CN101402688A - Fusion protein, encoding gene and uses thereof - Google Patents

Abstract

The invention discloses a fusion protein, coded genes thereof and an application thereof. The fusion protein comprises urate oxidase and albumin. The urate oxidase is non-human urate oxidase and is preferably selected as urate oxidase from microorganisms; the albumin is the protein having more than 90 percent of homology with human serum albumin and is preferably selected as human serum albumin. The fusion protein has the following advantages as: (1) the immunogenicity is extremely low in respect of a human body; (2) the half-life of the urate oxidase in the human body can be prolonged; (3) medicaments can be supplied repeatedly to improve the effect of treating diseases; (4) the original activity of the urate oxidase for decomposing uric acid can be maintained; and (5) potential side effect or toxicity accompanying with the use of urate oxidase can be reduced, which mainly reduces the generation of antibodies and anaphylactic reaction.

Description

A kind of fusion rotein and encoding gene thereof and application

Technical field

The present invention relates to a kind of fusion rotein and encoding gene thereof and application.

Background technology

Urico-oxidase (Uricase or Urate Oxidase, UOX EC 1.7.3.3) is a key enzyme of purine metabolism approach, can form wallantoin by the catalysis uric acid.Uric acid is the final product of different biological purine metabolisms with wallantoin, and both solvability difference are very big, and uric acid solubleness in vivo is very low, be about 11mg/dl water, and wallantoin is its 13.5 times, is about 147mg/dl water.Uric acid accumulation in vivo can cause gout and urate nephropathy (Duk-HeeKang, Takahiko Nakagawa, Lili Feng et al.A Role for Uric Acid in the Progression ofRenal Disease[J] .J.Am.Soc.Nephrol., 2002,13:2888-2897).There are some researches show, uric acid accumulation in vivo is relevant with the dysfunction of histoorgans such as the heart, kidney, with the gentle inductive high lithemia of uricase inhibitor mouse model, symptoms such as Renal artery disease, injury of the kidney and hypertension occurred, the alleviation and the disappearance of symptom have proved that uric acid is the reason place of these diseases behind the removal inhibitor.Studies show that artery disease may promote that the propagation of smooth muscle cell is relevant with uric acid.

Urate oxidase gene (UOX) is an ancient gene, be present in (Xiangwei Wu in prokaryotic organism and the eukaryote, Cheng Chi Lee, Donna M.Muzny, Et Al.Urate oxidase:Primary structureand evolutionary implications[J] .Proc.Natl.Acad.Sci., 1989,86:9412-9416).It is not high to derive from different biological UOX dna homologs, for example, sequence homology between the UOX of subtilis (Bacillus subtilis) and soybean is 25%, sequence homology between the UOX of flavus (Aspergillus flavus) and soybean is 29%, sequence homology between the UOX of B.subtilis and A.flavus is 36%, but the amino acid residue sequence that constitutes avtive spot is all guarded in all species.

The active forfeiture of UOX finds in many sibships kind far away, in birds, terrestrial Reptilia, cyclostomes, insect and ring insects except that Diptera.In human and birds, lack activated UOX, thereby excrete with the final product of uric acid as purine metabolism, high lithemia disease and associated urarthritis (Masako Oda have been caused, Yoko Satta, Osamu Takenaka, et al.Loss of Urate Oxidase Activityin Hoinoids and its Evolutionary Implications[J] .Mol.Biol.Evol., 2002,19 (5): 640-653), other Mammals of the concentration ratio of uric acid is high 50 times in the human serum.The rat that knocks out the UOX gene has caused serious high lithemia disease and urate nephropathy, and 50% rat is dead before 4 ages in week.There are some researches show that all there is a single nonsense mutation in the UOX gene from the common ancestors to the modern, make to have caused the active forfeiture of UOX by proteic translation process premature termination.In primate, Prosimii and old world monkey have shown the UOX activity with respect to rat and rabbit medium level; By comparison, Genus Homo and some New World monkeies then do not show any can detected UOX activity.People's UOX gene is 96% with the homology of the UOX gene of old world monkey, illustrates to have an accident in evolution, causes people's UOX gene silencing; New World monkey and people's sibship is than its near with old world monkey, so the sudden change of the unlikely UOX with the people of the inactivation of the UOX of New World monkey is the same.

The intravital high lithemia of people may be caused by several reasons, along with the increase at age, and bodily fuctions's decline, the uraturia ability that causes urinating weakens, and uric acid deposits in uriniferous tubules and Collecting duct, has caused gout.In addition, for tumour patient, the mass mortality of the tumour cell that causes in the therapeutic process makes intravital uric acid concentration sharply raise, and promptly so-called tumor lysis syndrome (Tumor Lysis Sydrome, TLS).The most normal children acute leukemia and the lymphoma of coming across of TLS, adult's tumour patient also can occur once in a while, is accompanied by the quick hyperplasia of marrow and lymph in the therapeutic process, can cause the damage of renal failure and other organ usually.

The drug main that is used for the treatment of high lithemia disease clinically will comprise Zyloric, halogenation urine, hydration or dialysis etc.By suppressing the activity of XOD, Zyloric has reduced the generation of uric acid, but increased the burden of uric acid precursor (xanthine and xanthoglobulin) to kidney, and the solubleness of xanthine in urine is lower than uric acid, therefore Zyloric treatment high lithemia disease usually causes xanthine ephrosis and calculus, and original uric acid is not subjected to the influence of Zyloric in the body, can only lean on patient self to discharge.Halogenation and hydration can only partly increase the excretion of uric acid, are not the ideal methods, may need to dialyse frequently.

UOX is oxidized to wallantoin by the direct catalysis of enzyme with uric acid, and the meltage of the latter in urine is multiplied with respect to the former, use the effective ways (Annemans that high risk patient that UOX treats TLS clinically is proved to be to prevent TLS, K.Moeremans, M.Lamotte, et al.Pan-European multicentre economicevaluation of recombinant urate oxidase (rasburicase) in prevention and treatment ofhyperuricaemia and tumour lysis syndrome in haematological cancer patients[J] .SupportCare Cancer, 2003,11:249-257; Stanton C.Goldman, John S.Holcenberg, Jerry Z.Finklestein, et al.A randomized comparison between rasburicase and allopurinol inchildren with lymphoma or leukemia at high risk for tumor lysis[J] .Blood, 2001,97 (10): 2998-3003).In the TLS high risk patient who randomly draws, treat with Zyloric, reorganization UOX, halogenation and hydration respectively, the result shows that reorganization UOX can make the uric acid level in the blood plasma significantly descend 2.6 times, and remained on below the 8mg/L within four hours; And can reach the level that reorganization UOX treats with patient's uric acid level after 96 hours of Zyloric treatment, but the burden that uric acid precursor-xanthine that produces and xanthoglobulin have increased the weight of kidney needs dialysis to remove sometimes.

Experiment shows that reorganization UOX can prevent effectively that the TLS symptom from appearring in high leukocytic children acute leukaemic in therapeutic process.Lot of data shows that aspect preventing and treat the TLS symptom that occurs in the tumour patient chemotherapy process, reorganization UOX is more effective and safety than Zyloric.

The injection urico-oxidase can of short durationly alleviate hyperuricemia and hyperuricuria at least.Because urico-oxidase is the human body extrinsic protein, duplicate injection reorganization UOX can produce the antibody of anti-UOX, has suppressed the activity of UOX.Can be by the surface of using soluble polymer shield enzyme molecule, reduce antigenicity and immunogenicity, and the transformation period (Davis of prolongation enzyme molecule, Y.K.Park, A.Abochowski, F.F.Davis, Hypouricaemic effect ofpolyethyleneglycol modified urate oxidase, Lancet 2 (1981) 281-283. Chinese patent CN1896231A).The UOX of PEG modification has at present entered the III clinical trial phase abroad.

Human serum albumin (HSA) is a kind of soluble and monomeric protein, constitutes half of Tot Prot in the blood.Lipid acid, steroid and hormone molecule etc. are carried and transmitted to albumin as a kind of underlying carrier, and its stable inert nature is to keep an important factor of blood pressure.Serum albumin is that a kind of spherical, nonglycosylated, molecular weight is the serum protein of 65kD.Human serum albumin gene is positioned on No. 4 karyomit(e), 16961 base pairs are arranged, be divided into 15 transcribed spacers, protein of the mRNA codified that behind the RNA processing and splicing, forms with 585 amino-acid residues, through the conversion processing of golgi body, removal guides polypeptide and is secreted into the extracellular this albumen again.Serum albumin has 35 cysteine residues, is a monomer that 17 disulfide linkage are arranged in blood.Utilize the method for microbial expression recombinant albumin in patent documentation, to disclose (EP330451; EP361991; CN1119352C).

Albumin is the main component in the blood, is 40 grams per liter blood at the intravital content of people, and life-span transformation period is 14-20 days.Utilize genetic engineering technique, with biologically active factors construction of fusion protein such as human serum albumin and human growth hormone, Interferon, rabbit or interleukin-2s and in yeast, express, at Chinese patent (CN1515591A; CN1269840C) open in, can improve the transformation period of biologically active factors in blood by merging with albumin.

Summary of the invention

The purpose of this invention is to provide a kind of fusion rotein and encoding gene thereof and application.

Fusion rotein provided by the present invention comprises urico-oxidase and albumin, described urico-oxidase can directly be connected described albuminous N-terminal or C-terminal and form fusion rotein, also can connect described albumin and described urico-oxidase to form fusion rotein by a connection peptides.

The length of described connection peptides is 2-100 amino acid, is preferably 5-50 amino acid, more preferably 5-20 amino acid.The length of connection peptides can be as short as described albumin macromolecule the steric hindrance that described urico-oxidase forms is kept minimum, as (G ₄S) _1-4Connection peptides helps combining of urico-oxidase and its substrate uric acid, but the adding of connection peptides may make fusion rotein produce extra immunogenicity as drug use the time, does not most preferably have connection peptides.

Above-mentioned fusion rotein both can be a secretion type expression albumen, also can be cell inner expression albumen, specifically can be secretion type expression albumen.

Described urico-oxidase is the urico-oxidase in inhuman source, as derive from microorganism (as flavus (Aspergillus flavus), candiyeast (Candida utilis), Bacillus subtilus (Bacillus sp.) etc.), plant (as soybean (Glycine max) root nodule etc.), no spinal animals (as fruit bat (Drosophila melanogaster)) or the urico-oxidase of spinal animals (as pig, ox, sheep, baboon) etc. is arranged, or its mutant.

Described albumin is the albumen that has 90% above homology with human serum albumin, specifically can be human serum albumin.

Described fusion rotein specifically can be following 1), 2), 3) or 4) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein;

3) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

4) with the aminoacid sequence of sequence in the sequence table 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 3) deutero-protein.

Above-mentioned 2) or 4) but the fusion rotein synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of fusion rotein above-mentioned 2) can by with sequence in the sequence table 1 from 5 ' dna sequence dna shown in the end 13-2703 bit base in the codon of one or several amino-acid residue of disappearance, and/or the missense mutation of carrying out one or several base pair obtains.The encoding gene of fusion rotein above-mentioned 4) can by with sequence in the sequence table 3 from 5 ' dna sequence dna shown in the end 10-2772 bit base in the codon of one or several amino-acid residue of disappearance, and/or the missense mutation of carrying out one or several base pair obtains.

The encoding gene of above-mentioned fusion rotein also belongs to protection scope of the present invention.

The encoding gene of above-mentioned fusion rotein specifically can be following 1)-6) in arbitrary described gene:

1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 13-2703 position deoxyribonucleotide;

2) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 1 and the dna molecular of encoding said fusion protein;

3) with 1) gene have the homology more than 90% and the dna molecular of encoding said fusion protein;

4) its encoding sequence be in the sequence table sequence 3 from 5 ' terminal 10-2772 position deoxyribonucleotide;

5) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 3 and the dna molecular of encoding said fusion protein;

6) with 4) gene have the homology more than 90% and the dna molecular of encoding said fusion protein.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The above-mentioned fusion rotein encoding gene total length that increases or arbitrary segmental primer are right, and the recombinant vectors, transgenic cell line or the reorganization bacterium that contain above-mentioned fusion rotein encoding gene also belong to protection scope of the present invention.

Available existing expression vector establishment contains the recombinant expression vector of described fusion rotein encoding gene, specifically can be the pPIC plasmid.

Described host is including, but not limited to Mammals (as people, monkey, mouse, rabbit etc.), fish, insect, plant, yeast, fungi and bacterium etc., be preferably yeast belong, belong to (Saccharomyces), Pichia (Pichia), debaryomyces hansenii (Hansenula), genus kluyveromyces (Kluyveromyces), Candida (Candida), spore garden yeast belong (Tarulaspora) and Schizosaccharomyces etc. including, but not limited to yeast saccharomyces cerevisiae; More preferably be Pichia pasteur bacterial classification (Pichia pastoris).

The present invention is prepared into fusion rotein with the urico-oxidase and the human serum albumin fusion in inhuman source, experimental result shows, the urico-oxidase in this fusion rotein and inhuman source has in the similar body and the external biological activity, and immunogenicity greatly reduces, can solve the allos urico-oxidase and be used for the immunogenicity problem that the human body therapy process is produced, can be used for preparing the medicine of treatment hyperuricemia and gout.Fusion rotein of the present invention has the following advantages: 1) extremely low for the human body immunogenicity; 2) can prolong urico-oxidase in the intravital transformation period of people; 3) can repeat administration, improve the treatment of diseases effect; 4) keep the original uricolytic activity of urico-oxidase; 5) reducing side effect or the toxicity of following when the potential urico-oxidase uses, mainly is to reduce production of antibodies and reduce anaphylaxis.

Description of drawings

Fig. 1 is the building process that contains the recombinant vectors of fusion rotein encoding gene

Fig. 2 is expression and the purification result of fusion rotein HSA-UO in yeast

Fig. 3 is that fusion rotein HSA-UO and rUO are in the intravital immunogenicity determining result of rat

Embodiment

The preparation of embodiment 1, albumin and urico-oxidase fusion rotein

1, the acquisition of human serum albumin encoding gene

With people's tire liver cDNA library (available from Clontech company, production code member 637230) is template, and the design primer amplification does not contain the human serum albumin gene (HSA) of signal coding sequence, and concrete primer is as follows:

HSA5：5′-TCG CTCGAGAAAAGAGATGCACACAAGAGTGAGG-3′(Xho?I)，

HSA3：5′-TT GGATCCACCTCCACCGGAACCTCCACCTCCTAAGCCTAAGGCAGCTTGACTTG-3′(BamH?I)。

Pcr amplification system: people's tire liver cDNA 1.0 μ L, each 0.5 μ L of HSA5 primer and HSA3 primer, dNTP 2 μ L, KOD DNA Polymerase 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄2 μ L, ddH ₂O 35 μ L, cumulative volume 50 μ L.The PCR reaction conditions is: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 2min, totally 30 circulations.

Pcr amplification product is carried out agarose gel electrophoresis detect, the result shows, obtains the band of about 1700bp; Purifying also reclaims this band and checks order, and sequencing result shows that the nucleotide sequence of the band that is obtained is the encoding gene of human serum albumin, with its called after HSA.

With people's tire liver cDNA library (available from Clontech company, production code member 637230) is template, and the design primer amplification contains the human serum albumin gene (SHSA) of signal peptide, and concrete primer is as follows:

SHSA5：5′-GAC GGATCCACCATGAAGTGGGTAACCTTTATTTCC-3′(BamH?I)，

SHSA3：5′-TT GAATTCACCTCCACCGGAACCTCCACCTCCTAAGCCTAAGGCAGCTTGACTTG-3′(EcoR?I)。

Pcr amplification system: people's tire liver cDNA 1.0 μ L, each 0.5 μ L of SHSA5 primer and SHSA3 primer, dNTP 2 μ L, KOD DNA Polymerase 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄2 μ L, ddH ₂O 35 μ L, cumulative volume 50 μ L.The PCR reaction conditions is: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 2min, totally 30 circulations.

Pcr amplification product is carried out agarose gel electrophoresis detect, the result shows, obtains the band of about 1800bp; Purifying also reclaims this band and checks order, and sequencing result shows that the nucleotide sequence of the band that is obtained is the encoding gene of the human serum albumin that contains signal peptide, with its called after SHSA.

2, the acquisition of urico-oxidase encoding gene

With the plasmid pET-UO that contains the Aspergillus flavus uricoxidase gene (Wu Weili, Fang Hongqing etc. the expression and purification of urico-oxidase and activity form are identified.Institute of Military Medical Science Institute periodical, 2005,29 (2): 124-126.) be template, design primer amplification UOX gene, concrete primer is as follows:

AFU5：5′-CGT AGATCTATGTCAGCAGTAAAAGCAGCAC-3′(Bgl?II)

AFU3：5′-TG GAATTCTTACAATTTAGACTTCTGAGAGGAAC-3’(EcoRI)。

Pcr amplification system: pET-UO plasmid 1.0 μ L, each 0.5 μ L of AFU5 primer and AFU3 primer, dNTP 2 μ L, KOD DNA Polymerase 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄2 μ L, ddH ₂O 35 μ L, cumulative volume 50 μ L.The PCR reaction conditions is: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations.

Pcr amplification product is carried out agarose gel electrophoresis detect, the result shows, obtains the band of about 900bp; Purifying also reclaims this band and checks order, and sequencing result shows that the nucleotide sequence of the band that is obtained is the encoding gene of Aspergillus flavus uricoxidase, with its called after AFUOX.

In order to improve the expression level in yeast, according to yeast preference codon design optimization urate oxidase gene UOA, and entrust Shanghai to give birth to worker company and carry out synthetic, synthetic urate oxidase gene UOA is inserted on the pMD18 carrier, with the recombinant vectors called after pMD18-UOA that obtains.

3, the structure of recombinant vectors pPIC9-HSAL2UO (two GGGGS are arranged between HSA and the AFUOX)

The HSA that above-mentioned steps 1 is obtained is inserted into pIB2 carrier (the Irina B Sears that cuts through same enzyme behind XhoI and BamHI double digestion, Jamse O ' Connor, Olivia W Rossanese, Benjamin S Glick.A versatile setof vectors for constitutive and regulated gene expression in Pichia pastoris.Yeast, 14 (8) 783-790,1998) in, correct recombinant vectors called after pIB2-HSA is identified in order-checking.The multiple clone site of pIB2 carrier has Xho I, BamHI and EcoR I restriction enzyme site successively.The AFUOX gene that above-mentioned steps 2 is obtained is identified correct recombinant vectors called after pIB2-HSAL2UO through being inserted into behind Bgl II and the EcoR I double digestion in the pIB2-HSA carrier of the above-mentioned structure of BamH I and EcoR I double digestion with order-checking.PIB2-HSAL2UO is behind XhoI and EcoRI double digestion, obtain not the band signal peptide but have the human serum albumin of connection peptides and the fusion gene of urico-oxidase, with its called after HSAL2UO, the deoxyribonucleotide sequence of HSAL2UO is shown in sequence in the sequence table 1, and the aminoacid sequence of the fusion rotein of its coding is shown in sequence in the sequence table 2.The fusion gene HSAL2UO of above-mentioned acquisition is inserted in the pPIC9 carrier (available from Invitrogen company, catalog number (Cat.No.) K1710-01) through XhoI and EcoRI double digestion, with the recombinant vectors called after pPIC9-HSAL2UO that obtains.The concrete building process of recombinant vectors pPIC9-HSAL2UO as shown in Figure 1.

4, the structure of recombinant vectors pPIC9-SHSAL2UOA

The SHSA that above-mentioned steps 1 is obtained is inserted into pIB2 carrier (the Irina B Sears that cuts through same enzyme respectively behind BamHI and EcoRI double digestion, Jamse O ' Connor, Olivia W Rossanese, Benjamin S Glick.Aversatile set of vectors for constitutive and regulated gene expression in Pichia pastoris.Yeast, 14 (8) 783-790,1998) in, will identify correct recombinant vectors called after pIB2-SHSA through order-checking.

Behind the recombinant vectors pMD18-UOA usefulness Bgl II and EcoRI double digestion with above-mentioned steps 2 acquisitions, reclaim the UOA gene fragment of about 900bp, be inserted in the pIB2-HSA carrier that the above-mentioned steps 3 through BamHI and EcoRI double digestion obtains, will identify correct recombinant vectors called after pIB2-HSAL2UOA through order-checking.

PIB2-HSAL2UOA reclaims the purpose fragment of about 1700bp behind NdeI (being arranged in the HSA gene) and EcoRI double digestion, be inserted into pIB2-SHSA carrier, will identify correct recombinant vectors called after pIB2-SHSAL2UOA through order-checking through NdeI (being arranged in the HSA gene) and EcoRI double digestion.

PIB2-SHSAL2UOA is through BamHI and EcoRI double digestion, reclaim the small segment of about 2800bp, obtain the human serum albumin that has signal peptide and have connection peptides and the fusion gene of urico-oxidase, with its called after SHSAL2UOA, the deoxyribonucleotide sequence of SHSAL2UOA is shown in sequence in the sequence table 3, and the aminoacid sequence of the fusion rotein of its coding is shown in sequence in the sequence table 4.This gene is inserted into through in the pPIC9 carrier (available from Invitrogen company, catalog number (Cat.No.) K1710-01) of BamHI and EcoRI double digestion, will identifies correct recombinant vectors called after pPIC9-SHSAL2UOA through order-checking.The concrete building process of recombinant vectors pPIC9-SHSAL2UOA as shown in Figure 1.

5, recombinant plasmid pPIC9-HSAL2UO and pPIC9-SHSAL2UOA are changed over to respectively express corresponding fusion proteins in the pichia spp

Recombinant plasmid pPIC9-HSAL2UO that above-mentioned steps 3 and step 4 are obtained and pPIC9-SHSAL2UOA carry out after the linearizing respectively with Sal I that electricity is converted among the Pichia yeast GS115, with the reorganization bacterium separate application of acquisition at MD substratum (the basic nitrogenous source of 1.34% quality percentage composition yeast, 4 * 10 ^-5% quality percentage composition vitamin H, 1% quality percentage composition glucose and 2% quality percentage composition agar powder) on the flat board, cultivate more than 2 days for 30 ℃.The advanced performing PCR of the mono-clonal that grows is identified, and then all PCR positive colonies are carried out expression screening according to the requirement in the Invitrogen company operational manual, obtain the bacterial strain of high expression level.Concrete screening method is as follows: the uric acid solution of getting 3ml40 μ mol/L, add the above-mentioned culture supernatant that contains the reorganization bacterium of recombinant plasmid pPIC9-HSAL2UO or pPIC9-SHSAL2UOA of 100 μ l respectively, after room temperature or 37 ℃ are placed 5min, with 0.3ml quality percentage composition is 20% KOH termination reaction, measures the variation of 293nm place light absorption value.Light absorption value changes more greatly, shows that the urico-oxidase activity in the fusion rotein that the reorganization bacterium expresses is high more, and corresponding Expression of Fusion Protein level is also high more.

6, the expression of fusion rotein HSAUOA and purifying

The reorganization bacterium of the high expression level HSAUOA fusion rotein that above-mentioned steps 5 screening is obtained is inoculated in the triangular flask that the 200mLBMG substratum is housed and breeds, with the fermented liquid that obtains as seed liquor; Above-mentioned seed liquor is forwarded to again 3L basic medium (40g/L glycerine, 18g/L K are housed ₂SO ₄, 15g/L MgSO ₄.7H ₂O, 4.13g/L KOH, 0.9g/L CaSO ₄.2H ₂O, 13.3mL/L H ₃PO ₄) the 5L fermentor tank in, ferment under the condition of 30 ℃ of pH 5.0.Fermenting, I finishes mutually after 20 hours, and adding the quality percentage composition by the flow velocity stream of 0.8ml/min is 5% glycerine 100ml.Secondary fermentation II finished mutually in 2 hours, transferred culture temperature to 20 ℃, transferred pH to 6.5, added the 3g soy peptone again in substratum, added methyl alcohol by the flow velocity stream of 0.2ml/min and carried out inducing culture, finished fermentation, centrifugal collection supernatant after 90 hours.

Adding ammonium sulfate volumetric molar concentration of ammonium sulfate to the supernatant liquor in above-mentioned supernatant liquor is 1.2M, mixed solution pre-equilibration Phenyl post with 25mM phosphoric acid buffer (PH7.0)+1.2M ammonium sulfate composition, last sample, then with 25mM phosphoric acid buffer (PH7.0) gradient elution that contains 1.2M to 0M ammonium sulfate, collect active peak, be concentrated into 10-15ml.The mixed solution pre-equilibration Superdex200 post of using 25mM phosphoric acid buffer (PH7.0)+0.15M sodium-chlor to form again with sample on the above-mentioned concentrated solution, is collected active peak.The albumen of collecting is carried out SDS-PAGE, and the result as shown in Figure 2.Among Fig. 2, Marker is the molecular weight of albumen standard, and 1 is the SDS-PAGE result of above-mentioned fermented liquid supernatant, the SDS-PAGE result at 2, the 3 active peaks of collecting for hydrophobic chromatography, the SDS-PAGE result at the 4 active peaks of collecting for gel-filtration.

Embodiment 2, fusion rotein are in the intravital immunogenic mensuration of rat

Male SD rat is divided into two groups, be respectively HSAUOA fusion rotein group and rUO protein groups (rUO proteic preparation process is referring to Wu Weili, Fang Hongqing etc. the expression and purification of urico-oxidase and activity form are identified.Institute of Military Medical Science Institute periodical, 2005,29 (2): 124-126.), every group of five rats, the HSAUOA fusion rotein of difference intravenous injection the foregoing description 1 preparation and the rUO albumen for preparing according to above-mentioned literature method.The dosage of HSAUOA fusion rotein group is the 0.9mg/kg body weight, and the dosage of rUO group is the 0.3mg/kg body weight.Be administered once weekly, around the successive administration.Respectively at before the administration first time and blood sampling in 14,21,28 and 35 days after the administration for the first time, separation of serum, it is to be measured to be put in 4 ℃ of refrigerators, and observes continuously 2 months.

By elisa plate, adopt the ELISA method to measure antibody titers with the rUO bag, three repetitions are established in experiment, and after rat vein was injected HSAUOA fusion rotein and rUO albumen continuously, the time dependent result of antibody titers was shown in Fig. 3 and table 1.As can be seen from Table 1, behind the intravenous injection HSAUOA fusion rotein, the antibody horizontal that produces in the rat body is starkly lower than the antibody horizontal (P＜0.05) that produces in the rat body behind the intravenous injection rUO, and behind the continuous intravenous injection rUO, rat internal antibody level is continuous rising trend, and behind the continuous intravenous injection HSAUOA fusion rotein, the intravital antibody horizontal of rat is not seen obvious rising.

As can be seen from Figure 3,14d, 21d, 28d and 35d behind the HSAUOA fusion rotein of injection the foregoing description 1 preparation, the antibody titers that produces in the rat body is respectively 20,120,100 and 40; 14d, 21d, 28d and 35d behind the injection rUO albumen, the antibody titers that produces in the rat body is respectively 416,1000,9600 and 32000.

Antibody titers behind table 1 rat continuous intravenous injection HSAUOA and the rUO

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agggcggacc?ttgccaagta?tatctgtgaa?aatcaagatt?cgatctccag?taaactgaag 840

gaatgctgtg?aaaaacctct?gttggaaaaa?tcccactgca?ttgccgaagt?ggaaaatgat 900

gagatgcctg?ctgacttgcc?ttcattagct?gctgattttg?ttgaaagtaa?ggatgtttgc 960

aaaaactatg?ctgaggcaaa?ggatgtcttc?ctgggcatgt?ttttgtatga?atatgcaaga 1020

aggcatcctg?attactctgt?cgtgctgctg?ctgagacttg?ccaagacata?tgaaaccact 1080

ctagagaagt?gctgtgccgc?tgcagatcct?catgaatgct?atgccaaagt?gttcgatgaa 1140

tttaaacctc?ttgtggaaga?gcctcagaat?ttaatcaaac?aaaattgtga?gctttttgag 1200

cagcttggag?agtacaaatt?ccagaatgcg?ctattagttc?gttacaccaa?gaaagtaccc 1260

caagtgtcaa?ctccaactct?tgtagaggtc?tcaagaaacc?taggaaaagt?gggcagcaaa 1320

tgttgtaaac?atcctgaagc?aaaaagaatg?ccctgtgcag?aagactatct?atccgtggtc 1380

ctgaaccagt?tatgtgtgtt?gcatgagaaa?acgccagtaa?gtgacagagt?caccaaatgc 1440

tgcacagaat?ccttggtgaa?caggcgacca?tgcttttcag?ctctggaagt?cgatgaaaca 1500

tacgttccca?aagagtttaa?tgctgaaaca?ttcaccttcc?atgcagatat?atgcacactt 1560

tctgagaagg?agagacaaat?caagaaacaa?actgcacttg?ttgagcttgt?gaaacacaag 1620

cccaaggcaa?caaaagagca?actgaaagct?gttatggatg?atttcgcagc?ttttgtagag 1680

aagtgctgca?aggctgacga?taaggagacc?tgctttgccg?aggagggtaa?aaaacttgtt 1740

gctgcaagtc?aagctgcctt?aggcttagga?ggtggaggtt?ccggtggagg?tggatctatg 1800

tcagcagtaa?aagcagcacg?ctacggcaag?gacaatgtcc?gcgtctacaa?ggttcacaag 1860

gacgagaaga?ccggtgtcca?gacggtgtac?gagatgaccg?tctgtgtgct?tctggagggt 1920

gagattgaga?cctcttacac?caaggccgac?aacagcgtca?ttgtcgcaac?cgactccatt 1980

aagaacacca?tttacatcac?cgccaagcag?aaccccgtta?ctcctcccga?gctgttcggc 2040

tccatcctgg?gcacacactt?cattgagaag?tacaaccaca?tccatgccgc?tcacgtcaac 2100

attgtctgcc?accgctggac?ccggatggac?attgacggca?agccacaccc?tcactccttc 2160

atccgcgaca?gcgaggagaa?gcggaatgtg?caggtggacg?tggtcgaggg?caagggcatc 2220

gatatcaagt?cgtctctgtc?cggcctgacc?gtgctgaaga?gcaccaactc?gcagttctgg 2280

ggcttcctgc?gtgacgagta?caccacactt?aaggagacct?gggaccgtat?cctgagcacc 2340

gacgtcgatg?ccacttggca?gtggaagaat?ttcagtggac?tccaggaggt?ccgctcgcac 2400

gtgcctaagt?tcgatgctac?ctgggccact?gctcgcgagg?tcactctgaa?gacttttgct 2460

gaagataaca?gtgccagcgt?gcaggccact?atgtacaaga?tggcagagca?aatcctggcg 2520

cgccagcagc?tgatcgagac?tgtcgagtac?tcgttgccta?acaagcacta?tttcgaaatc 2580

gacctgagct?ggcacaaggg?cctccaaaac?accggcaaga?acgccgaggt?cttcgctcct 2640

cagtcggacc?ccaacggtct?gatcaagtgt?accgtcggcc?gttcctctca?gaagtctaaa 2700

ttgtaa 2706

<210>2

<211>897

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>2

Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly?Glu

1 5 10 15

Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln

20 25 30

Gln?Cys?Pro?Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr?Glu

35 40 45

Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys

50 55 60

Ser?Leu?His?Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu

65 70 75 80

Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro

85 90 95

Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn?Leu

100 105 110

Pro?Arg?Leu?Val?Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe?His

115 120 125

Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg

130 135 140

Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg

145 150 155 160

Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala

165 170 175

Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser

180 185 190

Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu

195 200 205

Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro

210 215 220

Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys

225 230 235 240

Val?His?Thr?Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp

245 250 255

Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser

260 265 270

Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser?His

275 280 285

Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser

290 295 300

Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala

305 310 315 320

Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg

325 330 335

Arg?His?Pro?Asp?Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr

340 345 350

Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His?Glu

355 360 365

Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro

370 375 380

Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu

385 390 395 400

Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro

405 410 415

Gln?Val?Ser?Thr?Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys

420 425 430

Val?Gly?Ser?Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys

435 440 445

Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His

450 455 460

Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser

465 470 475 480

Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr

485 490 495

Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala?Asp

500 505 510

Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala

515 520 525

Leu?Val?Glu?Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu

530 535 540

Lys?Ala?Val?Met?Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys

545 550 555 560

Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val

565 570 575

Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly?Leu?Gly?Gly?Gly?Gly?Ser?Gly?Gly

580 585 590

Gly?Gly?Ser?Met?Ser?Ala?Val?Lys?Ala?Ala?Arg?Tyr?Gly?Lys?Asp?Asn

595 600 605

Val?Arg?Val?Tyr?Lys?Val?His?Lys?Asp?Glu?Lys?Thr?Gly?Val?Gln?Thr

610 615 620

Val?Tyr?Glu?Met?Thr?Val?Cys?Val?Leu?Leu?Glu?Gly?Glu?Ile?Glu?Thr

625 630 635 640

Ser?Tyr?Thr?Lys?Ala?Asp?Asn?Ser?Val?Ile?Val?Ala?Thr?Asp?Ser?Ile

645 650 655

Lys?Asn?Thr?Ile?Tyr?Ile?Thr?Ala?Lys?Gln?Asn?Pro?Val?Thr?Pro?Pro

660 665 670

Glu?Leu?Phe?Gly?Ser?Ile?Leu?Gly?Thr?His?Phe?Ile?Glu?Lys?Tyr?Asn

675 680 685

His?Ile?His?Ala?Ala?His?Val?Asn?Ile?Val?Cys?His?Arg?Trp?Thr?Arg

690 695 700

Met?Asp?Ile?Asp?Gly?Lys?Pro?His?Pro?His?Ser?Phe?Ile?Arg?Asp?Ser

705 710 715 720

Glu?Glu?Lys?Arg?Asn?Val?Gln?Val?Asp?Val?Val?Glu?Gly?Lys?Gly?Ile

725 730 735

Asp?Ile?Lys?Ser?Ser?Leu?Ser?Gly?Leu?Thr?Val?Leu?Lys?Ser?Thr?Asn

740 745 750

Ser?Gln?Phe?Trp?Gly?Phe?Leu?Arg?Asp?Glu?Tyr?Thr?Thr?Leu?Lys?Glu

755 760 765

Thr?Trp?Asp?Arg?Ile?Leu?Ser?Thr?Asp?Val?Asp?Ala?Thr?Trp?Gln?Trp

770 775 780

Lys?Gln?Phe?Ser?Gly?Leu?Gln?Glu?Val?Arg?Ser?His?Val?Pro?Lys?Phe

785 790 795 800

Asp?Ala?Thr?Trp?Ala?Thr?Ala?Arg?Glu?Val?Thr?Leu?Lys?Thr?Phe?Ala

805 810 815

Glu?Asp?Asn?Ser?Ala?Ser?Val?Gln?Ala?Thr?Met?Tyr?Lys?Met?Ala?Glu

820 825 830

Gln?Ile?Leu?Ala?Arg?Gln?Gln?Leu?Ile?Glu?Thr?Val?Glu?Tyr?Ser?Leu

835 840 845

Pro?Asn?Lys?His?Tyr?Phe?Glu?Ile?Asp?Leu?Ser?Trp?His?Lys?Gly?Leu

850 855 860

Gln?Asn?Thr?Gly?Lys?Asn?Ala?Glu?Val?Phe?Ala?Pro?Gln?Ser?Asp?Pro

865 870 875 880

Asn?Gly?Leu?Ile?Lys?Cys?Thr?Val?Gly?Arg?Ser?Ser?Gln?Lys?Ser?Lys

885 890 895

Leu

<210>3

<211>2781

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

ggatccacca?tgaagtgggt?aacctttatt?tcccttcttt?ttctctttag?ctcggcttat 60

tccaggggtg?tgtttcgtcg?agatgcacac?aagagtgagg?ttgctcatcg?gtttaaagat 120

ttgggagaag?aaaatttcaa?agccttggtg?ttgattgcct?ttgctcagta?tcttcagcag 180

tgtccatttg?aagatcatgt?aaaattagtg?aatgaagtaa?ctgaatttgc?aaaaacatgt 240

gttgctgatg?agtcagctga?aaattgtgac?aaatcacttc?ataccctttt?tggagacaaa 300

ttatgcacag?ttgcaactct?tcgtgaaacc?tatggtgaaa?tggctgactg?ctgtgcaaaa 360

caagaacctg?agagaaatga?atgcttcttg?caacacaaag?atgacaaccc?aaacctcccc 420

cgattggtga?gaccagaggt?tgatgtgatg?tgcactgctt?ttcatgacaa?tgaagagaca 480

tttttgaaaa?aatacttata?tgaaattgcc?agaagacatc?cttactttta?tgccccggaa 540

ctccttttct?ttgctaaaag?gtataaagct?gcttttacag?aatgttgcca?agctgctgat 600

aaagctgcct?gcctgttgcc?aaagctcgat?gaacttcggg?atgaagggaa?ggcttcgtct 660

gccaaacaga?gactcaagtg?tgccagtctc?caaaaatttg?gagaaagagc?tttcaaagca 720

tgggcagtag?ctcgcctgag?ccagagattt?cccaaagctg?agtttgcaga?agtttccaag 780

ttagtgacag?atcttaccaa?agtccacacg?gaatgctgcc?atggagatct?gcttgaatgt 840

gctgatgaca?gggcggacct?tgccaagtat?atctgtgaaa?atcaagattc?gatctccagt 900

aaactgaagg?aatgctgtga?aaaacctctg?ttggaaaaat?cccactgcat?tgccgaagtg 960

gaaaatgatg?agatgcctgc?tgacttgcct?tcattagctg?ctgattttgt?tgaaagtaag 1020

gatgtttgca?aaaactatgc?tgaggcaaag?gatgtcttcc?tgggcatgtt?tttgtatgaa 1080

tatgcaagaa?ggcatcctga?ttactctgtc?gtgctgctgc?tgagacttgc?caagacatat 1140

gaaaccactc?tagagaagtg?ctgtgccgct?gcagatcctc?atgaatgcta?tgccaaagtg 1200

ttcgatgaat?ttaaacctct?tgtggaagag?cctcagaatt?taatcaaaca?aaattgtgag 1260

ctttttgagc?agcttggaga?gtacaaattc?cagaatgcgc?tattagttcg?ttacaccaag 1320

aaagtacccc?aagtgtcaac?tccaactctt?gtagaggtct?caagaaacct?aggaaaagtg 1380

ggcagcaaat?gttgtaaaca?tcctgaagca?aaaagaatgc?cctgtgcaga?agactatcta 1440

tccgtggtcc?tgaaccagtt?atgtgtgttg?catgagaaaa?cgccagtaag?tgacagagtc 1500

accaaatgct?gcacagaatc?cttggtgaac?aggcgaccat?gcttttcagc?tctggaagtc 1560

gatgaaacat?acgttcccaa?agagtttaat?gctgaaacat?tcaccttcca?tgcagatata 1620

tgcacacttt?ctgagaagga?gagacaaatc?aagaaacaaa?ctgcacttgt?tgagcttgtg 1680

aaacacaagc?ccaaggcaac?aaaagagcaa?ctgaaagctg?ttatggatga?tttcgcagct 1740

tttgtagaga?agtgctgcaa?ggctgacgat?aaggagacct?gctttgccga?ggagggtaaa 1800

aaacttgttg?ctgcaagtca?agctgcctta?ggcttaggag?gtggaggttc?cggtggaggt 1860

ggatctatgt?ccgctgttaa?ggctgctaga?tacggtaagg?acaacgttag?agtttacaag 1920

gttcacaagg?acgagaagac?cggtgttcaa?accgtttacg?agatgaccgt?ttgtgttttg 1980

ttggagggtg?agatcgagac?ctcctacacc?aaggctgaca?actccgttat?cgttgctacc 2040

gactccatca?agaacaccat?ctacatcacc?gctaagcaaa?acccagttac?cccaccagag 2100

ttgttcggtt?ccatcttggg?tacccacttc?atcgagaagt?acaaccacat?ccacgctgct 2160

cacgttaaca?tcgtttgtca?cagatggacc?agaatggaca?tcgacggtaa?gccacaccca 2220

cactccttca?tcagagactc?cgaggagaag?agaaacgttc?aagttgacgt?tgttgagggt 2280

aagggtatcg?acatcaagtc?ctccttgtcc?ggtttgaccg?ttttgaagtc?caccaactcc 2340

caattctggg?gtttcttgag?agacgagtac?accaccttga?aggagacctg?ggacagaatc 2400

ttgtccaccg?acgttgacgc?tacctggcaa?tggaagcaat?tctccggttt?gcaagaggtt 2460

agatcccacg?ttccaaagtt?cgacgctacc?tgggctaccg?ctagagaggt?taccttgaag 2520

accttcgctg?aggacaactc?cgcttccgtt?caagctacca?tgtacaagat?ggctgagcaa 2580

atcttggcta?gacaacaatt?gatcgagacc?gttgaatact?ccttgccaaa?caagcactac 2640

ttcgagatcg?acttgtcctg?gcacaagggt?ttgcaaaaca?ccggtaagaa?cgctgaggtt 2700

ttcgctccac?aatccgaccc?aaacggtttg?atcaagtgta?ccgttggtag?atcctcccaa 2760

aagtccaagt?tgtaagaatt?c 2781

<210>4

<211>921

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>4

Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala

1 5 10 15

Tyr?Ser?Arg?Gly?Val?Phe?Arg?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala

20 25 30

His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu

35 40 45

Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val

50 55 60

Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp

65 70 75 80

Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp

85 90 95

Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala

100 105 110

Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln

115 120 125

His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val

130 135 140

Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys

145 150 155 160

Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro

165 170 175

Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys

180 185 190

Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu

195 200 205

Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys

210 215 220

Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val

225 230 235 240

Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser

245 250 255

Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly

260 265 270

Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile

275 280 285

Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu

290 295 300

Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp

305 310 315 320

Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser

325 330 335

Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly

340 345 350

Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val

355 360 365

Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys

370 375 380

Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu

385 390 395 400

Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys

405 410 415

Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu

420 425 430

Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val

435 440 445

Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His

450 455 460

Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val

465 470 475 480

Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg

485 490 495

Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe

500 505 510

Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala

515 520 525

Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu

530 535 540

Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys

545 550 555 560

Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala

565 570 575

Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe

580 585 590

Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly

595 600 605

Leu?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Met?Ser?Ala?Val?Lys

610 615 620

Ala?Ala?Arg?Tyr?Gly?Lys?Asp?Asn?Val?Arg?Val?Tyr?Lys?Val?His?Lys

625 630 635 640

Asp?Glu?Lys?Thr?Gly?Val?Gln?Thr?Val?Tyr?Glu?Met?Thr?Val?Cys?Val

645 650 655

Leu?Leu?Glu?Gly?Glu?Ile?Glu?Thr?Ser?Tyr?Thr?Lys?Ala?Asp?Asn?Ser

660 665 670

Val?Ile?Val?Ala?Thr?Asp?Ser?Ile?Lys?Asn?Thr?Ile?Tyr?Ile?Thr?Ala

675 680 685

Lys?Gln?Asn?Pro?Val?Thr?Pro?Pro?Glu?Leu?Phe?Gly?Ser?Ile?Leu?Gly

690 695 700

Thr?His?Phe?Ile?Glu?Lys?Tyr?Asn?His?Ile?His?Ala?Ala?His?Val?Asn

705 710 715 720

Ile?Val?Cys?His?Arg?Trp?Thr?Arg?Met?Asp?Ile?Asp?Gly?Lys?Pro?His

725 730 735

Pro?His?Ser?Phe?Ile?Arg?Asp?Ser?Glu?Glu?Lys?Arg?Asn?Val?Gln?Val

740 745 750

Asp?Val?Val?Glu?Gly?Lys?Gly?Ile?Asp?Ile?Lys?Ser?Ser?Leu?Ser?Gly

755 760 765

Leu?Thr?Val?Leu?Lys?Ser?Thr?Asn?Ser?Gln?Phe?Trp?Gly?Phe?Leu?Arg

770 775 780

Asp?Glu?Tyr?Thr?Thr?Leu?Lys?Glu?Thr?Trp?Asp?Arg?Ile?Leu?Ser?Thr

785 790 795 800

Asp?Val?Asp?Ala?Thr?Trp?Gln?Trp?Lys?Gln?Phe?Ser?Gly?Leu?Gln?Glu

805 810 815

Val?Arg?Ser?His?Val?Pro?Lys?Phe?Asp?Ala?Thr?Trp?Ala?Thr?Ala?Arg

820 825 830

Glu?Val?Thr?Leu?Lys?Thr?Phe?Ala?Glu?Asp?Asn?Ser?Ala?Ser?Val?Gln

835 840 845

Ala?Thr?Met?Tyr?Lys?Met?Ala?Glu?Gln?Ile?Leu?Ala?Arg?Gln?Gln?Leu

850 855 860

Ile?Glu?Thr?Val?Glu?Tyr?Ser?Leu?Pro?Asn?Lys?His?Tyr?Phe?Glu?Ile

865 870 875 880

Asp?Leu?Ser?Trp?His?Lys?Gly?Leu?Gln?Asn?Thr?Gly?Lys?Asn?Ala?Glu

885 890 895

Val?Phe?Ala?Pro?Gln?Ser?Asp?Pro?Asn?Gly?Leu?Ile?Lys?Cys?Thr?Val

900 905 910

Gly?Arg?Ser?Ser?Gln?Lys?Ser?Lys?Leu

915 920

Claims (10)

1, a kind of fusion rotein comprises urico-oxidase and albumin, and described urico-oxidase is the urico-oxidase in inhuman source, is preferably the urico-oxidase that derives from microorganism; Described albumin is the albumen that has 90% above homology with human serum albumin, is preferably human serum albumin.

2, fusion rotein according to claim 1 is characterized in that: described fusion rotein also comprises connection peptides.

3, fusion rotein according to claim 1 and 2 is characterized in that: described fusion rotein is following 1), 2), 3) or 4) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein;

3) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

4) with the aminoacid sequence of sequence in the sequence table 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 3) deutero-protein.

4, the encoding gene of arbitrary described fusion rotein among the claim 1-3.

5, encoding gene according to claim 4 is characterized in that: described encoding gene is following 1)-6) in arbitrary described gene:

1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 13-2703 position deoxyribonucleotide;

2) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 1 and the dna molecular of encoding said fusion protein;

3) with 1) gene have the homology more than 90% and the dna molecular of encoding said fusion protein;

4) its encoding sequence be in the sequence table sequence 3 from 5 ' terminal 10-2772 position deoxyribonucleotide;

5) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 3 and the dna molecular of encoding said fusion protein;

6) with 4) gene have the homology more than 90% and the dna molecular of encoding said fusion protein.

6, the recombinant vectors, transgenic cell line or the reorganization bacterium that contain claim 4 or 5 described genes.

7, transgenic cell line according to claim 6 is characterized in that: the described cell that sets out is Mammals, fish, insect, plant or microorganism cells, is preferably microorganism cells.

8, reorganization bacterium according to claim 6 is characterized in that: the described bacterium that sets out is the bacterial strain that yeast saccharomyces cerevisiae belongs to (Saccharomyces), Pichia (Pichia), debaryomyces hansenii (Hansenula), genus kluyveromyces (Kluyveromyces), Candida (Candida), spore garden yeast belong (Tarulaspora) or Schizosaccharomyces; Be preferably Pichia pasteur bacterial classification (Pichia pastoris).

9, total length or arbitrary segmental primer of amplification claim 4 or 5 described genes are right.

10, described fusion rotein of claim 1-3 or claim 4 or 5 described genes treat and/or prevent application in hyperuricemia and the gout medicine in preparation.

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