CN101875923A - Fusion protein of human serum albumin and urate oxidase and preparation method thereof - Google Patents
Fusion protein of human serum albumin and urate oxidase and preparation method thereof Download PDFInfo
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- CN101875923A CN101875923A CN2009102154465A CN200910215446A CN101875923A CN 101875923 A CN101875923 A CN 101875923A CN 2009102154465 A CN2009102154465 A CN 2009102154465A CN 200910215446 A CN200910215446 A CN 200910215446A CN 101875923 A CN101875923 A CN 101875923A
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Abstract
The invention discloses a fusion protein of human serum albumin and urate oxidase and a preparation method thereof, and the fusion protein is characterized in that the fusion protein of the human serum albumin and the urate oxidase consists of a human serum albumin amino acid sequence, a urate oxidase amino acid sequence and a connecting peptide, the sequence of the fusion protein is formed by connecting the human serum albumin amino acid sequence with the urate oxidase amino acid sequence through the connecting peptide, the human serum albumin amino acid sequence is arranged at an N terminal of the fusion protein, and the urate oxidase amino acid sequence is arranged at a C terminal of the fusion protein; or the human serum albumin amino acid sequence is arranged at the C terminal of the fusion protein, the urate oxidase amino acid sequence is arranged at the N terminal of the fusion protein, and the two protein amino acid sequences are connected through the connecting peptide. The fusion protein of the human serum albumin and the urate oxidase has long half-life and is a urate oxidase novel preparation.
Description
Technical field
The present invention relates to a kind of fusion rotein, relate in particular to fusion rotein of a kind of human serum albumin and urico-oxidase and preparation method thereof.
Background technology
Purine is the important component part of organism genetic material nucleic acid, nucleic acid metabolism produces purine nucleotides, latter's degradation production xanthine produces uric acid under the effect of XOD, uric acid can further (Urate Oxidase be decomposed into wallantoin, allantoic acid, CO2 and NH3 under effect UOX) at urico-oxidase.Human and some primate bodies are interior to lack activated urico-oxidase, so uric acid is the final product of human and these animal purine metabolisms.Uric acid solubleness is lower, is decomposed into the higher wallantoin of solubleness after the oxidasic oxidation of dialuric acid, makes that the nucleic acid metabolism product is easier to excrete.If hyperuricemia appears in lasting rising of uric acid concentration in blood and the urine, often cause disease to take place, wherein more representational is gout.
Gout is a kind of chronic disease that the uric acid metabolism disorder causes, urate crystal is its pathogenic factor in joint and soft tissue deposition, is accompanied by inflammation, and some patient develops into joint injury or osteoarthropathy at last.Along with improving constantly of standard of living, hyperuricemia and gout sickness rate obviously increase.EPDML investigation shows, to the nineties in last century, increased by 3 times in the period of Britain's patient with gout 20 abroad, U.S.'s patient with gout increases by 80% in the period of 1990 to 1999, Canada more than 30 years old the male sex and more than 50 years old women's gout sickness rate reached that the male sex reaches 9% more than 2%, 80 years old.China's gout sickness rate is 0.3%, and existing more than 400 ten thousand patient with gout have acute attack more than 200 every year, and nearly 1,000,000 people's gouts disable.Actively control hyperuricemia and gout Hazard Factor are to reduce the key of gout outbreak, also are one of important steps that reduces cardiovascular disorder danger.
Gout treatment mainly uses gout inflammation interference medicament and uric acid resisting chemistry medicine at present, and representative drugs has colchicine, NSAID (non-steroidal anti-inflammatory drug), adrenocortical hormone, uric acid decorporation medicine, suppresses the uricogenesis medicine.Colchicine has the specificity of alleviating the gout inflammation, low dose oral medication repeatedly normally, but this medicine has serious gastrointestinal reaction.Other antiphlogiston also all plays the effect that improves symptom, but side effect is all very big.Probenecid, sulfinpyrazone, narcaricin are the medicine for improving uric acid excretion of using always, Zyloric suppresses the formation of uric acid, these medicines all need to take the long period and keep result of treatment, their side effect incidence is identical, and it is the most serious with Zyloric, the normal irritated syndromes of other purine that takes place reaches 10%, and these mortality reach 25%.Recently Kai Fa novel xanthine oxidase inhibitor Febuxostat effect is better than Zyloric, but needs long-term medication.The present methods of treatment of some patient with gout is inoperative, and acute arthritis often takes place these intractable patient with gout, and the tophi that urate causes appears in chronic joint deformity, the forfeiture of patient body mobility.These serious patient with gout especially need effective treatment means with mitigate the disease, improve the quality of living.
Developed reorganization urico-oxidase product (Rasburicase) abroad, urico-oxidase can be the external wallantoin of the higher easier eliminating of solubleness specifically with uricolysis.The reorganization urico-oxidase clinical indication of external listing is: be used for preventing and treating the hyperuricemia that leukemia of children, lymphatic cancer and the contingent tumor lysis syndrome of malignant solid tumor treatment cause.The main side effect of this product is that immune response, generation antibody and anaphylaxis take place, and secondly is that the transformation period is short.For patient with gout, can't long-term prescription.For urico-oxidase is used for patient with gout, be necessary to adopt the protein slow release method to prolong its transformation period, reduce antigenicity.
(Polyethlene glycol is a kind of water-soluble high-molecular substance of nontoxic, non-immunogenicity PEG) to polyoxyethylene glycol, can pass through covalent bonds mode modifying protein.Proteinic Pegylation modify be overcome protein immunogenic-kind of effective ways, polyethyleneglycol modified pharmaceutical grade protein not only can reduce immunogenicity and toxicity but also can obviously improve the stability of pharmaceutical grade protein, reduce the drainage of protein molecule, the transformation period in the extension body by kidney.The polyethyleneglycol modified urico-oxidase Pegloticase of U.S. Savient company exploitation finishes phase iii clinical trial in August, 2008 and submits the new drug application for registration to U.S. FDA, test-results shows that this product has the obvious treatment effect to gout, but antibody can appear in 88% patient, cause the reduction of curative effect, 5% patient has anaphylaxis.And activated polyglycol costs an arm and a leg, and makes that the price of this series products is very high.
(Human Serum Albumin is the method for another prolongation protein transformation period that receives much attention for the fusion protein technology of carrier-albumin integration technology (Albumin fusion technology) HSA) with human serum albumin.Because HSA non-enzymatic activity and immunogenicity, human compatibility is good, molecular weight big (being about 66kDa), transformation period is about 19d, microbial fermentation expression amount height [Chuang VT, Kragh-Hansen U, Otagifi M.Phammceutical strategies utilizingrecombinant human serum albumin[J] Pharm Res, advantage such as 2002,19 (5): 569-577], but polypeptide/protein medicaments molecular weight prolong drug transformation period increased by the construction of fusion protein technology.
Summary of the invention
The purpose of this invention is to provide a kind of urico-oxidase new formulation, particularly human serum albumin of long half time and the fusion rotein of urico-oxidase.
The fusion rotein of human serum albumin of the present invention and urico-oxidase is made up of human serum albumin aminoacid sequence, urico-oxidase aminoacid sequence and connection peptides, and the fusion rotein sequence is connected by connection peptides by human serum albumin aminoacid sequence, urico-oxidase aminoacid sequence.
The human serum albumin aminoacid sequence of the fusion rotein of human serum albumin of the present invention and urico-oxidase and urico-oxidase aminoacid sequence can be its mutant.
Human serum albumin of the present invention and urico-oxidase fusion rotein, the aminoacid sequence of human serum albumin are at the N of fusion rotein end, and the urico-oxidase aminoacid sequence is at the C of fusion rotein end; Perhaps, the aminoacid sequence of human serum albumin is at the C of fusion rotein end, and the urico-oxidase aminoacid sequence is at the N of fusion rotein end, and two proteinic aminoacid sequences connect by connection peptides.
Inventor's serum albumin and urico-oxidase fusion rotein connection peptides aminoacid sequence are [GlyGlyGlyGlySer] n, and wherein n is the integer of 1-10.
The connection peptides aminoacid sequence of preferred inventor's serum albumin and urico-oxidase fusion rotein is [GlyGlyGlyGlySer] n, and wherein n is the integer of 1-3.
The urico-oxidase aminoacid sequence of inventor's serum albumin and urico-oxidase fusion rotein is fungi or the microorganism urico-oxidase and the mutant thereof in flavus, Arthrobacter globiformis or candiyeast source, perhaps, the urico-oxidase aminoacid sequence is to derive from Mammals urico-oxidase sequence and mutant thereof; Perhaps, the urico-oxidase aminoacid sequence is urico-oxidase sequence and the mutant thereof that derives from the invertebrates of fruit bat; Perhaps, the urico-oxidase aminoacid sequence is reorganization urico-oxidase and the mutant thereof that derives from plant, or the mosaic of these sequence different fragments.
Preferred human serum albumin of the present invention and urico-oxidase fusion rotein, urico-oxidase are Aspergillus flavus uricoxidase.
The present invention also is provided as the primed DNA sequence that obtains coding any described human serum albumin of claim of claim 1-6 and urico-oxidase antigen-4 fusion protein gene.
The present invention further provides the engineering strain that contains described human serum albumin of claim 7 and urico-oxidase fusion rotein dna sequence dna.
The present invention also provides a kind of method for preparing human serum albumin and urico-oxidase fusion rotein: human cloning serum albumin and urate oxidase gene, splicing obtains human serum albumin and urico-oxidase antigen-4 fusion protein gene, construction recombination plasmid, transformed yeast host bacterium, screening obtains engineering strain, the culturing engineering bacterium is induced human serum albumin and urico-oxidase Expression of Fusion Protein, and separation and purification obtains human serum albumin and urico-oxidase fusion rotein.
The recombinant plasmid transformed pichia spp that the present invention makes up, the expression product that cultivate to obtain can carry out separation and purification through various bionic downstreams technological method and obtain product, comprising ultrafiltration, saltout, in the precipitation, centrifugal, dialysis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reversed phase chromatography, sieve chromatography technology one or more.
Description of drawings
Fig. 1 is that recombinant plasmid pPIC9-HSA-UOX makes up synoptic diagram;
Fig. 2 is the SDS-PAGE electrophoretic analysis of expression product.
1 swimming lane: standard protein molecular weight (97.4,66.2,43,31,20,14.4kd);
2～4 swimming lanes: purifying HAS-UOX.
Specific embodiment
The clone of embodiment 1:HSA cDNA
Human serum albumin coding gene sequence according to the Genebank report designs primer HSA1 and HSA2 respectively, and sequence is seen sequence table 1 and table 2.With polymerase chain reaction (Polymerase Chain Reaction, PCR) method obtains HSA cDNA from people liver tire cDNA library, wherein primer HSA2 has also added the sequence of coding GlyGlyGlyGlySer joint peptide at its 3 ' end except that the 3 ' end sequence that comprises the HSA gene.The PCR condition is: in the 100ul reaction system, add 1ul people liver tire cDNA library, the HSA1 of 20umol/L and each 3ul of HSA2 primer, the dNTP of 2mmol/L, 10ul, 10X reaction buffer 10ul, Taqplusl archaeal dna polymerase 5U.With the 9600PCR instrument of Perk-Elmer company, the PCR condition is 94 ℃ of sex change 1 minute, anneals 1 minute for 55 ℃, and 72 ℃ were extended 3 minutes, and after 35 circulations, 72 ℃ were extended 10 minutes again.Show the band of an expection big or small (1.8KD) by gel batteries analytical reaction thing, PCR product battery purifying reclaims the object tape that test kit reclaims the about 1.8KD of purifying with dna fragmentation.
The acquisition of embodiment 2:UOX gene
With plasmid pET-32a-rasburicase is template, and design is seen sequence table 3 and 4 as primer UOX1 and UOX2.With primer UOX1 and UOX2 amplification, promptly obtain having the urate oxidase gene of 5 peptide linkers and BamH I restriction enzyme site.The PCR condition is: 94 ℃ of pre-sex change 5min, carry out 30 circulations with 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 40s then, and last 72 ℃ of 10min, 4 ℃ of 10min termination reactions reclaim gene segment.
The acquisition of embodiment 3:HSA-UOX fusion gene
The UOX gene fragment that obtains is reclaimed as template, with the HSA fragment as another template.With primer HSA1 and UOX2 primer amplification, promptly obtain to have the HSA-UOX fragment of BamH I and EcoR I restriction enzyme site.The PCR condition is: 94 ℃ of pre-sex change 5min, carry out 30 circulations with 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 4min then, and last 72 ℃ of 10min, 4 ℃ of 10min termination reactions reclaim gene segment.
Embodiment 4: the structure of recombinant plasmid pPIC9-HSA-UOX and expression
With BamH I/EcoR I double digestion PCR product and pPIC9 carrier, electrophoresis purifying, reclaim gene fragment and carrier that enzyme is cut, connect with the T4DNA ligase enzyme then, to connect product transformed into escherichia coli DH5 α, identify behind 37 ℃ of cultivation 16h, and select the positive colony order-checking.The plasmid construction flow process is seen accompanying drawing one.
After Bgl II linearizing, electric shock is transformed in the pichia spp GS115 host bacterium with the expression vector pPIC9-HSA-UOX that builds, with selective medium MD screening transformant.Positive transformant is inoculated in the 5mL YPD liquid nutrient medium, and 30 ℃ of overnight incubation are transferred in the BMGY liquid nutrient medium again, add 0.5% methyl alcohol behind the 24h and induce, and induce centrifugal collection supernatant behind about 60h, carry out the SDS-PAGE electrophoretic analysis.
Embodiment 5: the separation and purification of expression product
The culture supernatant ultrafiltration and concentration is centrifugal, and the sample supernatant liquor is that the ultrafiltration pipe of 30kd concentrates through holding back relative molecular mass, helps reducing sample volume to carry out molecular sieve purification.0.1mol/L, the phosphate buffered saline buffer balance Sephadex G-75 molecular sieve of pH8.0, collect protein peak behind the last sample 60min, the target protein peak appears about 90min.
Embodiment 6: human serum albumin and urico-oxidase fusion rotein
Human serum albumin after the separation and purification and urico-oxidase fusion rotein being carried out the SDS-PAGE electrophoretic analysis, the results are shown in accompanying drawing two, is 101kd through the molecular weight of scanning analysis human serum albumin and urico-oxidase fusion rotein.The analysis of the immunity marking, fusion rotein and human serum albumin antibody, urico-oxidase antibody are positive.
The activity of urico-oxidase contains uric acid 0.18 μ mol behind the analysis purifying in the 3ml buffer solution system, and 8.9,30 ℃ of insulations of pH detect wavelength 292nm, and it is a unit of enzyme activity that per minute is oxidized to the required enzyme amount of allantoic acid with 1 μ mol uric acid.The enzyme 1 μ L that adds 1mg/mL, reaction finishes the back and surveys absorption value, calculates zymolytic uric acid according to typical curve, and the calculating specific enzyme activity is 4.1U/mg.
Sequence table
＜110〉Hangzhou Biodoor Biotechnology Co., Ltd.
＜120〉fusion rotein of human serum albumin and urico-oxidase
<160>4
<210>1
<211>42
<212>DNA
＜213〉artificial sequence
<400>1
HSA1：5’GC?TTC?GAAACC?ATG?ATG?AAG?TGG?GTAACC?TTTATT?TCC?CTT?3’
<210>1
<211>41
<212>DNA
＜213〉artificial sequence
<400>2
HSA2：5’TA?GGATCC?ACC?ACC?ACC?TAA?GCC?TAA?GGC?AGC?TTG?ACT?TGC?3’
<210>1
<211>39
<212>DNA
＜213〉artificial sequence
<400>3
UOX1：5’GGT?GGT?GGT?GGATCC?ATG?TCC?GCG?GTT?AAA?GCA?GCC?CGC?3’
<210>1
<211>41
<212>DNA
＜213〉artificial sequence
<400>4
UOX2：5’TCT?GGATCC?TTA?CAATTTAGA?CTT?CAG?A?3’
Claims (10)
1. human serum albumin and urico-oxidase fusion rotein, it is characterized in that: the fusion rotein of human serum albumin and urico-oxidase is made up of human serum albumin aminoacid sequence, urico-oxidase aminoacid sequence and connection peptides, and the fusion rotein sequence is connected by connection peptides by human serum albumin aminoacid sequence, urico-oxidase aminoacid sequence.
2. human serum albumin according to claim 1 and urico-oxidase fusion rotein is characterized in that: the aminoacid sequence of human serum albumin is at the N of fusion rotein end, and the urico-oxidase aminoacid sequence is at the C of fusion rotein end; Perhaps, the aminoacid sequence of human serum albumin is at the C of fusion rotein end, and the urico-oxidase aminoacid sequence is at the N of fusion rotein end, and two proteinic aminoacid sequences connect by connection peptides.
3. human serum albumin according to claim 2 and urico-oxidase fusion rotein is characterized in that: the connection peptides aminoacid sequence is [GlyGlyGlyGlySer]
n, wherein n is the integer of 1-10.
4. human serum albumin according to claim 3 and urico-oxidase fusion rotein is characterized in that: the connection peptides aminoacid sequence is [GlyGlyGlyGlySer]
n, wherein n is the integer of 1-3.
5. according to any described human serum albumin of claim of claim 1-4 and urico-oxidase fusion rotein, it is characterized in that: the urico-oxidase aminoacid sequence is fungi or the microorganism urico-oxidase and the mutant thereof in flavus, Arthrobacter globiformis or candiyeast source, perhaps, the urico-oxidase aminoacid sequence is to derive from Mammals urico-oxidase sequence and mutant thereof; Perhaps, the urico-oxidase aminoacid sequence is urico-oxidase sequence and the mutant thereof that derives from the invertebrates of fruit bat; Perhaps, the urico-oxidase aminoacid sequence is reorganization urico-oxidase and the mutant thereof that derives from plant, or the mosaic of these sequence different fragments.
6. human serum albumin according to claim 5 and urico-oxidase fusion rotein is characterized in that: urico-oxidase is an Aspergillus flavus uricoxidase.
7. according to any described human serum albumin of claim of claim 1-6 and urico-oxidase fusion rotein, it is characterized in that: the molecular weight of fusion rotein is 90kd-105kd.
8. according to any described human serum albumin of claim of claim 1-6 and urico-oxidase fusion rotein, it is characterized in that: the specific enzyme activity of fusion rotein is 3.0-8.0EAU/mg.
9. method for preparing human serum albumin as claimed in claim 1 and urico-oxidase fusion rotein, it is characterized in that: human cloning serum albumin and urate oxidase gene, splicing obtains human serum albumin and urico-oxidase antigen-4 fusion protein gene, construction recombination plasmid, transformed yeast host bacterium, screening obtains engineering strain, the culturing engineering bacterium, induce human serum albumin and urico-oxidase Expression of Fusion Protein, separation and purification obtains human serum albumin and urico-oxidase fusion rotein.
10. as the preparation method of a kind of human serum albumin as described in the claim 9 and urico-oxidase fusion rotein, it is characterized in that: the method for separation and purification comprises ultrafiltration, saltouts, in the precipitation, centrifugal, dialysis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reversed phase chromatography, sieve chromatography technology one or more.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178464A (en) * | 2013-05-22 | 2014-12-03 | 李殿明 | Preparation method of recombinant human albumin-uricase fusion protein |
| WO2017152399A1 (en) * | 2016-03-09 | 2017-09-14 | 兰州大学 | Fusion protein of vasoactive intestinal peptide and preparation method and use thereof |
| CN109022282A (en) * | 2018-08-08 | 2018-12-18 | 大连大学 | A kind of screening technique producing marine low temperature urate oxidase bacterial strain |
| WO2023024066A1 (en) * | 2021-08-27 | 2023-03-02 | 中国科学院深圳先进技术研究院 | Proximity labeling complex, proximity labeling method, and intermolecular interaction analysis method |
-
2009
- 2009-12-31 CN CN2009102154465A patent/CN101875923A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178464A (en) * | 2013-05-22 | 2014-12-03 | 李殿明 | Preparation method of recombinant human albumin-uricase fusion protein |
| WO2017152399A1 (en) * | 2016-03-09 | 2017-09-14 | 兰州大学 | Fusion protein of vasoactive intestinal peptide and preparation method and use thereof |
| CN109022282A (en) * | 2018-08-08 | 2018-12-18 | 大连大学 | A kind of screening technique producing marine low temperature urate oxidase bacterial strain |
| WO2023024066A1 (en) * | 2021-08-27 | 2023-03-02 | 中国科学院深圳先进技术研究院 | Proximity labeling complex, proximity labeling method, and intermolecular interaction analysis method |
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Application publication date: 20101103 |