN101928704A CN101928704A CN 201010117931 CN201010117931A CN101928704A CN 101928704 A CN101928704 A CN 101928704A CN 201010117931 CN201010117931 CN 201010117931 CN 201010117931 A CN201010117931 A CN 201010117931A CN 101928704 A CN101928704 A CN 101928704A
- Authority
- CN
- China
- Prior art keywords
- aspergillus flavus
- oxidase
- peg
- flavus uricoxidase
- urico
- 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
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a polyethylene glycol modifier of aspergillus flavus urate oxidase, wherein polyethylene glycol and the aspergillus flavus urate oxidase are connected through a covalent bond, the subunit of each aspergillus flavus urate oxidase molecule is averagely combined with 2-10 polyethylene glycol molecules, the average molecular weight of the polyethylene glycol molecule is 4-9kd, and the enzymatic specific activity of the polyethylene glycol modified aspergillus flavus urate oxidase is 5.0-10.0U/mg. A preparation method thereof is realized by adopting the following technical scheme: recombinant aspergillus flavus urate oxidase protein with the purity of larger than 95 percent is modified by adopting the activated polyethylene glycol molecule with the average molecular weight of 4-9kd for 0.1-10 hours at the pH value of 5-10 and the temperature of 4-37 DEG C, and the mass ratio of the polyethylene glycol molecule to the aspergillus flavus urate oxidase is 3:1-20:1. The polyethylene glycol modified aspergillus flavus urate oxidase realizes the balance in the aspects of high specific activity, long half life, low immunogenicity, high stability, low cost and the like and has industrialization value.
Description
Technical field
Relate to a kind of Aspergillus flavus uricoxidase modifier for the treatment of gout and hyperuricemia, relate in particular to high Aspergillus flavus uricoxidase carbowax modifier of a kind of activity and preparation method thereof.
Background technology
The final product of human body purine metabolism is a uric acid, has the species of active urico-oxidase different with other oneself expressions, and uric acid can not be to get rid of external wallantoin easily by further oxygenolysis.Under the influence of factors such as disease, heredity or diet, uric acid can accumulate in blood, urine and tissue juice, concentration raises gradually, when having surpassed its solubleness, will increase the kidney burden, cause the damage of renal function, particularly secular uric acid level raises, can cause urate crystal in the relatively not too abundant joint of blood flow, subcutis deposition, form uratoma.Irregular gout outbreak, not only patient is very painful, also might cause joint deformity, disable.
The treatment gout is mainly used anti-inflammatory drug and uric acid resisting medicine at present, representative drugs has colchicine, NSAID (non-steroidal anti-inflammatory drug), adrenocortical hormone, Zyloric, probenecid, bromobenzene Malong etc., these medicines all need to take medicine for a long time, and have significant side effects.Urico-oxidase (Urate Oxidase, UOX) can effectively decompose uric acid is the external wallantoin of easier eliminating, the mode of action of its uric acid reducing level is different with other drug, performance uric acid resisting effect rapidly after the administration, side effect is little.The approval prototype reorganization urico-oxidase Rasburicase of U.S. food Drug Administration was used for the treatment of the hyperuricemia that the pediatric tumor chemicotherapy causes in 2002.There was urico-oxidase to be used for the clinical study report of gout treatment in recent years, particularly can not tolerate patient (the Rasburicase for tophaceus gout treatment of Zyloric treatment, Ribeiro A et al, ActaReumatol Port.2009 Jul-Sep; 34 (3): 551-4; Urate oxidase (rasburicase) for treatment ofsevere acute gout:a case report, Wipfler-Frei β muth E.et al, Clin Exp Rheumatol.2009Jul-Aug; 27 (4): 658-660), obtained good result of treatment.But prototype reorganization urico-oxidase is the transformation period weak point in vivo, and immunogenicity is strong, has limited its application in gout treatment.
(Polyethlene glycol PEG) modifies urico-oxidase and reduces immunogenicity, the existing many parts of reports of prolong half-life with polyoxyethylene glycol.Among the Chinese patent CN1322141A, the contriver has done summary to early stage research, sees specification sheets 6-8 page or leaf " table 1: the feature of PEG-urico-oxidase in the previous research ".Above-mentioned table 1 has been summed up 14 parts of previous research files altogether, wherein adopts 11 parts in the document of false monofilament yeast source place urico-oxidase, derives from 1 part in the document of the urico-oxidase of pork liver, does not report 2 parts in the document in urico-oxidase source.The PEG molecule type that the listed document of table 1 adopts comprises each 1 part of the linear PEG of 5KD (9 parts), the PEG of 2*5KD branch (3 parts) and 0.7,7.5, the linear PEG of 10KD.The listed document of table 1 has disclosed the proteinic characteristic of stock rule of PEG modification activities generally, and promptly along with the increasing of PEG degree of modification, the activity of the urico-oxidase of being modified is with respect to the active reduction of natural enzyme, and the immunogenicity of modified outcome reduces simultaneously.But above-mentioned document does not disclose a kind of technical scheme, and residual enzyme activity, transformation period, the immunogenicity of the PEG-urico-oxidase of this technical scheme preparation can satisfy the requirement of clinical application.
In CN1322141A, the contriver discloses the urico-oxidase activity of modifying and has remained on the modification protocols of prototype enzyme more than 75%, and the degree of modification of PEG (in the quantity of each urico-oxidase subunit link coupled PEG molecule) is difference with the PEG molecule of the urico-oxidase on different sources ground, different molecular weight.For example, the chimeric UOX of pig-baboon, PEG molecular weight are at 2-10 bar/subunit, and enzymic activity all can remain on more than 75%, yet soybean UOX only when 2/subunit, could keep the activity more than 75%; For flavus UOX,, can keep the activity more than 75% when the PEG of 5KD is that the PEG of 12/subunit or 30KD is 7 a/subunit; For Arthrobacter globiformis UOX, then only when the PEG of 30KD is 2/subunit, could keep the activity more than 75%.Contriver's result of study has disclosed a kind of trend, and along with the increase of degree of modification, the activity of urico-oxidase is tending towards descending generally.What contriver's result of study showed is on the other hand: the PEG of macromolecule (10-30KD) can keep higher activity with respect to PEG (5KD) modifier of small molecular weight.Contriver's result of study also shows, with respect to single candiyeast, flavus, soybean source place urico-oxidase, derives from the chimeric urico-oxidase of urico-oxidase, especially one boar-baboon of pig, can keep higher enzymic activity after PEG modifies.See Fig. 1-Fig. 5, embodiment 4-12.
Savient Pharmaceuticals Inc. is developing the chimeric urico-oxidase new drug of pig-baboon that PEG modifies, and is used for gout treatment.This product is modified with the PEG of molecular weight 10KD, and each urico-oxidase subunit is in conjunction with 9 ± 1 PEG molecules (Briefing Document for KRYSTEXXA
TM(pegloticase) for IV infusion, BLA 125293, Savient Pharmaceuticals Inc., Page 27 of 161), the product specific activity is 95% (Pegloticase, a polyethylene glycol conjugate of uricase for thepotential intravenous treatment of gout, Biggers K of its prototype protein-active (2.3-5.6U/mg), Scheinfeld N., Curr Opin Investig Drugs. 2008Apr; 9 (4): 422-9).
The PEG that Chinese patent CN1561390A discloses the urico-oxidase that derives from candiyeast modifies, contriver's result of study shows, in conjunction with the PEG-urico-oxidase of average 19 molecular weight 5KDPEG, specific activity is 5.8IU/mg, and the intravital transformation period of mouse is 6 hours; And in conjunction with the PEG-urico-oxidase of similar number 20KDPEG, specific activity is 7.8IU/mg, and the intravital transformation period of mouse is 3 days.
Chinese patent CN101327327A discloses polyethyleneglycol modified Aspergillus flavus uricoxidase and preparation technology's thereof patent, and the employing molecular weight is that the polyoxyethylene glycol of 20kd is modified by halfcystine, and the product activity is not done report.According to (1996) Cloning such as YasujiKoyama, Sequence Analysis, and Expression in Escherichia coli of theGene Encoding the Candida utilis Urate Oxidase (Uricase), J.Biochem.120,969-973, each subunit of Aspergillus flavus uricoxidase molecule has three free cysteines, wherein Cys103 is near the molecular surface active centre, and two other halfcystine Cys35 and Cys290 are distributed in the duct, inside of tetramer molecule.Therefore, with the halfcystine of macromole PEG modification Aspergillus flavus uricoxidase molecular surface, PEG molecules influence substrate makes enzymic activity reduce greatly with combining of enzyme.If carrying out multiple spot modifies, then at first need to make intramolecular two halfcystines to come out, the sex change reagent of employing saboteur inner hydrophobic reactive force such as high concentration urea, Guanidinium hydrochloride can make halfcystine fully expose and modify, but enzymic activity correspondingly can lose.
Chinese patent CN101302501A discloses the PEG-urico-oxidase that N-terminal is modified, wherein urico-oxidase is the reorganization urico-oxidase that derives from flavus, PEG is that molecular weight is the N-terminal selectivity PEG of 20KD, 40KD, the efficient of modification reaction reaches 61%, 39% prototype albumen does not obtain modifying, therefore the yield of modified outcome is lower, preparation cost height.
The urico-oxidase that PEG modifies in the prior art, what have is active on the low side, so dosage will improve, and urico-oxidase is as the heterologous protein of human body, higher dosage means the risk increasing of medication; Prior art is thought with the long and/or active maintenance of the PEG modified outcome transformation period of macromolecule better, but there are problems such as cost height in the PEG of macromolecule; The modifying method modification efficient that has is not high and/or product stability is undesirable.Therefore seek a kind of enzymic activity height, long half time, immunogenicity is low, product stability good and cost is low urico-oxidase carbowax modifier has very important significance for gout and chronic hyperuricemia patient's treatment.
Summary of the invention
The purpose of this invention is to provide a kind of active high, immunogenicity is low, the polyethyleneglycol modified urico-oxidase preparation of good stability, is used for the demand that gout and chronic hyperuricemia particularly can not be accepted the patient with gout of other drug treatment.
For achieving the above object, the present invention has adopted following technical scheme:
The carbowax modifier of Aspergillus flavus uricoxidase, polyoxyethylene glycol is connected with covalent linkage with Aspergillus flavus uricoxidase, each Aspergillus flavus uricoxidase molecule subunit is on average in conjunction with 2～10 peg molecules, the molecular-weight average of peg molecule is 4kd-9kd, and the specific enzyme activity of polyethyleneglycol modified Aspergillus flavus uricoxidase is 5.0-10.0U/mg.
One preferred aspect, polyethyleneglycol modified Aspergillus flavus uricoxidase is compared with prototype enzyme, the titre that Aspergillus flavus uricoxidase carbowax modifier animal injection back produces antibody is 1: 320～1: 1280.
One preferred aspect, polyethyleneglycol modified Aspergillus flavus uricoxidase is compared with prototype enzyme, the transformation period in animal body of Aspergillus flavus uricoxidase carbowax modifier is 62 hours.
The Aspergillus flavus uricoxidase that the present invention selects for use is the recombined Aspergillus flavus uricoxidase that adopts escherichia expression system to obtain by genetic engineering technique.
The molecular-weight average of the PEG that the present invention adopts is 4kd-9kd.The molecular-weight average of the PEG that Aspergillus flavus uricoxidase modifier of the present invention is especially selected for use is 5kd-8kd.Particularly preferred Aspergillus flavus uricoxidase modifier of the present invention, the molecular-weight average of PEG is 5kd.
The urine flavicidic acid oxydase modifier that the present invention obtains, specific enzyme activity is 5.0-10.0U/mg.Particularly preferred Aspergillus flavus uricoxidase modifier, specific enzyme activity are 8.0-10.0U/mg.
In order to realize the present invention, thing activated PEG molecule passes through amine ester bond covalent coupling to the Aspergillus flavus uricoxidase molecule.
The present invention also provides a kind of preparation method of Aspergillus flavus uricoxidase carbowax modifier, polyoxyethylene glycol is connected by covalent linkage with Aspergillus flavus uricoxidase, each Aspergillus flavus uricoxidase molecule subunit is on average in conjunction with 4～6 peg molecules, this method realizes by the following technical solutions: purity is greater than 95% recombined Aspergillus flavus uricoxidase albumen, adopting molecular-weight average is that the activated polyethylene glycol molecule of 4kd-9kd is in pH value 5～10,4～37 ℃ of temperature, 0.1～10 hour time modified, and the mass ratio of peg molecule and Aspergillus flavus uricoxidase is 3: 1～20: 1.
Preferred the present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and the molecular-weight average of the PEG that selects for use is 5kd～8kd.
Particularly preferred the present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and the molecular-weight average of the PEG that selects for use is 5kd.
The present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and activated PEG molecule or mono methoxy PEG molecule are coupled on the urico-oxidase molecule by amine ester covalent linkage.
The preparation method of Aspergillus flavus uricoxidase carbowax modifier of the present invention, activated PEG are PEG nitro phenyl ester or PEG succinimide ester.
The preparation method of Aspergillus flavus uricoxidase carbowax modifier of the present invention, preferred activated PEG are any one in PEG propionic ester, PEG acetic ester and the PEG carbonic ether.
The present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and the pH value value of preferred modification reaction is controlled at 8.0-9.2.
The present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and the temperature of preferred modification reaction is 10-25 ℃.
The present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and the preferred modification reaction time is 0.5-2 hour.
The present invention prepares Aspergillus flavus uricoxidase carbowax modifier method, and the mass ratio of preferred peg molecule and urico-oxidase is 5: 1～15: 1.
Result of study of the present invention unlike the prior art.The present invention's polyethyleneglycol modified Aspergillus flavus uricoxidase of 5KD, when degree of modification was the 2-10/ subunit, specific activity was 8-10U/mg; Transformation period has increased 5-10 doubly than prototype enzyme simultaneously, and transformation period T1/2 is 60 hours in the chicken body of being in; Immunogenicity is than the obvious reduction of prototype enzyme, and the cavy anaphylaxis significantly is lower than prototype enzyme.Simultaneously, the Aspergillus flavus uricoxidase good stability that 5KD of the present invention is polyethyleneglycol modified, production cost is lower.Therefore, polyethyleneglycol modified Aspergillus flavus uricoxidase of the present invention realized height ratio work, the long half-lift, aspects such as reduced immunogenicity, high stability, low cost equilibrium, have industrialization value.The present invention is compared with the prior art and sees Table one.
Be better than small molecular weight PEG modification though prior art demonstrates the PEG modification of macromolecule generally, the PEG of 10KD-30KD molecular weight modifies as preferred technical scheme, and the present invention is not limited to any existing theory and hypothesis.
Table one, this patent are compared with the prior art
| This patent | CN1322141A table 1 is quoted | CN1322141A | CN1561390A | CN101327327A | CN101302501A | ||
| Enzyme source | Flavus | Single candiyeast etc. | Single candiyeast, Arthrobacter globiformis, pig-baboon, flavus, soybean | Single candiyeast | Flavus | Flavus | |
| The PEG molecular weight | 5KD, 10KD, 20KD 5KD are preferred | 5KD, 2*5KD | 5KD, 10KD, 30KD | 5KD, 20KD | 20KD | 20KD, 40KD | |
| Decorating site | Methionin | Methionin | Methionin | Methionin | Halfcystine | N-terminal amino | |
| Degree of modification | Each subunit is in conjunction with 4-6 PEG molecule, preferably 5. | Flavus: 5KD12 bar; 30KD7 bar soybean: 5KD, 30KD are 2; Arthrobacter globiformis: 30KD is 2; Single candiyeast: 5KD6 bar; The 30KD6 bar; 10KD9 bar pig-baboon: various molecular weight 2-10 all can | Article 19 ,/subunit | ||||
| Active | 9.6U/mg protoenzyme 18.7U/mg | More than 75%/protoenzyme: | 5KD:5.8U/mg 20KD:7.8U/mg protoenzyme: about 10U/mg | Do not have | 8-12U/mg | ||
| | Chicken | 60 hours | 5KD:6 hour 20KD:3 days | ||||
| Immunogenicity | Reduce than prototype albumen | Fig. 7 and explanation: behind the multiple dosing, 24 hours residual enzyme activity of administration is represented |
Description of drawings
The escherichia coli expression Aspergillus flavus uricoxidase of accompanying drawing 1, purifying;
1, standard protein molecular weight (97.4,66.2,43,31,20,14.4kd);
The escherichia coli expression urico-oxidase of 2-4, purifying.
Accompanying drawing 2, polymolecular PEG modify urico-oxidase electrophoretic analysis result;
1 and 8 swimming lanes: standard protein molecular weight (97.4,66.2,43,30,20.1,14.4kd);
2,3 and 4 swimming lanes: unmodified Aspergillus flavus uricoxidase;
5 swimming lanes: 5k PEG modifies Aspergillus flavus uricoxidase;
6 swimming lanes: 10k PEG modifies Aspergillus flavus uricoxidase;
7 swimming lanes: 20k PEG modifies Aspergillus flavus uricoxidase,
Accompanying drawing 3, different molecular weight PEG modifier duration of efficacy in animal body relatively;
Serum uric acid level after accompanying drawing 4, the animal long term administration;
Accompanying drawing 5, PEG and urico-oxidase different ratios modified product electrophoretic analysis result;
1 swimming lane: PEG and Aspergillus flavus uricoxidase mass ratio are 10: 1;
2 swimming lanes: PEG and Aspergillus flavus uricoxidase mass ratio are 7: 1;
3 swimming lanes: PEG and Aspergillus flavus uricoxidase mass ratio are 5: 1;
4 swimming lanes: PEG and Aspergillus flavus uricoxidase mass ratio are 3: 1;
5 swimming lanes: unmodified Aspergillus flavus uricoxidase.
The different pH value modified product electrophoretic analysis results that accompanying drawing 6, PEG modify urico-oxidase;
1-8 swimming lane modification reaction pH value is respectively 6,6.5,7,7.5,8,8.5,9,9.2.
Accompanying drawing 7, PEG modify urico-oxidase different time product electrophoretic analysis result;
The 1-5 swimming lane modification time is respectively 0.5,1,2,4,8hr.
Accompanying drawing 8, urico-oxidase and PEG modifier thereof are at different pH value stabilizations;
Accompanying drawing 9, urico-oxidase and the stability of PEG modifier under differing temps thereof;
Accompanying drawing 10, urico-oxidase and the PEG modifier rat test of pesticide effectiveness thereof;
The intravital activity change curve of chicken of being in after accompanying drawing 11, the urico-oxidase administration;
Accompanying drawing 12, the urico-oxidase PEG modifier intravital activity change curve of chicken of being in;
Accompanying drawing 13, PEG acetic ester are modified urico-oxidase;
1 swimming lane: the mass ratio of activated PEG and urico-oxidase is 4: 1;
2 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 6: 1;
3 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 8: 1;
4 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 10: 1;
5 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 15: 1.
Accompanying drawing 14, PEG carbonic ether are modified urico-oxidase;
1 swimming lane: the mass ratio of PEG carbonic ether and urico-oxidase is 15: 1;
2 swimming lanes: the mass ratio of PEG carbonic ether and urico-oxidase is 10: 1;
3 swimming lanes: the mass ratio of PEG carbonic ether and urico-oxidase is 8: 1;
4 swimming lanes: the mass ratio of PEG carbonic ether and urico-oxidase is 6: 1;
5 swimming lanes: the mass ratio of PEG carbonic ether and urico-oxidase is 4: 1.
Accompanying drawing 15, PEG nitro phenyl ester are modified urico-oxidase.
1 swimming lane: the mass ratio of activated PEG and urico-oxidase is 10: 1;
2 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 8: 1;
3 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 6: 1;
4 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 4: 1;
5 swimming lanes: the mass ratio of activated PEG and urico-oxidase is 2: 1.
Specific embodiment
Further specify the present invention below in conjunction with specific embodiment, but and non-limiting the present invention.The preparation of embodiment one, Aspergillus flavus uricoxidase and determination of activity
1, the preparation of Aspergillus flavus uricoxidase
The preparation method of recombined Aspergillus flavus uricoxidase comprises the steps: the acquisition of (1), Aspergillus flavus uricoxidase gene; (2) structure of expression vector and transformed into escherichia coli engineering bacteria: synthetic Aspergillus flavus uricoxidase cDNA fragment is inserted into the restriction enzyme site of expression vector plasmid, then direct transformed into escherichia coli host bacterium; (3), the screening of positive colony, cultivation, abduction delivering: filter out positive colony and on substratum, cultivate, target protein is expressed through inducing; (4), the separation of fusion rotein, purifying: with colibacillus engineering microorganism collection, broken bacterium, through the centrifugal bacterium liquid supernatant that obtains brokenly, then to broken bacterium liquid supernatant chromatographic technique purifying; (5), the enzyme of fusion rotein is cut and further separation and purification.
The acquisition of Aspergillus flavus uricoxidase gene is synthetic and by the design primer by full gene, PCR obtains again, the expression vector plasmid is pET-32a, the host bacterium is e. coli bl21 (DE3), restriction enzyme site is Nde I/BamH I, do resistance screening with penbritin, the temperature of culture medium culturing is controlled at 37, the abduction delivering working concentration is that the IPTG of 0.5～1.0mmol/L is as inductor, chromatographic technique is ion exchange chromatography, sieve chromatography, hydrophobic chromatography and affinity chromatography, and it is enteropeptidase that enzyme is cut used enzyme.
Aspergillus flavus uricoxidase cDNA sequence according to bibliographical information (Genebank A32839, A32840, A32841), synthetic 30 sections complementary oligonucleotide, and hold at 5 ' end and 3 ' and to introduce restriction enzyme site Nde I, BamH I respectively, press the ordinary method of molecular cloning, at first handle 30min for 37 ℃ with T4 phage polynucleotide kinase, each oligonucleotide fragment of phosphorylation mixes to wait mole, 94 ℃ of sex change 5min, immediately 65 ℃ annealing 10min, add the T4 ligase enzyme then, 16 ℃ of connections are spent the night.
The pET-32a plasmid reclaims big fragment with Nde I, BamH I double digestion, be connected with synthetic Aspergillus flavus uricoxidase cDNA fragment, 20 μ L reaction system gene fragments and the big segmental ratio of carrier are 10: 1, add T4DNA ligase enzyme 300 units, 15 ℃ of connections are spent the night, and get 10 μ L and connect product direct transformed into escherichia coli host bacterium BL21 (DE3) competent cell, coat the amicillin resistance flat board, 37 ℃ of overnight incubation obtain engineering bacteria.
Penbritin is done resistance screening, obtains positive colony pET-32a-Uox.Extract plasmid, identify with restriction enzyme.Positive transformant carries out sequential analysis with universal primer, and cloned sequence and implementation sequence are in full accord as a result.The inoculation positive colony is cultivated, and induces through the IPTG of 1.0mmol/L.
Get 100ml and cultivate the centrifugal collection thalline of bacterium liquid, suspend with 10ml 20mmol/L PB damping fluid again, carry out ultrasonication, collect then and go up cleer and peaceful precipitation, get supernatant 50ul, handle back 12%SDS-PAGE electrophoretic analysis, last sample 20ul with 2X SDS-PAGE sample-loading buffer, terminal objective albumen mainly is present in brokenly in the supernatant liquor behind the bacterium, and the expression amount of target protein is more than 30%.
With the broken bacterium supernatant liquor ion exchange chromatography separation and purification of collecting, its process makes its specific conductivity less than 5mS/cm for being that 8.5 20mmol/L Tris.CL damping fluid dilutes with pH value.Damping fluid balance SP post with same is washed till baseline with level pad behind the last sample, with 0.01～0.5mol/LD NaCL gradient elution, collects the target protein peak.
To make the concentration of target protein reach 1mg/mL after the dilution of ion-exchange target protein peak, sample cuts damping fluid (50mmol/L Tris.CL, 150mmol/L NaCL, 2.5mmol/L CaCL to enteropeptidase
2, pH value 7.5) dialyse and carry out buffer-exchanged, add the enteropeptidase of 20U/mL then, 16 ℃ of incubated overnight enzymes are cut, the 12%SDS-PAGE electrophoretic analysis.Target protein after enzyme is cut is added (NH
4)
2SO
4Making final concentration is 1.0-1.5mol/L, uses 1.2mol/L (NH on the sample
4)
2SO
4, 20mmol/L PB, the damping fluid equilibrated Phenyl-Sepharose Fast Flow post of pH value 7.4 is washed and is dragged by reducing the salt concn gradient, collects the target protein peak.
The hydrophobic chromatography peak that obtains is through Sephadex G-25 desalination, and balance liquid is 20mmol/L PB, pH value 7.4.Be the Aspergillus flavus uricoxidase of polishing purification after the sample desalination, SDS-PAGE analysed preparation purity the results are shown in Figure one, and sample purity is greater than 95%.
2, the determination of activity of Aspergillus flavus uricoxidase
Get and survey the damping fluid 2.0ml that lives, add uric acid storage liquid 1.5ml, the rearmounted 30 ℃ of water-bath balance 5min of mixing, in above-mentioned reaction solution, add the 1.0ml trial-product, mix the back in 30 ℃ of reaction 5min, reaction finishes the back and adds 0.5ml reaction terminating liquid termination reaction, measures optical density at wavelength 292nm place with ultraviolet visible spectrophotometry.Get and survey the damping fluid 2.0ml that lives, add uric acid storage liquid 1.5ml and 0.5ml reaction terminating liquid, add the 1.0ml trial-product again, mix the back in 30 ℃ the reaction 5min after as reaction tubes uric acid initial value, get and survey live damping fluid 3.5ml and 0.5ml reaction terminating liquid, add the 1.0ml trial-product again, mix the back as sample blank.
Get uric acid reference liquid 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml and place test tube respectively, add respectively and survey the damping fluid 4.0ml that lives, 3.5ml, 3.0ml, 2.5ml, 2.0ml to make final volume be 4.5ml.The rearmounted 30 ℃ of water-bath balance 5min of mixing.Add the 0.5ml reaction terminating liquid, measure absorption value.Measure 4.5ml and survey the damping fluid of living, 30 ℃ of water-bath balance 5min add the 0.5ml reaction terminating liquid as blank.The gained data are used to specify typical curve.
Corresponding its optical density of typical curve uric acid concentration is asked linear regression equation, will record trial-product reaction tubes optical density substitution linear regression equation, promptly gets the concentration of trial-product reaction tubes uric acid.Uric acid reduction value that decomposition causes according to enzyme and protein concentration calculate the specific activity of enzyme.The specific activity of the Aspergillus flavus uricoxidase of purifying is 18.7U/mg.
Embodiment two, different molecular weight PEG modify urico-oxidase
1, the preparation of modified outcome
The escherichia coli expression Aspergillus flavus uricoxidase purity of purifying is greater than 95%, and concentration 2.0mg/ml with G25 post exchange buffering liquid, prepares level pad 0.1mol/L Na
2PO
4-20mmol/L NaCl, flow velocity 10ml/min treats to go up sample after the post effluent liquid reaches balance, collects protein peak.The mono methoxy PEG propionic ester of different molecular weight is joined by mass ratio in the urico-oxidase that changes buffer system at 7: 1, and stirring at room is modified termination reaction after 2 hours.With the ultra-filtration membrane of 50KD with urico-oxidase PEG reaction mixture to 20mmo/L PB, the 8.0 damping fluid ultrafiltration of pH value are removed free PEG and are urico-oxidase PEG modifier stoste.
Modified outcome is carried out SDS-PAGE analyze, electrophoresis result shows the urico-oxidase of this modifier for polymolecular PEG modification, sees figure two.
Measure the proteic PEG modification rate in back of modifying with reference to (1966) Determination of free amino groups in proteins byTrinitrobenzenesulfonic acid.Analytical Biochemistry (14:328-336) methods such as Habeeb A.F..With 20mmol/L PB damping fluid, pH value 8.0 dialysis treatment urico-oxidases and PEG modify urico-oxidase, accurately measure protein concn, accurately are mixed with 1.0mg/ml.Accurately measure 1, urico- oxidase solution 0,40,80,120,200,300, the 400 μ l of 0mg/ml place test tube respectively, and the 20mmol/L PB damping fluid that adds pH value 8.0 adds the NaHCO of pH value 9.16 then to 1ml
3-Na
2CO
3Damping fluid 1ml adds 0.1%TNBS solution 1ml, thermal agitation on mixer oscillator again.PEG to 1mg/ml modifies the above operation of urico-oxidase solution repetition.40 ℃ of reaction 2hr add 10%SDS solution 1ml then respectively, add 1N HCl 0.5ml more respectively.Measure the absorption value of OD335nm, the drawing standard curve.Calculate the albumen slope of albumen slope/unmodified of modifying.
PEG modifies the total quantity of quantity=[1-(PEG modifies the slope/urico-oxidase slope of urico-oxidase)] * urico-oxidase primary amine residue of urico-oxidase primary amine residue.
Modification rate=[1-(PEG modifies the slope/urico-oxidase slope of urico-oxidase)] * 100%.
Experimental result sees Table two.
The modification degree measurement result of table two, PEG urico-oxidase
2, the active determination in vitro of modified outcome
Measure the enzymic activity of modified outcome according to the method for embodiment 1, the results are shown in Table three.
Table three, urico-oxidase PEG polymolecular modification activities result
| Numbering | 1? | 2? | 3? | 4? |
| Molecular weight polyethylene glycol | 5kd? | 10kd? | 20kd? | 40kd? |
| The urico-oxidase specific activity | 9.6U/mg? | 4.1U/mg? | 2.0U/mg? | 0.7U/mg? |
| Activity residual percentage ratio | 51.3％? | 21.9％? | 10.7％? | 3.7％? |
3, duration of efficacy compares in the body of modified outcome
With the SD rat is the experimental animal modeling, and relatively molecular weight is the PEG of 5k, 10k, the 20k reorganization urico-oxidase uric acid resisting effect of the modifying difference of time length in animal body.10 animals of blank group, not administration; 10 animals of model group, the VITAMIN B4 of dosage 150mg/kg/day and, the Tibutol modeling of 250mg/kg/day; 10 animals of positive controls are used the 150mg/kg/day VITAMIN B4, treat with the 20mg/kg/day allopurinol in the time of the modeling of 250mg/kg/day Tibutol; 10 animals of 5kPEG urico-oxidase group are used the 150mg/kg/day VITAMIN B4, the modeling of 250mg/kg/day Tibutol, and 5kPEG modifies urico-oxidase dosage 0.5mg/kg; 10 animals of 10kPEG urico-oxidase group, 150mg/kg/day VITAMIN B4, the modeling of 250mg/kg/day Tibutol, 10kPEG urico-oxidase therapeutic dose 0.5mg/kg; Use the dosage treatment of 20kPEG urico-oxidase 0.5mg/kg in the time of 10 animal modelings of 20kPEG urico-oxidase group.Before the administration, the blood sampling of rat eye socket, anticoagulant heparin, the initial value of mensuration blood plasma uric acid concentration.. three groups of PEG urico-oxidase treatment treated animal dosages are 0.5mg/kg, are administered once.The treatment that is administered once every day of allopurinol group, irritate stomach and give modeling agent VITAMIN B4 and Tibutol half an hour after the administration, and the blood plasma uric acid concentration is measured in posterior orbit blood sampling in two hours, experimental result is seen accompanying drawing three, and it is longer that the product that 5kPEG modifies is kept time of drug effect in animal body.
4, the situation of animal generation antibody after the modified outcome administration
Purpose: relatively different molecular weight 5k, 10k, 20kPEG modify reorganization urico-oxidase long term injections after produce the situation of antibody.
Test method: laboratory animal is the SD rat, 10 of every treated animals, the not administration of blank group, model group 150mg/kg/day VITAMIN B4 and the modeling of 250mg/kg/day Tibutol, irritate the stomach treatment with the 150mg/kg/day Zyloric in the time of the positive controls modeling, 5kPEG urico-oxidase group, 10kPEG urico-oxidase group and 20kPEG urico-oxidase group dosage are 0.5mg/kg.Before the administration, the blood sampling of rat eye socket, anticoagulant heparin, the initial value of mensuration blood plasma uric acid concentration.The blood plasma uric acid concentration is measured in posterior orbit blood sampling in two hours.
Three groups of PEG urico-oxidases of beginning medication treatment group dosage is 0.5mg/kg.The Zyloric gastric infusion, irritate stomach and give modeling agent VITAMIN B4 and Tibutol half an hour after the administration.The blood plasma uric acid concentration is measured in posterior orbit blood sampling in two hours.
Rechallenge after 40 days, three groups of PEG urico-oxidase treatment group dosages are 0.5mg/kg.Allopurinol irritate stomach give with.Irritate stomach and give modeling agent VITAMIN B4 and Tibutol half an hour after the administration.The blood plasma uric acid concentration is measured in posterior orbit blood sampling in two hours.
Cross and 2 weeks be mixed into emulsion with the PEG urico-oxidase of three kinds of molecular weight respectively with complete freund adjuvant, subcutaneous injection goes into respectively to organize rat.Three groups of PEG urico-oxidase treatment group dosages are 0.5mg/kg.
The three groups of PEG urico-oxidase treatment group dosages in 4 week backs are 0.5mg/kg.Allopurinol irritate stomach give with.Irritate stomach and give modeling agent VITAMIN B4 and Tibutol half an hour after the administration.The plasma antibody titre is measured in posterior orbit blood sampling in two hours, and test-results sees Table four, and animal blood uric acid measurement result is seen accompanying drawing four.
The result shows, booster immunization after the urico-oxidase medication of three kinds of molecular weight PEG modifications, and not obviously difference of animal's antibody titre after the medication again, promptly macromolecule PEG does not show clear superiority after modifying urico-oxidase aspect immunogenicity.PEG modification urico-oxidase group uric acid level was lower after the blood uric acid measurement result showed generation antibody, and antibody does not influence the activity of enzyme
Plasma antibody titre after table four, the medication of SD rat
Comparative Examples one, unit molecule PEG modify urico-oxidase
The mono methoxy PEG propionic ester of different molecular weight is joined by mass ratio in the urico-oxidase of buffer system pH value 6.0 at 5: 1, and stirring at room is modified termination reaction after 1 hour.With the ultra-filtration membrane of 50KD with urico-oxidase PEG reaction mixture to 20mmo/L PB, free PEG is removed in the 8.0 damping fluid ultrafiltration of pH value, obtains the urico-oxidase that unit molecule PEG modifies.
Measure the enzymic activity of modified outcome according to the method for embodiment 1, determination of activity the results are shown in Table five.
Table five, urico-oxidase PEG unit molecule modification activities result
| Numbering | 5? | 6? | 7? | 8? |
| Molecular weight polyethylene glycol | 5kd? | 10kd? | 20kd? | 40kd? |
| The urico-oxidase specific activity | 13.9U/mg? | 10.7U/mg? | 10.1U/mg? | 8.2U/mg? |
| Activity residual percentage ratio | 74.3％? | 57.2％? | 54.1％? | 43.9％? |
The 5-8 sample of the 1-4 sample of embodiment 2 preparation and Comparative Examples 1 preparation is placed the accelerated test of carrying out study on the stability in a day in 37 ℃, and the active reservation situation of sample sees Table six.
Table six, PEG urico-oxidase stability accelerated test result
| Numbering | 1? | 2? | 3? | 4? | 5? | 6? | 7? | 8? |
| Enzymic activity (U/mg) | 8.9? | 3.1? | 1.2? | 0? | 0.8? | 3.3? | 1.8? | 2.2? |
Stability accelerated test result shows that the Aspergillus flavus uricoxidase of single-point PEG modification is 37 ℃ of very fast forfeitures of enzymic activity, and multiple spot is modified sample and is had the Aspergillus flavus uricoxidase that the active 5K molecular weight of high enzyme PEG modifies and can keep stable preferably, placed one day for 37 ℃, activity can also keep 92.7%.
Comparative Examples two, polyethyleneglycol modified urico-oxidase molecule halfcystine
Escherichia coli expression Aspergillus flavus uricoxidase purity is greater than 95%, and concentration 2.0mg/ml is with G25 post exchange buffering liquid, preparation level pad 0.05mol/L PB-20mmol/L NaCl, flow velocity 10ml/min treats to go up sample after the post effluent liquid reaches balance, collects protein peak.With molecular weight is that the mono methoxy PEG maleimide of 5k, 10k, 20k joins in the urico-oxidase by mass ratio at 2: 1, stirring at room reaction 8 hours, the electrophoretic analysis modified outcome is also measured enzymic activity, enzymic activity is lower than 1.0U/mg as a result, modified outcome is that unit molecule PEG modifies, show that PEG modifies that the activity of urico-oxidase is subjected to very big influence behind the halfcystine, activity is lost substantially, and near the halfcystine illustrate in PEG and the activity combines combining of interference substrate and enzyme afterwards.
The mass ratio of mono methoxy PEG maleimide and urico-oxidase is brought up to 5: 1 join in the urico-oxidase, stirring at room reaction 8 hours, the electrophoretic analysis modified outcome is the unit molecule modifier, enzymic activity 0.3U/mg.
In order to make PEG fully modify halfcystine in the urico-oxidase molecule, with 0.05mol/L PB--8mol/L Urea as level pad with G25 post exchange buffering liquid, the protein peak of collecting, mass ratio by 5k mono methoxy PEG maleimide and urico-oxidase joins in the urico-oxidase at 7: 1, stirring at room reaction 10 hours, the electrophoretic analysis modified outcome is the polymolecular modifier, enzymic activity 0, and enzymic activity completely loses.
The optimization of embodiment three, PEG (5KD) and urico-oxidase modification reaction
1, the ratio of activated PEG and urico-oxidase is selected
Urico-oxidase concentration 2.0mg/mL gets 0.25mL urico-oxidase stoste, adds equal-volume 0.2mol/LNa
2HPO
4-0.02mol/L NaCL damping fluid is that final concentration of protein is 0.5mg/0.5mL, the molecular weight that adds 150mg/mL then respectively is activated PEG propionic acid NHS ester 15 μ L, 25 μ L, 35 μ L and the 50 μ L of 5kd, make the mass ratio of activated PEG propionic acid NHS ester and urico-oxidase reach 3: 1 respectively, 5: 1,7: 1 and 10: 1, mix rapidly, 4 hours termination reactions of room temperature reaction, the SDS-PAGE electrophoretic analysis is carried out in sampling, the results are shown in accompanying drawing five, 7: 1 and 10: 1 modifier homogeneity good.
2, activated PEG is modified the pH value selection of urico-oxidase
Urico-oxidase concentration 2.0mg/mL gets 0.25mL urico-oxidase stoste, and adding equal-volume pH value is respectively 6,6.5,7,7.5,8,8.5,9,9.2 0.2mol/L NaH
2PO
4-Na
2HPO
4-0.02mol/L NaCL damping fluid, making final concentration of protein is 0.5mg/0.5mL, the molecular weight that adds 150mg/mL then respectively is the activated PEG propionic acid NHS ester 25 μ L of 5kd, make the mass ratio of activated PEG propionic ester and urico-oxidase reach 5: 1 respectively, mix rapidly, 4 hours termination reactions of room temperature reaction, the SDS-PAGE electrophoretic analysis is carried out in sampling, the results are shown in accompanying drawing six, modified product was all once better in the scope of pH value 8-9.2.
3, the selection of modification reaction time
Urico-oxidase concentration 2.0mg/mL gets 0.25mL urico-oxidase stoste, adds equal-volume 0.2mol/LNaH
2PO
4-Na
2HPO
4-0.02mol/L NaCL damping fluid, making final concentration of protein is 0.5mg/0.5mL, the molecular weight that adds 150mg/mL then is the activated PEG propionic ester 30 μ L of 5kd, make the mass ratio of activated PEG propionic ester and urico-oxidase reach 5: 1 respectively, mix rapidly, 0.5,1,2,4,8 hour termination reaction of room temperature reaction, the SDS-PAGE electrophoretic analysis is carried out in sampling, the results are shown in accompanying drawing seven, the result shows that speed of response is very fast, and modified outcome was all once better after half an hour.
Embodiment four, PEG (5KD) multiple spot is modified the study on the stability of urico-oxidase
1, the pH value stabilization of urico-oxidase PEG modifier
Prepare the damping fluid of different pH value values: 50mmol/L NaAc-HAc, pH value 4.0; 50mmol/L NaAc-HAc, pH value 6.0; 50mmol/L PB, pH value 7.4; 50mmol/L PB, pH value 8.0; 50mmol/L Tris-HCL, pH value 9.0; 50mmol/L TEA, pH value 8.9; 50mmol/L Na
2CO
3-NaHCO
3, pH value 10.To recombinate urico-oxidase and PEG reorganization urico-oxidase respectively with 10 times of above-mentioned buffered soln dilutions, then sample is placed the different time sampling at 4 ℃ and carry out determination of activity with the concentration that corresponding damping fluid is diluted to 1 μ g/ml, the results are shown in accompanying drawing eight, the urico-oxidase activity change in different pH value scopes of as can be seen from the figure recombinating is bigger, and what active maintenance was best is in pH value 8.5.PEG reorganization urico-oxidase is more stable in the scope of pH value 7-10, and activity keeps more than 80% substantially.Same pH value condition, 4 ℃ to preserve after 1,6,36 day enzymic activity approaching, shows that 4 ℃ of shelf times are little to the enzymic activity influence.The forfeiture of enzymic activity is mainly caused by the variation of pH value under the acidic conditions.So the reorganization urico-oxidase is modified the tolerance increase of back albumen to the pH value through PEG, can adapt to wider pH value scope.
2, the temperature stability of urico-oxidase PEG modifier
Reorganization urico-oxidase stoste and PEGization reorganization urico-oxidase are respectively at 4 ℃, room temperature condition placement down, carry out determination of activity after the concentration that is diluted to 1 μ g/ml of taking a sample when arriving certain hour, the results are shown in accompanying drawing nine, after the urico-oxidase of as can be seen from the figure recombinating is placed 10 days at ambient temperature, active obviously decline, and have degraded to produce, and to place after 60 days, activity is very low.And PEGization reorganization urico-oxidase is after room temperature was placed 2 months, active decline.The reorganization urico-oxidase under 4 ℃ of conditions, places one month more stable, but along with increasing active decline storage period, and the degraded generation is arranged.PEGization reorganization urico-oxidase is placed the half a year activity and is still kept fine under 4 ℃ of conditions.This shows that the reorganization urico-oxidase is modified the back thermostability through PEG obviously to be increased.
Embodiment five, PEG (5KD) multiple spot is modified the rat test of pesticide effectiveness of urico-oxidase
The 5K PEG urico-oxidase of observing various dose is at the intravital duration of efficacy of SD rat, 10 of animal per groups, the not administration of blank group, model group 150mg/kg/day VITAMIN B4 and the modeling of 250mg/kg/day Tibutol, treat with the 20mg/kg/day allopurinol in the time of the positive controls modeling, three dosage of medication component, high dose group 2.47mg/kg 5KPEG urico-oxidase, middle dosage group 0.83mg/kg 5K PEG urico-oxidase, low dose group 0.27mg/kg 5K PEG urico-oxidase.Administration began test after the day before yesterday, basic plasma uric acid level was measured in every rat eye socket blood sampling, positive controls is irritated stomach and is given and the 20mg/kg/day allopurinol, high dose group, middle dosage group and low dose group tail vein injection are respectively given and 2.47mg/kg, 0.83mg/kg and 0.27mg/kg 5K PEG urico-oxidase, administration is irritated stomach and is given and modeling agent 150mg/kg/day VITAMIN B4,250mg/kg/day Tibutol after 0.5 hour.Give and modeling agent every rat eye socket blood sampling after 2 hours, measure plasma uric acid level.Every 24 hours, irritate stomach and give and positive drug and modeling agent then, plasma uric acid level is measured in blood sampling, and high, medium and low dosage group is respectively organized plasma uric acid level and compared end test when not having significant difference with model group, the results are shown in accompanying drawing ten.
Embodiment six, PEG (5KD) multiple spot is modified the cavy hypersensitive test of urico-oxidase
Relatively molecular weight 5kPEG modify the reorganization urico-oxidase and the originality protein medicine-feeding after the irritated situation of cavy, laboratory animal is an albino guinea-pig, every group of 6 animals, the not administration of physiological saline group; Albumin group priming dose 0.5mg/0.5ml/, booster dose 1.0mg/ml/ only; 5kPEG modify reorganization urico-oxidase low dose group priming dose 0.5mg/kg, 0.5ml/ only, booster dose 1.0mg/kg, 1.0ml/ are only; 5kPEG modify reorganization urico-oxidase high dose group priming dose 2.3mg/kg, 0.5ml/ only, booster dose 1.0ml/ is only; Originality albumen high dose group priming dose 0.3mg/0.5mL, 0.5mL/, booster dose 0.6mg/1mL, 1mL/ are only; Originality albumen low dose group priming dose 0.06mg/0.5mL, 0.5mL/, booster dose 0.12mg/1mL, 1mL/ are only.Every cavy according to dosage respectively abdominal injection give with albumin or be subjected to the reagent thing to carry out sensitization, weigh in before the administration, every the cavys in 2 week backs according to dosage respectively intravenous injection give with albumin or excited by the reagent thing, observe death condition or other allergic symptoms of respectively organizing cavy.The reaction of animals situation sees Table seven, and the result shows that urico-oxidase cavy allergic symptom after PEG modifies alleviates, and illustrates that immunogenicity reduces than prototype albumen.
Table seven, hypersensitive test reaction of animals situation
Embodiment seven, PEG (5KD) multiple spot is modified the pharmacokinetics of urico-oxidase
After choosing 6 mensuration of tame chicken serum basic value, be divided into 2 groups at random.Be subjected to test product respectively, urico-oxidase dosage 1.6mg/kg, 5kPEG modify Aspergillus flavus uricoxidase dosage 1.28mg/kg, press the dosage intravenous administration for every group.The different time vein is got blood after the administration, and the centrifugal collection serum in back that condenses naturally carries out enzyme activity assay.Method is seen the applicant's Chinese patent application 200610049079.2, the reduction of the uric acid absorption value that causes with enzyme reaction in the serum comes the active variation of reaction enzymes, urico-oxidase and modifier thereof the situation that the chicken activity in vivo changes of being in is seen accompanying drawing 11 and accompanying drawing 12, the result is 308 minutes through the drug half-life urico-oxidase prototype albumen T1/2 that software analysis calculates, it is 3697.495 minutes that 5kPEG modifies urico-oxidase T1/2, and the urico-oxidase transformation period has prolonged 12 times after modifying.
Embodiment eight, different activated PEG are modified urico-oxidase
1, the PEG acetic ester is modified urico-oxidase
Aspergillus flavus uricoxidase concentration 5.0mg/mL gets 1.0mL urico-oxidase solution, adds 4.0ml 0.2mol/LNa
2HPO
4It is 1.0mg/mL that-0.02mol/L NaCL damping fluid makes final concentration of protein, the activated PEG acetic ester that adds molecular weight then respectively and be 5kd makes the mass ratio of activated PEG acetic ester and Aspergillus flavus uricoxidase reach 4: 1～15: 1 respectively, mix rapidly, 2 hours termination reactions of room temperature reaction, the SDS-PAGE electrophoretic analysis is carried out in sampling, the results are shown in accompanying drawing 13, the homogeneity that sample is modified is good.
2, the PEG carbonic ether is modified urico-oxidase
Aspergillus flavus uricoxidase concentration 5.0mg/mL gets 1.0mL urico-oxidase solution, adds 4.0ml 0.2mol/LNa
2HPO
4It is 1.0mg/mL that-0.02mol/L NaCL damping fluid makes final concentration of protein, the activated PEG carbonic ether that adds molecular weight then respectively and be 10kd makes the mass ratio of activated PEG carbonic ether and Aspergillus flavus uricoxidase reach 4: 1～15: 1 respectively, mix rapidly, 2 hours termination reactions of room temperature reaction, the SDS-PAGE electrophoretic analysis is carried out in sampling, the results are shown in accompanying drawing 14,6: 1～10: 1 ratio of sample modification uniformity of sample is good.
3, PEG nitro phenyl ester is modified urico-oxidase
Aspergillus flavus uricoxidase concentration 5.0mg/mL gets 1.0mL urico-oxidase solution, adds 4.0ml 0.2mol/LNa
2HPO
4It is 1.0mg/mL that-0.02mol/L NaCL damping fluid makes final concentration of protein, the activated PEG nitro phenyl ester that adds molecular weight then respectively and be 20kd makes the mass ratio of activated PEG nitro phenyl ester and Aspergillus flavus uricoxidase reach 2: 1～10: 1 respectively, mix rapidly, 2 hours termination reactions of room temperature reaction, the SDS-PAGE electrophoretic analysis is carried out in sampling, the results are shown in accompanying drawing 15,4: 1～10: 1 ratio of sample modification uniformity of sample is good.
Claims (18)
1. the carbowax modifier of an Aspergillus flavus uricoxidase, polyoxyethylene glycol is connected with covalent linkage with Aspergillus flavus uricoxidase, each Aspergillus flavus uricoxidase molecule subunit is on average in conjunction with 2～10 peg molecules, it is characterized in that: the molecular-weight average of peg molecule is 4kd-9kd, and the specific enzyme activity of Aspergillus flavus uricoxidase carbowax modifier is 5.0-10.0U/mg.
2. the carbowax modifier of Aspergillus flavus uricoxidase as claimed in claim 1, it is characterized in that: compare with the Aspergillus flavus uricoxidase in conjunction with polyoxyethylene glycol not, the titre that Aspergillus flavus uricoxidase carbowax modifier animal injection back produces antibody is 1: 320～1: 1280.
3. the carbowax modifier of Aspergillus flavus uricoxidase as claimed in claim 1 is characterized in that: compare with the Aspergillus flavus uricoxidase in conjunction with PEG not, the transformation period in animal body of Aspergillus flavus uricoxidase carbowax modifier is 62 hours.
4. as the carbowax modifier of the described Aspergillus flavus uricoxidase of each claim of claim 1-3, it is characterized in that: Aspergillus flavus uricoxidase is the reorganization urico-oxidase that adopts escherichia expression system to obtain by genetic engineering technique.
5. as the carbowax modifier of the described Aspergillus flavus uricoxidase of each claim of claim 1-3, it is characterized in that: the molecular-weight average of polyoxyethylene glycol is 5kd-8kd.
6. the carbowax modifier of Aspergillus flavus uricoxidase as claimed in claim 5, it is characterized in that: the molecular-weight average of polyoxyethylene glycol is 5kd.
7. as the carbowax modifier of the described Aspergillus flavus uricoxidase of each claim of claim 1-3, it is characterized in that: the specific enzyme activity of Aspergillus flavus uricoxidase carbowax modifier is 6.0-10.0U/mg.
8. the carbowax modifier of Aspergillus flavus uricoxidase as claimed in claim 7, it is characterized in that: the specific enzyme activity of Aspergillus flavus uricoxidase carbowax modifier is 8.0-10.0U/mg.
9. the carbowax modifier of Aspergillus flavus uricoxidase as claimed in claim 1, it is characterized in that: peg molecule is coupled on the Aspergillus flavus uricoxidase molecule by amine ester covalent linkage.
10. the carbowax modifier of Aspergillus flavus uricoxidase as claimed in claim 1, it is characterized in that: peg molecule is branched.
11. the preparation method of the carbowax modifier of an Aspergillus flavus uricoxidase, it is characterized in that: purity is greater than 95% recombined Aspergillus flavus uricoxidase albumen, the employing molecular-weight average is that the activated polyethylene glycol molecule of 4kd-9kd was modified in pH value 5～10,4～37 ℃ of temperature, 0.1～10 hour time, and the mass ratio of peg molecule and Aspergillus flavus uricoxidase is 3: 1～20: 1.
12. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 11 is characterized in that: peg molecule passes through amine ester bond covalent coupling to the urico-oxidase molecule.
13. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 11 is characterized in that: activated polyethylene glycol is polyoxyethylene glycol nitro phenyl ester or polyoxyethylene glycol succinimide ester.
14. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 13 is characterized in that: the polyoxyethylene glycol succinimide ester is any one in polyoxyethylene glycol propionic ester, polyoxyethylene glycol acetic ester and the polyethylene glycol carbonates.
15. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 11 is characterized in that: the pH value of modification reaction is 8～9.2.
16. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 11 is characterized in that: the temperature of modification reaction is 10～15 ℃.
17. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 11 is characterized in that: the modification reaction time is 0.5～2 hour.
18. the preparation method of the carbowax modifier of a kind of Aspergillus flavus uricoxidase as claimed in claim 11 is characterized in that: the mass ratio of peg molecule and Aspergillus flavus uricoxidase is 5: 1～15: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010117931 CN101928704A (en) | 2010-03-04 | 2010-03-04 | Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010117931 CN101928704A (en) | 2010-03-04 | 2010-03-04 | Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101928704A true CN101928704A (en) | 2010-12-29 |
Family
ID=43368134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010117931 Pending CN101928704A (en) | 2010-03-04 | 2010-03-04 | Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101928704A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105412942A (en) * | 2015-12-23 | 2016-03-23 | 沈阳三生制药有限责任公司 | Pegylated recombined candida utilis urate oxidase freeze-drying injection |
| CN112852772A (en) * | 2021-01-19 | 2021-05-28 | 中国科学院过程工程研究所 | Urate oxidase based on intramolecular cross-linking and polyethylene glycol modification and preparation method thereof |
| CN114438048A (en) * | 2020-11-05 | 2022-05-06 | 重庆派金生物科技有限公司 | Urate oxidase preparation and application thereof |
| CN114438047A (en) * | 2020-11-05 | 2022-05-06 | 重庆派金生物科技有限公司 | Method for preparing polyethylene glycol modified urate oxidase |
| WO2024017379A1 (en) * | 2022-07-22 | 2024-01-25 | 派格生物医药(苏州)股份有限公司 | Preparation method for polyethylene glycol conjugate of active polypeptide or protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322141A (en) * | 1998-08-06 | 2001-11-14 | 山景药品公司 | PEG-urate oxidase conjugates and use thereof |
| CN1690197A (en) * | 2004-04-27 | 2005-11-02 | 杭州北斗生物技术有限公司 | Process for preparing aspergillus flavus urate oxidase |
| CN101302501A (en) * | 2007-05-10 | 2008-11-12 | 刘国安 | PEGylated uricoxidase compound, preparing method, preparation and use thereof |
-
2010
- 2010-03-04 CN CN 201010117931 patent/CN101928704A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322141A (en) * | 1998-08-06 | 2001-11-14 | 山景药品公司 | PEG-urate oxidase conjugates and use thereof |
| CN1690197A (en) * | 2004-04-27 | 2005-11-02 | 杭州北斗生物技术有限公司 | Process for preparing aspergillus flavus urate oxidase |
| CN101302501A (en) * | 2007-05-10 | 2008-11-12 | 刘国安 | PEGylated uricoxidase compound, preparing method, preparation and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《Pharm Res》 20031231 Lee H et al. N-terminal site-sepcific mono-PEGylation of epidermal growth factor 146-150 1-18 第20卷, 2 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105412942A (en) * | 2015-12-23 | 2016-03-23 | 沈阳三生制药有限责任公司 | Pegylated recombined candida utilis urate oxidase freeze-drying injection |
| CN105412942B (en) * | 2015-12-23 | 2019-02-26 | 沈阳三生制药有限责任公司 | The recombination candida utili urate oxidase freeze dried injection of Pegylation |
| CN114438048A (en) * | 2020-11-05 | 2022-05-06 | 重庆派金生物科技有限公司 | Urate oxidase preparation and application thereof |
| CN114438047A (en) * | 2020-11-05 | 2022-05-06 | 重庆派金生物科技有限公司 | Method for preparing polyethylene glycol modified urate oxidase |
| WO2022095973A1 (en) * | 2020-11-05 | 2022-05-12 | 杭州远大生物制药有限公司 | Urate oxidase preparation and use thereof |
| WO2022095974A1 (en) * | 2020-11-05 | 2022-05-12 | 杭州远大生物制药有限公司 | Method for preparing polyethylene glycol-modified urate oxidase |
| CN112852772A (en) * | 2021-01-19 | 2021-05-28 | 中国科学院过程工程研究所 | Urate oxidase based on intramolecular cross-linking and polyethylene glycol modification and preparation method thereof |
| WO2024017379A1 (en) * | 2022-07-22 | 2024-01-25 | 派格生物医药(苏州)股份有限公司 | Preparation method for polyethylene glycol conjugate of active polypeptide or protein |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102161984B (en) | The method of separating tetramer uric acid oxydiazing enzyme | |
| CN101194016B (en) | A variant form of urate oxidase and use thereof | |
| CN101812438B (en) | Arginine deiminase mutant and preparation and application thereof | |
| CN101137391B (en) | Uses of recombinant super-compound interferon | |
| EA026112B1 (en) | Stable composition comprising hyaluronidase and immunoglobulin, and methods of use thereof | |
| US20130052179A1 (en) | Arginine deiminase mutant and preparation and application thereof | |
| CN102827270A (en) | Pegylated exenatide ramification and use thereof | |
| CN101928704A (en) | Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof | |
| CN102145178A (en) | PEG interleukin 15 | |
| CN106554948A (en) | Saltant type uricase, the saltant type uricase of PEG modifications and its application | |
| CN104073482A (en) | Pegylated tissue kallikrein as well as preparation method and application thereof | |
| CN101198357A (en) | Human granulocyte-colony stimulating factor isoforms | |
| EP2123680B1 (en) | Recombinant chimeric protein of neutrophil inhibitory factor and hirugen and medicament composition thereof | |
| WO2012063984A1 (en) | Improved iduronate-2-sulfatase and use thereof | |
| WO2009086656A1 (en) | Y-type polyethylene glycol modified g-csf and preparation method and use thereof | |
| CN102260653B (en) | Preparation and application method of PEG recombinant pig-human urate oxidase fusion protein | |
| CN102295695A (en) | Recombinant human follicle stimulating hormone and preparation thereof | |
| CN101163716B (en) | Interleukin-6 polyethylene glycol conjugate and its preparing method and use | |
| TW202122409A (en) | Methods for production of human recombinant arginase 1 and uses thereof | |
| CN102731658B (en) | Tat PTD-Endostatin recombination protein, preparation method and application thereof | |
| CN101591649A (en) | Arginine deiminase that methoxy poly (ethylene glycol) is modified and preparation thereof and application | |
| CN101906158B (en) | Pegylation hypoglycemic polypeptide and preparation method and application thereof | |
| JP2023548207A (en) | Urate oxidase preparations and their applications | |
| CN101596320B (en) | Medicament for treating tumor and application thereof | |
| CN101906156B (en) | Structures and application of bifunctional protein and derivatives of bifunctional protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20101229 |