CN107254453B

CN107254453B - A kind of oxalate oxidase great-hearted under physiological ph conditions and its application

Info

Publication number: CN107254453B
Application number: CN201710606904.2A
Authority: CN
Inventors: 王怡; 汪小锋; 刘海峰; 汪卫
Original assignee: WUHAN KANGFUDE BIOTECHNOLOGY Co Ltd
Current assignee: WUHAN KANGFUDE BIOTECHNOLOGY Co Ltd
Priority date: 2014-12-22
Filing date: 2015-02-10
Publication date: 2019-10-25
Anticipated expiration: 2035-02-10
Also published as: CN107299089B; CN107299089A; CN104673765A; CN107254453A; CN104673765B

Abstract

The present invention provides a kind of oxalate oxidase great-hearted under physiological ph conditions and its applications.For its gene order as shown in SEQ ID NO.4, amino acid sequence belongs to gene engineering technology field as shown in SEQ ID NO.8.65.33% of Rate activity of oxalate oxidase under the conditions of pH7.4 not less than its maximum Rate activity, therefore, drug containing oxalate oxidase can be made injection or embed through enteric material, provide possibility to research and develop a kind of recombination oxalate oxidase drug suitable for treating the excessive disease of oxalic acid.The drug of the oxalate oxidase preparation prevention excessive disease of oxalic acid also can be used in the present invention.

Description

A kind of oxalate oxidase great-hearted under physiological ph conditions and its application

Technical field

The present invention relates to the albumen of a kind of oxalate oxidase gene great-hearted under physiological ph conditions and its coding with answer With more particularly to the oxalate oxidase reduce oxalosis disease patient blood or oxalate crystal growth in healthy urine concentration being used to prepare Application in drug belongs to gene engineering technology field.

Background technique

Stone in urinary system disease is the Major health disease in a kind of global range, and most stone in urinary system are by oxalic acid Calcium or calcium oxalate and phosphoric acid calcium component form.Many diseases are excessively high related with the intracorporal oxalic acid amount of people, such as primary top grass Excessively caused advanced stage kidney stone disease, cardiac conduction hinder for Aciduria, the second hyperoxaluria, autism, Vulvodynia, oxalic acid Hinder, Chron disease, inflammatory bowel disease, colitis, urinary calculus, asthma, chronic obstructive pulmonary disease, fibromyalgia, Zellweger syndrome etc. and other chronic diseases.Primary hyperoxaluria (primary hyperoxaluria, PH) Including I type, II type and type III are all glyoxylate metabolic disorder genetic disease caused by extremely rare gene mutation, the disease The missing of specific metabolic enzyme leads to oxalic acid building up in vivo in cause of disease liver: I type disease is because of glyoxylate transamination Enzyme missing causes, and II type disease is because glyoxalic acid/hydroxypyruvate reductase missing causes, and type III disease is because of the distinctive line of liver Plastochondria enzyme 4- hydroxyl -2- oxygen-glutaraldehyde contracting enzyme missing causes, and there are also the primary hyperoxaluria diseases of some non-three of the above types Disease, reason is just in further research.Primary hyperoxaluria disease is a kind of hereditary disease very serious, patients with terminal Intracorporal blood medium-height grass acid concentration is much higher than normal person's oxalic acid level (< 5 μm of ol/L), up to 100 μm of ol/L along with patient's kidney The failure of function, oversaturated calcium oxalate can deposit formation in kidney, thyroid gland, myocardium, bone, skin, blood vessel and eye Calculus damages human body vitals, threat to life.Second hyperoxaluria (secondary hyperoxaluria, SH) is one The food-borne oxalic acid excessive resorption of kind eats disease caused by a large amount of oxalic acid precursors, caused by reason includes enterogastric diseases, such as Enteritis, cystic fibrosis, short bowel syndrome are rich in oxalic acid food or oxalic acid precursor preferred diet.Second hyperoxaluria It is a kind of very common disease, the no primary hyperoxaluria of harm is serious, but high blood oxalic acid and urine concentration of oxalic acid Still chronic disease caused by patient can be allowed to suffer from oxalic acid excessively or advanced stage urinary tract system failure.

Human body is differed by 10~70% that the oxalic acid of food-borne absorption accounts for uropoiesis total oxalate, and the oxalic acid in food can be with It is absorbed by entire pipe intestinal digesting system, including stomach, small intestine and large intestine, therefore removes food in digesting and assimilating system organ In oxalic acid can effectively prevent oxalic acid and absorbed by gastrointestinal tract.Industry generally acknowledges, the excessively high calculus that will lead to of oxalic acid calcium concentration It is formed, therefore, the concentration for reducing oxalic acid helps to reduce the risk of calculus.

The chronic renal failure (CRF) and renal failure advanced stage (ESRF) patient for needing to dialyse for a long time are also held very much Easily lead to oxalosis.The renal function missing of such patient causes oxalic acid that can not be removed efficiently, while needing in dialysis procedure Vitamin C is injected as anti-oxidation medicine, and it can be metabolized in human body and be converted into oxalic acid.In such blood in patient body Oxalic acid is generally in 30-90 μm of ol/L.2006 annual datas show that the U.S. has 340,000 people to need to dialyse every year, and the number is also always Rise, therefore, such patient is also among the risk for suffering from the excessive disease of oxalic acid.

Zellweger syndrome (ZSD) patient also excessively has with oxalic acid very high relevance (83%), and ZSD is a kind of mistake Disease caused by oxide enzyme lacks, although the mechanism of patient's ZSD Oxalate metabolism exception is unclear, ZSD is in patient body Blood concentration of oxalic acid and primary hyperoxaluria patient be much the same.

It is lithogenous that oxalosis patient's shape can be significantly reduced almost without effective remedy measures so far Risk, wherein the main certain methods used limit food merely by oral oxalate-degrading enzyme or oxalate degrading bacteria The absorption of object mesoxalic acid.However, these methods can all encounter a common challenge, that is, the highly acid in gastric environment adds The pepsin of high vigor can degrade oxalate-degrading enzyme albumen, and trypsase and chymotrypsin in enteron aisle can also degrade oxalic acid drop Solve enzyme.Moreover, the optimum pH of most known oxalate-degrading enzyme is faintly acid, so in the enteron aisle of neutral meta-alkali Debility or vigor are very low.Oral oxalic acid decomposed bacteria equally encounters the challenge of the strong acid environment of stomach and the proteolysis of enteron aisle, nothing Method forms dominant microflora and plays drop oxalic acid effect.

There are mainly two types of oxalate-degrading enzymes in nature: oxalate decarboxylase and oxalate oxidase (www.brenda- Enzymes.org/), however not being found a kind of oxalate decarboxylase has degradation oxalic acid vigor in physiological environment (pH7.4).The Once there is the albumen of oxalate oxidase vigor from barley by clearly verifying, play an important role in Barley Seeds during Germination, Therefore the germinal protein that is otherwise known as (germin protein).Similar (the germ- of the germinal protein once reported in all documents Like) oxalate oxidase has the vigor of degradation oxalic acid in acid condition.Have article (Biochem.J., 1962,85, 33) report Bougainvillea spectabilis blade has oxalate oxidase vigor under the conditions of pH7.4, but it extracts solution and does not have oxalate oxidase living Power.Report has great-hearted oxalate oxidase under pH > 7, and amino acid sequence is unknown, the description of WO2011/066282 patent A kind of enzyme of oxalate-degrading enzyme in fungi, patent Example 5 is not purified out, without any sequence yet Information, inventor herein also once attempts to purify the enzyme, but does not succeed.Makoto K et al. also once delivered an article (Journal of the Institute of Brewing, vol 115,2009,232-237) says it from one in article The oxalate oxidase of kind barley seed and fructus hordei germinatus is vibrant under neutral ph.But inventor herein extracted identical kind Barley in oxalate oxidase, it is found that it has no oxalate oxidase vigor in pH7.4.Therefore, it finds under physiological ph conditions Great-hearted oxalate oxidase, and its amino acid sequence is obtained, realize that genetic engineering recombinant expression, industrialized production utilize it The remedy measures for reducing the oxalic acid content in human body are still very important.

Summary of the invention

In order to overcome the shortcomings in the prior art, the application has screened thousands of kinds of plant samples, tests it in physiological pH item Whether there is oxalate oxidase vigor under part (pH7.4), screens beet stem, banana skin, Bougainvillea spectabilis, cordate telosma, four-o'clock, house lizard Wood, candidate's species such as manioca, the gene order of coding oxalate oxidase is then cloned by gene cloning means, passes through base Because engineering recombinant expression system (Escherichia coli, Pichia pastoris, tobacco and pea) recombinantly expresses, the correctness of gene order is verified And the vigor property of its oxalate oxidase.

The present invention provides one group under the conditions of 7.4 pH great-hearted oxalate oxidase, can degrade oxalic acid or oxalic acid Salt, the consistency of any one of gene order and SEQ ID NO.1-4 at least 50%.

The present invention provides one group under the conditions of 7.4 pH great-hearted oxalate oxidase, can degrade oxalic acid or oxalic acid Salt, any one of gene order and SEQ ID NO.1-4 at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100% consistency.

It is a further object to provide one group under the conditions of 7.4 pH great-hearted oxalate oxidase, can degrade Oxalic acid or oxalates, the consistency of any one of amino acid sequence and SEQ ID NO.5-8 at least 50%.

The present invention also provides one group under the conditions of 7.4 pH great-hearted oxalate oxidase, can degrade oxalic acid or oxalic acid Salt, any one of amino acid sequence and SEQ ID NO.5-8 have at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100% consistency.

The above oxalate oxidase can be from beet stem, banana skin, Bougainvillea spectabilis, cordate telosma, four-o'clock, house lizard wood, manioca It extracts and obtains in equal plant species.

The present invention also provides one group under the conditions of 7.4 pH great-hearted oxalate oxidase, can degrade oxalic acid or oxalic acid Salt is the polyethyleneglycol modified oxalate oxidase with different molecular weight.

The molecular weight of the polyethylene glycol is 2kD, 5kD, 10kD, 20kD, 4 arm -20kD, 30kD, 40kD or 4 arms - Any one in 40kD.

The present invention provides a kind of expression vector comprising oxalate oxidase gene, wherein oxalate oxidase gene sequence with The consistency of any one of SEQ ID NO.1-4 at least 50%.

The present invention provides a kind of expression vector comprising oxalate oxidase gene, wherein oxalate oxidase gene sequence with Any one of SEQ ID NO.1-4 at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100% consistency.

The present invention provides a kind of recombinant expression strain, includes the above expression vector, and can generate oxalate oxidase.

The present invention provides a kind of genetically modified plants strain, Activities of Some Plants (including blade, root are spent) or plant cell, turn Change has the expression vector containing the above oxalate oxidase gene sequence.

The present invention also provides the oxalate oxidases to reduce the drug of oxalosis disease patient concentration of oxalic acid in preparation In application.The oxalosis disease includes primary hyperoxaluria, two type hyperoxalurias, zellweger disease, Or chronic urinary system damageability disease etc..

Since the oxalate oxidase has enzyme activity under the conditions of 7.4 pH, the drug containing oxalate oxidase can be with Injection is made or is embedded through enteric material.

One group of oxalate oxidase provided by the invention, its Rate activity under the conditions of pH7.4 is not less than its maximum Rate activity 20%, and its be uniquely being disclosed of finding up to now and what is be verified have work in pH7.4 or pH7.0~8.0 The oxalate oxidase of power.A kind of recombination oxalic acid oxygen suitable for treating the excessive disease of oxalic acid is researched and developed in being found to be for the oxalate oxidase Change enzyme drug and provides possibility.The drug of the oxalate oxidase preparation prevention excessive disease of oxalic acid also can be used in the present invention.

Detailed description of the invention

Fig. 1 is yeast expression vector Z α -5102 map.

Fig. 2 is yeast expression vector GAPZ α -5102 map.

Fig. 3 is yeast expression vector Z α -5601 map.

Fig. 4 is yeast expression vector GAPZ α -5601 map.

Fig. 5 is yeast expression vector Z α -30640 map.

Fig. 6 is the expression that SDS-PAGE analyzes Pichia pastoris X33 medium-height grass acid oxidase Z α -5102.

Fig. 7 is the expression that SDS-PAGE analyzes Pichia pastoris X33 medium-height grass acid oxidase Z α -5601.

Fig. 8 is the expression that SDS-PAGE analyzes Pichia pastoris X33 medium-height grass acid oxidase Z α -30640.

Fig. 9 is the expression that SDS-PAGE analyzes Pichia pastoris X33 medium-height grass acid oxidase GAPZ α -5601.

Figure 10 is plant expression vector map.

Figure 11 is the colour developing of tobacco leaf mesoxalic acid oxidase.

Figure 12 is oxalate oxidase viability examination, pH 7.4.

Contain 2mM oxalic acid in the test tube of left side, is free of oxalic acid in the test tube of right side.

Figure 13 is influence result figure of the oxalate oxidase of different molecular weight PEG modification in rat model to blood oxalic acid.

Figure 14 be different molecular weight PEG modification oxalate oxidase in rat model to urine oxalic acid influence result figure.

Figure 15 is that SD rat, antibody is immunized in the B5102 sample that oxalate oxidase B5102 and different molecular weight PEG is modified Potency result figure.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

[embodiment 1] produces the screening of oxalate oxidase plant and vigor is tested

Inventor herein is from Medicinal Plants of Guangxi garden, Kunming botanical garden, plants in Wuhan Botanical Garden and extra large Subtropical Botanical Garden Over thousands of kind of plant sample is acquired, the screening experiment of oxalate oxidase is carried out.

When producing oxalate oxidase foliage filter screening, sample first passes through processing, and treatment process is as follows: by a certain amount of plant sample (leaf, root, stem or flower) is shredded with scissors, is put into the quantitative beaker for filling pure water, is suspension with high-shear homogenizer homogenate (particle < 100 μm), homogenate are divided into soluble fraction (supernatant) and insoluble part (precipitating), insoluble part after being centrifuged It is resuspended with pure water, the two is respectively intended to survey the vigor of degradation oxalic acid.

Oxalate oxidase vigor test method has following two:

(1) HPLC method: the oxalic acid solution (being adjusted to pH 7.4 with NaOH) of the 12mM of 40 μ l samples and 360 μ l is taken to mix Afterwards, it reacts 48 hours for 37 DEG C, reaction solution adds terminate liquid (100 μ l 1.5M sulfuric acid solution) to terminate reaction.Reaction solution is through high speed centrifugation Removal precipitating, supernatant are crossed after 0.4 μm of film and measure oxalic acid content with HPLC.One enzyme-activity unit is defined as every in the above system Minute reduces enzyme amount required for 1 micromole's oxalic acid.

(2) development process surveys oxalate oxidase vigor:

20 μ l sample solutions and 580 μ l vigor developing solutions (contain 10mM oxalic acid；The chloro- 2- hydroxy benzene sulfonic acid of 4mM 3,5- bis- Sodium (DHBS)；The horseradish peroxidase of 2U；The 4-AA (4-AA) of 1mM；The phosphate buffer of 50mM, pH6.0 Or 7.4), 37 DEG C of reaction 30min monitor OD with microplate reader₄₉₂Absorbance.One enzyme-activity unit is defined as in above-mentioned reactant Enzyme amount required for 1 micromole's oxalic acid is reduced in system per minute.

By screening, it has been found that banana skin (banana peel), beet stem (beet stem), Bougainvillea spectabilis blade (Bougainvillea spectabilis leaves), four-o'clock tender shoots (Mirabilis jalapa young leaf), night Come spiceleaf piece (Telosma cordatum (Brum.f.)), manioca blade (Jatropha carcas L.leaf), house lizard wood Plant samples such as (Sauropus androgynus (L.) leaf) have specific oxalate oxidase vigor in the environment of pH7.4.

The clone of [embodiment 2] banana and beet oxalate oxidase gene

(1) oxalate oxidase crude product is extracted in beet stem and banana skin sample

A certain amount of beet stem and banana skin sample are weighed, is put into the beaker for filling pure water after chopping, through high-speed homogenization Machine homogenate is particle less than 100 μm, centrifugation removal supernatant (debility or extremely low oxalate oxidase vigor), and precipitating is with containing The buffer solution (2mM PBS, pH7.0) of surfactant (1% thiosulfonic acid NaTDC) and sucrose (50%w/v) is resuspended Afterwards, it extracting 2~5 days for 4 DEG C, the crude extract of oxalate oxidase collects elution samples peak through Q-sepharose chromatography, after The surfactant and salt component in sample are removed through dialysis.Oxalate oxidase sample still has after Native-PAGE is separated Oxalate oxidase activity, by identifying sample elution peak and oxalic acid oxygen after the oxalate oxidase vigor colour developing of Native-PAGE glue Change enzyme band, after the separation of SDS-PAGE glue, after oxalate oxidase band is cut in the analysis test of commission China Agricultural University The heart does nitrogen end (N-terminal) amino acid sequencing and the analysis of MS-MS mass spectrum sequence.

(2) genome sequence is retrieved

According to N-terminal sequencing result, by the protein sequence of nitrogen end in the banana announced and the open gene of beet T-blastn retrieval is carried out in group sketch, candidate gene order is obtained, is then compared with mass spectrographic the sequencing results, Determine target oxalate oxidase gene sequence.

(3) gene cloning

Special primer (table 1) is designed according to fixed oxalate oxidase gene DNA sequence dna, in banana and the cDNA of beet PCR amplification is carried out in library, whether sequencing identification is cloned into oxalate oxidase gene after PCR product is carried out purification and recovery.This Experiment is cloned into B5100, B5102, B5601 from beet altogether, oxalate oxidases candidate's base such as M30640 from banana Because of (as shown in SEQ ID NO.1-4).Also it is cloned into a plurality of germin-like gene order simultaneously: 108,122,303, 1013 (as shown in SEQ ID NO.9-12).

1 oxalate oxidase cloning primer of table

PCR system (50 μ l):

PCR program:

[embodiment 3] E. coli system expression recombination oxalate oxidase

(1) building of plasmid

Oxalate oxidase (OxOx) B5100, B5102, B5601, M30640 gene cloning is built into pAT carrier (load Body is derived from pET-32a carrier), recombinant plasmid is then transferred to E.coli Origami B competent cell.

(2) shaking flask is expressed in a small amount

By B5100, B5102, B5601, the glycerol strain of M30640 is coated on LB agarose plate (containing 100 μ g/ml ammonia benzyls Antibiotic), 37 DEG C of inversion overnight incubations to have monoclonal bacterial plaque appearance.Picking single colonie is inoculated in 200ml Shake flask medium (culture medium of LB containing 50ml, 100 μ g/ml ammonia benzyl antibiotic), 220rpm, 37 DEG C of cultures, OD₆₀₀To 0.6~0.8, MnCl is added₂ The final concentration of 0.6mM of final concentration of 1mM, IPTG carries out inducing expression, and after inducing 5h, 9500g is centrifuged 15min and collects thallus, bacterium Weight is suspended from 50mM arginine solution, and ice-bath ultrasonic is crushed 30min, and washing 2 times is resuspended with 50mM arginine, in inclusion body Having 80% is oxalate oxidase albumen.Thallus yield is under the fermentation condition up to 0.2~0.6g/L.

(3) 7L fermentor great expression

By B5100, B5102, B5601, M30640 gene monoclonal bacterial plaque accesses (the LB culture containing 200ml of 500ml shaking flask The ammonia benzyl antibiotic of base and 100 μ g/ml), it is incubated overnight, as seed liquor.Seed liquor is accessed 7L's by 0.5% inoculum concentration Fermentor (the basal medium containing 3.5L: glycerol 12g/L；Yeast powder 12g/L), 200rpm, 37 DEG C of cultures, 10% ammonium hydroxide tune It saves pH to stablize after 6.85, about 8h, starts at the uniform velocity to add supplemented medium (30g/L glycerol, 60g/L yeast powder), work as OD₆₀₀It reaches When to 20, IPTG to final concentration of 0.8mM is added, continues to induce 20h, tank is put and collects thallus.Thallus yield is in the fermentation condition Lower reachable 1~5g/L.

(4) inclusion body protein washing and renaturation

Inclusion body washing:

Coli somatic cell washing buffer (50mM Tris, 50mM NaCl, 5mM EDTA, 5mM DTT, 1%TritonX-100,2M urea are resuspended with hydrochloric acid tune pH8.0), and nuclease then is added according to 1:1000 (Benzonase, the MgCl containing final concentration of 0.5mM₂), 15min is stirred, the thinning laggard horizontal high voltage homogeneous of bacterium solution is high-pressure homogeneous Condition be 1100bar, repeatedly be crushed 4 times.Clasmatosis liquid is subjected to 8000g and is centrifuged 10min, abandons supernatant, collects inclusion body Precipitating.Inclusion body is sufficiently resuspended with suitable wash buffer, and stir about 30min, 8000g are centrifuged 10min, is abandoned supernatant and is collected Precipitating washs repeatedly and inclusion body is resuspended with pure water afterwards three times, and 8000g is centrifuged 10min after being sufficiently resuspended, and obtains inclusion body.SDS- PAGE electrophoretic analysis purity of protein, if renaturation can be reached and require (purity > 80%).

Renaturing inclusion bodies:

Oxalate oxidase inclusion body uses rapid dilution method renaturation: washing qualified solubilization of inclusion bodies in 8M urea liquid In, after measuring protein concentration, 1ml/min is at the uniform velocity added drop-wise to rapid dilution renaturation, final concentration of protein in the renaturation solution of pre-cooling and is 100 μ g/ml, magnetic agitation mixing are placed on 4 DEG C of renaturation for 24 hours.Renaturation solution formula be it is set forth below any one:

1. 20mM Tris,300mM NaCl,1mM MnCl₂,1mM GSH/0.2mM GSSG,pH8.0

2. 20mM Tris,300mM NaCl,1mM MnCl₂,1mM GSH/0.2mM GSSG,400mM arginine, pH8.0

3. 20mM Tris,300mM NaCl,1mM MnCl₂,1mM GSH/0.2mM GSSG,100mMα- cyclodextrin,pH8.0

4. 20mM Tris,300mM NaCl,1mM MnCl₂, 1mM GSH/0.2mM GSSG, 2% β- cyclodextrin,pH8.0

5. 20mM Tris,300mM NaCl,1mM MnCl₂, 1mM GSH/0.2mM GSSG, 40%sucrose, pH8.0

6. 20mM Tris,300mM NaCl,1mM MnCl₂, 1mM GSH/0.2mM GSSG, 40%glucose, pH 8.0

7. 20mM Tris,1mM MnCl₂,1mM GSH/0.2mM GSSG,pH8.0

8. 20mM Tris,1mM MnCl₂,1mM GSH/0.2mM GSSG,400mM arginine,pH8.0

9. 20mM Tris,1mM MnCl₂,1mM GSH/0.2mM GSSG,50mM betaine,pH8.0

10. 20mM Tris,1mM MnCl₂,50mM betaine,pH8.0

11. 20mM Tris,0.5-5mM MnCl₂,1mM GSH/0.2mM GSSG,50mM betaine,pH8.0

12. 20 mM Tris,1mM MnCl₂, 1mM GSH/0.2mM GSSG, 50 mM betaine, pH4.0-10.0

13. 20 mM Tris,1mM MnCl₂,50 mM betaine,0.05mM 1-hexanol,50mM acetamide,300 mM KCl,pH8.0

Refolded protein is through phenyl sepharose purifying resin, and vigor is up to 0.1~15U/mg albumen.

Initialism:

1, GSH glutathione 6, sucrose sucrose

2, GSSG oxidized form of glutathione 7, glucose glucose

3, Arginine arginine 8, betaine glycine betaine

4, α-cyclodextrin alpha-cyclodextrin 9, hexanol hexanol

5, β-cyclodextrin beta-cyclodextrin 10, acetamide acetamide

[embodiment 4] Bichi yeast system expresses oxalate oxidase

(1) vector construction

By B5100, B5102, B5601, M30640 gene is cloned by Xho I and Not I restriction enzyme site into pPICZ α B And pGAPZ α A carrier, the recombinant plasmid of building are Z α -5102, GAPZ α -5102, Z α -5601, GAPZ α -5601, Z α - 30640 (Fig. 1~5).Recombinant plasmid Z α -5102, Z α -30640, Z α -5601 are linearized with Pme I (Mss I) restriction endonuclease, GAPZa-B5102, GAPZ α -5601, GAPZ α -30640 are linearized with Bln I (Avr II).Carrier after linearisation is logical Electricity is crossed to turn mode and convert Pichia pastoris X33 strain to be expressed.

Table 2. constructs pPICZ α B and pGAPZ α A expression vector Primer and sequence

PCR system (50ul):

PCR program:

(2) protein expression

With YPDS culture medium (10g/L yeast powder, 20g/L peptone, 20g/L glucose, 1M sorbierite, 20g/L agar, Containing 100 μ g/L zeocin antibiotic) positive colony of plate screening, then the YPDS plate (1mg/ml with high concentration antibiotic Zeocin the screening of copy number) is carried out, the clone of the high copy number screened is expressed with shaking flask to be verified.

The YPD culture medium of the clone access 4mL of Z α -5102, Z α -30640, the Z α -5601 of the high copy screened, 28 DEG C culture 18~20h, by 4% inoculum concentration switching 500ml shaking flask (culture medium containing 50ml) expression culture medium BMGY (yeast powder 1% (w/v), peptone 2% (w/v), 100mM kaliumphosphate buffer pH 6.0, no amino yeast nitrogen 1.34% (w/v) are sweet 1% (w/v) of oil, biotin 0.04% (w/v)), 28 DEG C, after 220rpm grows 18~20h, OD₆₀₀3.0~6.0 are reached, centrifugation BMMY culture medium (yeast powder 1% (w/v), peptone 2% (w/v), 100mM kaliumphosphate buffer at are resuspended in after collecting thallus PH 6.0, no amino yeast nitrogen 1.34% (w/v), 1.0% (v/v) methanol, biotin 0.04% (w/v), 5mM MnCl₂) Carry out inducing expression.Inducing expression temperature is 28 DEG C, adds methanol daily to final concentration of 1.0%, 90~120h of continuous induction. Culture takes supernatant for determination of color oxalate oxidase vigor and SDS-PAGE identification and analysis after 9500g is centrifuged.

The GAPZa-B5102 of the high copy screened, GAPZ α -5601, GAPZ α -30640 bacterial strain, switching are cultivated in BYPD Base (10g/l yeast powder, 20g/l peptone, 20g/l glucose, biotin 400 μ g/l, MnCl₂5mM and 100mM phosphoric acid Potassium buffer, pH 6.0), 28 DEG C, 220rpm shake culture adds 2% glucose after 48h, after cultivating 72h, collects culture Object centrifugation, supernatant are analyzed for oxalate oxidase vitality test and SDS-PAGE.

The results show that four kinds of oxalate oxidases (B5100, B5102, B5601, M30640) can in Yeast system at Function expression, yield are 0.01~0.1mg/ml culture medium, and SDS-PAGE is analysis shows that purpose band is 24KD (Fig. 6~9), colour developing The test of method vigor shows that it has oxalic acid degradation vigor at pH7.4.

[embodiment 5] botanical system expresses oxalate oxidase

(1) vector construction

By B5102, B5601, M30640 gene introduces Xba I and Kpn I restriction enzyme site by PCR amplification, then through this PHTE carrier is inserted into two sites, obtains pHTE-5601, pHTE-5102and pHTE-30640 (Figure 10).Positive recombinant plasmid It is transferred in Agrobacterium tumefaciems GV3101 after bacterium solution PCR and endonuclease digestion verifying with freeze thawing recovery method.

Table 3 constructs plant expression vector Primer and sequence

PCR system (50 μ l):

PCR program:

(2) transformation of tobacco blade carries out transient expression

This uncured tobacco of wild type (N.benthamiana) (16/8h illumination/dark, 25 DEG C) in greenhouse is cultivated 5~8 weeks. Conversion there are into B5102, B5601, the Agrobacterium strain of M30640 gene is inoculated in (rifampin containing 25mg/L in 3ml LB culture medium And the kanamycins of 50mg/L), 28 DEG C, 220rpm is incubated overnight to OD₆₀₀> 1.2 are used as seed liquor.Seed liquor is in 1:100 ratio It accesses 2L LB culture medium (rifampin containing 25mg/L, 20 μM of acetosyringones of 50mg/L kanamycins and), 220rpm, 28 DEG C It cultivates to OD₆₀₀For 0.8~1.2 (about 18h).5000rpm is centrifuged 5min, removes supernatant, the bacterium of 5 times of volumes of bacterial sediment Re-suspension liquid (10mM 2- (N- morpholine) ethane sulfonic acid sodium (MES) pH 5.5,10mM MgSO₄And 100mM acetosyringone), Incubated at room temperature 4h.

Agrobacterium bacterium solution is injected in tobacco leaf by 1ml or 3ml syringe and is infected, each injection point is generally about Having 7 square centimeters of range can be infected to each blade need to infect several points.Plant after infecting is placed in a greenhouse growth 7 Normal watering and fertilising is given once daily in it or so.

(3) blade oxalate oxidase vigor test experiments

The tobacco leaf cultivated 7 days after infecting is harvested for the test of oxalate oxidase vigor.Test method are as follows: infection court Surrounding vanes (containing negative control group) are cut to the discoid of diameter about 1cm, are centrifuged after vanes high-speed homogenization, supernatant precipitating Be placed in oxalate oxidase vitality test buffer (40mM succinic acid, 2.5mM oxalic acid, 5U/ml horseradish peroxidase, The chloro- 1- naphthols of 0.6mg/ml 4-, 60% (V/V) ethyl alcohol, pH 5.0), it is incubated at room temperature colour developing overnight, does not show enzyme activity in supernatant, Oxalate oxidase vigor is presented in precipitating.

It similarly infects method and infects expression experiment for pea seedlings.

Two kinds of expression systems of tobacco and pea the results show that for expression 3 oxalate oxidase genes all have grass Acid oxidase vigor (Figure 11, Figure 12), expression quantity are about the fresh blade of 0.01~5mg/g, but the overwhelming majority grass expressed Acid oxidase is present in the solid material of blade, insoluble or cannot effectively dissolve out.

The vigor test of [embodiment 6] oxalate oxidase (Bacillus coli expression) under different ph values

Test method:

10 μ l oxalate oxidase solutions be added to 190 μ l vigor test solution in (800mg/L 4-AA, 4.8mM sodium 3,5-dichloro-2-hydroxybenzenesulfonate, 10U/ml horseradish peroxidase, 5mM oxalic acid, different pH buffers), it mixes 37 DEG C of 10~60min of incubation are placed on, microplate reader reads OD₄₉₂Light absorption value.

Different pH buffer compositions: the buffer of pH4.5~6.0 selects the citrate buffer solution of 50mM, and pH6.0~8.0 is selected With the phosphate buffer of 50mM.

Test result shows that the maximum vigor of oxalate oxidase 5100 is 18.2U/mg, and 5102 maximum vigor are 19.5U/mg, 5601 maximum vigor are 18.7U/mg, and 30640 maximum vigor are 14U/mg.Compare for convenience, we are by four kinds of enzymes in difference Vigor under pH value is scaled relative activity and arranges into table 3:

The opposite Rate activity at various ph values of 3 four kinds of oxalate oxidases of table

The PEGylated modification of [embodiment 7] oxalate oxidase

The washing of oxalate oxidase inclusion body:

Bacillus coli cells with wash buffer (50mM Tris, 50mM NaCl, 5mM EDTA, 5mM DTT, 1% TritonX-100,2M urea are sufficiently resuspended (concentration about 30g/L) with hydrochloric acid tune pH8.0), nucleic acid then are added according to 1:1000 Enzyme (benzonase, the MgCl of final concentration of 0.5mM₂), stir 15min, the thinning laggard horizontal high voltage homogeneous of bacterium solution.It is high-pressure homogeneous Condition be 1100bar, repeatedly be crushed 4 times.The centrifugation of clasmatosis liquid, 8000g, 10min abandon supernatant, collect precipitating.

Inclusion body cleaning:Inclusion body is sufficiently resuspended with suitable wash buffer, stir about 30min, 8000g centrifugation 10min abandons supernatant and collects precipitating, washs repeatedly and inclusion body is resuspended with pure water afterwards three times, and 8000g is centrifuged 10min after being sufficiently resuspended, Obtained inclusion body weighing, SDS-PAGE analyze inclusion body protein purity, if can reach renaturation and require (purity > 80%).

Q column denaturation purifying oxalate oxidase inclusion body:

The qualified inclusion body (purity > 80%) of washing is dissolved in 8M urea liquid (20mM Tris-HCl, 8M urea, pH 8.0), 13000g is centrifuged 10min, discards precipitating, supernatant loading and the Q column (5 column volumes of equilibrium liquid balance) balanced, with washing De- liquid B (8M urea, 30g/L NaCl, Tris-HCl, pH8.0) elution 6 column volumes (CV) elutes impurity protein, uses elution instead Liquid C (8M urea, 160g/L NaCl, Tris-HCl, pH8.0) elution, collects target sample peak, through Q column purification, oxalic acid oxidation For enzyme inclusion body protein purity up to 95%, sample after purification is concentrated into renaturation reality of the 5mg/ml for next step with super filter tube It tests.

Oxalate oxidase inclusion body protein renaturation

It is operated by being described in embodiment 3.

Phenyl sepharose purifying resin

Before loading, Phenyl sepharose column is flat with equilibrium liquid (10mM Tris-HCl, 500mM NaCl, pH 8.0) Weigh 5 column volumes (CV).NaCl to final concentration of 0.5M is added in oxalate oxidase B5102 after renaturation, 0.45 μ is crossed after mixing The filter membrane of m is clarified.Clarified solution loading and the Phenyl sepharose pillar balanced, with wash buffer (10mM Tris- HCl, 250mM NaCl, pH 8.0) use instead after 1.5 column volumes of washing eluent (10mM Tris-HCl, 20% Isopropanol, pH 7.0) elution.Target protein sample peak is collected, and NaCl to 50mM is added to carry out salt precipitation, after saltouing Sample is centrifuged 10min through 12000g, discards supernatant, and precipitating is redissolved in borate buffer (10mM Na₂B₄O₇~H₃BO₃,pH 9.0) the PEGylated modification in, for the later period.It purifies obtained sample and carries out SDS-PAGE analysis purity of protein, oxalate oxidase Vigor test evaluation renaturation effect.

The PEGylated modification of oxalate oxidase

The oxalate oxidase B5102 albumen borate buffer for being dissolved in borate solution adjusts concentration to 2mg/ml.It presses 10 multiple proportions column investment mPEG-SC (the Methoxy PEG Succinimidyl of the molal quantity of lysine on B5102 albumen Carboxymethyl Ester) modifier carries out PEGylated modification reaction into the B5102 protein solution of mild magnetic agitation, it mixes 28 DEG C of reaction 6h after even are added the glycine equal with mPEG mole and terminate reaction.We tested 2kD, 5kD, 10kD, The PEG modifier of 4 arm -40kD of 20kD, 4 arm -20kD, 30kD, 40kD and.

PEG modification after B5102 sample with molecular-exclusion chromatography (GE HiLoad Superdex 16/600GL, USA) into Row isolates and purifies.Molecular-exclusion chromatography uses phosphate buffer (10mM K before loading₂HPO₄~KH₂PO₄, pH 7.4) carry out it is flat Weigh 5 column volumes, carries out loading by the 5% of column volume, is then eluted again with phosphate buffer, flow velocity 1ml/min, collect sample Product peak.

The oxalate oxidase of [embodiment 8] different molecular weight PEG modification reduces blood oxalic acid and urine oxalic acid in rat model Experiment

30 5 week old SD male rats, weight is about 130~150g, is bought from Hubei Province's animal experimental center.Rat is raised It supports and adapts to be randomized into rat generation after a week in independent gas supply cage (IVC) system (18~26 DEG C of temperature, humidity 40~70%) It thanks in cage, is divided into 5 groups, wherein one group is control group, four groups are experimental group, every group of 6 rats.After the laundering period, control group With the blood oxalic acid of experimental group rat and urine oxalic acid it is stable after, the oxalic acid of 3 kinds of different molecular weight PEG (20K, 30K, 40K) modification Oxidase B 5102 is administered into different experimental group rats by tail vein injection mode, wherein one group of experimental group rat injects phase For physiological saline with dose volume as placebo, drug administration dosage is 0.2mg/ days/time, successive administration 3 days, control group Rat is not cooked any treatment processing.Control rats feed conventional mouse grain and drinking-water, and experimental group rat feeds conventional mouse grain and 1% second Glycol drinking-water.After administration, the blood oxalic acid and urine oxalic acid of all rats carry out detection monitoring daily.It is acquired daily by tail vein 0.3ml blood collects serum after 5000g is centrifuged 5min, hydrochloric acid tune is added for measuring blood concentration of oxalic acid, freshly harvested blood PH to 2.0~4.0 is saved, vitamin C in serum (Vc) is prevented to be converted into oxalic acid.The every 12h of urine (8:30am and 8:30pm) is collected Once, and with salt acid for adjusting pH to 1.5~2.0, Vc is prevented to be converted into oxalic acid.The blood oxalic acid and urine oxalic acid of all rats are tested It surveys, effect experiment can be carried out after stablizing.

The measurement of blood oxalic acid uses Amplex red (10-acetyl-3,7-dihydroxyphenoxazine) luciferase mark Method detection, testing process are as follows:

(1) blood serum sample after hydrochloric acid acidification is centrifuged 30min ultrafiltration with 10K ultrafiltration membrane centrifuge tube 5000g and removes serum egg It is white, 4 DEG C of temperature.

(2) ultrafiltrate is added in 96 hole fluorescence ELISA Plates (all black is opaque), 6 parallel holes of each sample spot, preceding 3 Oxalic acid reaction reagent (100mM citrate buffer solution, pH5.4,10 μM of Amplex red, 1U/mL HRP, 0.1U/ is added in a hole ML oxalate oxidase), background calibration reagent (100mM citrate buffer solution, pH5.4,10 μM of Amplex are added in rear 3 holes Red, 1U/mL HRP), 25 DEG C of incubation 30min after concussion mixes.

(3) fluorescent value is detected with fluorescence microplate reader (excitation wavelength: 538nm, wavelength of transmitted light: 590nm).

(4) according to the net fluorescent value of sample (oxalic acid test group Mean Fluorescence-background calibration group Mean Fluorescence) and oxalic acid Standard curve fluorescent value is compareed, and the concentration of oxalic acid in sample is calculated.

The oxalic acid assay kit that concentration of oxalic acid measurement uses U.S. Trinity company is urinated, concrete operation step is strictly pressed It is executed according to operational manual.

The results show that administration group (20K, 30K, 40K PEG-OxOx-B5102) can drop 40% compared with placebo ~50% blood concentration of oxalic acid, while 20%~40% urine concentration of oxalic acid dropping.(Figure 13 and Figure 14)

The measurement of [embodiment 9] PEG-OxOx immunogenicity

The B5102 sample of oxalate oxidase B5102 and different molecular weight PEG (2k, 5k, 10k, 20k, 30k, 40k) modification Immunogenic evaluation is carried out by tail vein injection SD rat.Injection dosage is 0.2mg/ times, once a week, persistent immunological four In week, different time points take blood serum sample to measure antibody titer, assessment immunogenicity (immune preceding 0 before immune for the first time and after immune It, the 7th day, the 14th day, the 21st day, the 28th day, the 45th day, the 60th day).

It is as follows to measure process:

(1) with coating buffer (50mM carbonate/bicarbonate buffer, pH9.6) dilution PEG modification B5102 protein sample is put in 96 hole elisa Plates holes of the supreme absorption of 100 μ l to 10 μ g/ml, is placed in 4 DEG C overnight with masking foil package Coating.

(2) coating plate is placed in room temperature, empties liquid and pats dry residual liquid, with 300 μ l cleaning solution (1.5mM KH₂PO₄； 8.1mM Na₂HPO₄·12H₂O；136mM NaCl；2.7mM KCl；It 0.05%Tween-20) cleans twice.

(3) each 300 μ l confining liquid (1.5mM KH of Kong Zhongjia₂PO₄；8.1mM Na₂HPO₄·12H₂O；136mM NaCl； 2.7mM KCl；5% skimmed milk power), it is incubated for 1h.

(4) it empties liquid and pats dry residual liquid, cleaned twice with 300 μ l eluents.

(5) blood serum sample (50 times, 100 times, 200 times, 400 times, 800 times ... ...) of gradient dilution are done with cleaning solution to mix After even plus 100 μ l are into every enzyme mark hole, 37 DEG C of incubation 1h or incubation at room temperature 3h.

(6) it empties liquid and pats dry residual liquid, cleaning solution is filled it up in each hole, empty liquid and pat dry residual liquid, It is repeated 3 times.

(7) with cleaning solution by the goat anti rat antibody (secondary antibody) that HRP is coupled be diluted to optimal concentration (generally 1/5000~ 1/20000) 100 μ l secondary antibodies, and into each instrument connection are added, are incubated at room temperature 1h.

(8) it empties liquid and pats dry residual liquid, eluent is filled it up in each hole, empty liquid and pat dry residual liquid, It repeats 3~5 times.

(9) by 6ml TMB reagent A (0.5mM EDTA-Na；5mM citric acid；10% glycerol；0.04%tetramethyl ) and 6ml reagent B (165mM sodium acetate benzidine；8.3mM citric acid；0.06%30%-H₂O₂) mix.

(10) each 100 μ l TMB developing solution of Kong Zhongjia, after being incubated at room temperature 30min, each 100 μ l of Kong Zhongjia terminates reaction Liquid (2M H₂SO₄)。

(11) microplate reader measures OD₄₅₀Light absorption value.

The results show that the antibody titer of oxalate oxidase reached peak at the 28th day.28th day result (Figure 15) Oxalate oxidase is shown after PEG is modified, the antibody titer of oxalate oxidase is remarkably decreased, and with modification PEG molecular weight Increase, fall is bigger, 20k, 30k, and the oxalate oxidase of 40kPEG modification is nearly no detectable antibody.

SEQUENCE LISTING

<110>Wuhan Kangfude Biotechnology Co., Ltd.

<120>a kind of oxalate oxidase great-hearted under physiological ph conditions and its application

<160> 34

<170> PatentIn version 3.3

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aaagggttta atattccctc agacacaaac aacactcaaa gagcagaagc cacactagta 180

gatgtcaatc gatttccagc acttaacaca ttaggtgtag ccatggctcg tgtagacttt 240

gcgtcctttg gcctaaacac acctcatttg caccctcgtg gttctgagat attcgcggtg 300

ctagagggga ctttatatgc cggcattgtc accaccgatt acaagctttt cgacacggtg 360

ttgagaaagg gtgacatgat tgttttccct caaggcttaa tccacttcca gcttaatctt 420

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tttcaggtag atgagatggt gattcagcaa cttcgatctc agttttcagg tcaaaacata 600

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aaagggttta atattccctc agacacaaac aacactcaaa gagcagaagc cacactagta 180

gatgtcaatc gatttccagc acttaacaca ttaggtgtag ccatggctcg tgtagacttt 240

gcgtcctttg gcctaaacac acctcatttg caccctcgtg gttctgagat attcgcggtc 300

ctagagggga ctttatatgc cggcattgtc accaccgata ataagctttt cgacacggtg 360

ttgagaaagg gtgacatgat tgttttccct caaggcttaa tccacttcca gcttaatctt 420

ggcaagacag atgctcttgc tattgcctct tttgggagcc aatttcctgg acgagttaat 480

gttgctaatg gtgtctttgg aactacgcca caaattttgg atgatgtact tacccaagcg 540

tttcaggtag ataagatggt gattgagcaa cttcgatctc agttttcagg tccaaacaca 600

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aaagggttta atatacctgc agacacaaac aacactcaag gagcaagtgc cacactagtg 180

gacattactc tattccctgc agttaacaca caaggagtct ccatggctcg tgtggacttt 240

gcgccatttg gactaaacac ccctcattta catcctcgtg gctcagaggt tttcgcagtg 300

atggaaggga ttatgtatgc tggttttgtg accactgatt ataagctcta tgatacaatt 360

ataaaaaagg gtgatattat tgtgtttcca caaggtctaa ttcatttcca acttaatctt 420

gggaagacag atgctttagc aattgcctca tttgggagcc aaaatccagg gagaattaat 480

atcgctgaca gtgtgtttgg tactactccg cgtgttctag atgatgtgct taccaaagga 540

tttcaaatcg atgagttgtt ggtcaagcaa cttcgttctc agttttctac tgataatata 600

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tttgatccga gtcctctcca agacttttgc gttgctgact acgactccaa cgtgtttgtg 60

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ttagacaagc ccgcgagcac cgccaacgag cttggcgcaa acatcactct cgtcaacgtg 180

gaacgactcc caggcctcaa ctcccttggc gtcgccatgt ctcgcatcga ctacgcgccc 240

ttcggtctca accctcctca ctcgcatcca cgatcgtcgg agatactgca cgtggcggaa 300

ggaacgctct acgccggctt cgtcacctcc aacacggaaa acggcaacct tctcttcgct 360

aagaagctga agaagggcga cgcgtttgtg ttccccaggg gcctcataca cttccagttc 420

aacatcgggg acaccgatgc ggtggcgttc gctaccttcg gcagccagag cccgggtctc 480

gtcaccaccg ccaacgcact gttcggatcg aagccgccca tcgctgatta cattcttgcc 540

caggccgtgc agcttagcaa gacgaccgtg ggctggcttc agcagcagca gtggttggac 600

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Ser Asp Pro Gly Leu Leu Gln Asp Phe Cys Val Gly Val Asn Asp Pro

1 5 10 15

Asp Ser Ala Val Phe Val Asn Gly Lys Phe Cys Lys Asn Pro Lys Asp

20 25 30

Val Thr Ile Asp Asp Phe Leu Tyr Lys Gly Phe Asn Ile Pro Ser Asp

35 40 45

Thr Asn Asn Thr Gln Arg Ala Glu Ala Thr Leu Val Asp Val Asn Arg

50 55 60

Phe Pro Ala Leu Asn Thr Leu Gly Val Ala Met Ala Arg Val Asp Phe

65 70 75 80

Ala Ser Phe Gly Leu Asn Thr Pro His Leu His Pro Arg Gly Ser Glu

85 90 95

Ile Phe Ala Val Leu Glu Gly Thr Leu Tyr Ala Gly Ile Val Thr Thr

100 105 110

Asp Tyr Lys Leu Phe Asp Thr Val Leu Arg Lys Gly Asp Met Ile Val

115 120 125

Phe Pro Gln Gly Leu Ile His Phe Gln Leu Asn Leu Gly Lys Thr Asp

130 135 140

Ala Leu Ala Ile Ala Ser Phe Gly Ser Gln Phe Pro Gly Arg Val Asn

145 150 155 160

Val Ala Asn Gly Val Phe Gly Thr Thr Pro Gln Ile Leu Asp Asp Val

165 170 175

Leu Thr Gln Ala Phe Gln Val Asp Glu Met Val Ile Gln Gln Leu Arg

180 185 190

Ser Gln Phe Ser Gly Gln Asn Ile Ser Ile Asn Thr Gly Arg Ser Ile

195 200 205

Leu Lys Leu Leu Thr Asp Val Ala

210 215

<210> 6

<211> 216

<212> PRT

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Ser Asp Pro Gly Leu Leu Gln Asp Phe Cys Val Gly Val Asn Asp Pro

1 5 10 15

Asp Ser Ala Val Phe Val Asn Gly Lys Phe Cys Lys Asn Pro Lys Asp

20 25 30

Val Thr Ile Asp Asp Phe Leu Tyr Lys Gly Phe Asn Ile Pro Ser Asp

35 40 45

Thr Asn Asn Thr Gln Arg Ala Glu Ala Thr Leu Val Asp Val Asn Arg

50 55 60

Phe Pro Ala Leu Asn Thr Leu Gly Val Ala Met Ala Arg Val Asp Phe

65 70 75 80

Ala Ser Phe Gly Leu Asn Thr Pro His Leu His Pro Arg Gly Ser Glu

85 90 95

Ile Phe Ala Val Leu Glu Gly Thr Leu Tyr Ala Gly Ile Val Thr Thr

100 105 110

Asp Asn Lys Leu Phe Asp Thr Val Leu Arg Lys Gly Asp Met Ile Val

115 120 125

Phe Pro Gln Gly Leu Ile His Phe Gln Leu Asn Leu Gly Lys Thr Asp

130 135 140

Ala Leu Ala Ile Ala Ser Phe Gly Ser Gln Phe Pro Gly Arg Val Asn

145 150 155 160

Val Ala Asn Gly Val Phe Gly Thr Thr Pro Gln Ile Leu Asp Asp Val

165 170 175

Leu Thr Gln Ala Phe Gln Val Asp Lys Met Val Ile Glu Gln Leu Arg

180 185 190

Ser Gln Phe Ser Gly Pro Asn Thr Ser Ile Asn Thr Gly Arg Ser Ile

195 200 205

Leu Lys Leu Leu Thr Asp Val Ala

210 215

<210> 7

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Ser Asp Pro Ala Pro Leu Gln Asp Phe Cys Ile Ala Val Asn Asp Pro

1 5 10 15

Asn Ser Ala Val Leu Val Asn Gly Lys Leu Cys Lys Asn Pro Lys Glu

20 25 30

Val Thr Ile Asp Asp Phe Leu Tyr Lys Gly Phe Asn Ile Pro Ala Asp

35 40 45

Thr Asn Asn Thr Gln Gly Ala Ser Ala Thr Leu Val Asp Ile Thr Leu

50 55 60

Phe Pro Ala Val Asn Thr Gln Gly Val Ser Met Ala Arg Val Asp Phe

65 70 75 80

Ala Pro Phe Gly Leu Asn Thr Pro His Leu His Pro Arg Gly Ser Glu

85 90 95

Val Phe Ala Val Met Glu Gly Ile Met Tyr Ala Gly Phe Val Thr Thr

100 105 110

Asp Tyr Lys Leu Tyr Asp Thr Ile Ile Lys Lys Gly Asp Ile Ile Val

115 120 125

Phe Pro Gln Gly Leu Ile His Phe Gln Leu Asn Leu Gly Lys Thr Asp

130 135 140

Ala Leu Ala Ile Ala Ser Phe Gly Ser Gln Asn Pro Gly Arg Ile Asn

145 150 155 160

Ile Ala Asp Ser Val Phe Gly Thr Thr Pro Arg Val Leu Asp Asp Val

165 170 175

Leu Thr Lys Gly Phe Gln Ile Asp Glu Leu Leu Val Lys Gln Leu Arg

180 185 190

Ser Gln Phe Ser Thr Asp Asn Ile Ser Thr Ser Thr Gly Arg Ser Phe

195 200 205

Leu Lys Leu Leu Ser Glu Thr Tyr

210 215

<210> 8

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<212> PRT

<213> banana peel

<400> 8

Phe Asp Pro Ser Pro Leu Gln Asp Phe Cys Val Ala Asp Tyr Asp Ser

1 5 10 15

Asn Val Phe Val Asn Gly Phe Ala Cys Lys Lys Ala Lys Asp Val Thr

20 25 30

Ala Asp Asp Phe Tyr Phe Thr Gly Leu Asp Lys Pro Ala Ser Thr Ala

35 40 45

Asn Glu Leu Gly Ala Asn Ile Thr Leu Val Asn Val Glu Arg Leu Pro

50 55 60

Gly Leu Asn Ser Leu Gly Val Ala Met Ser Arg Ile Asp Tyr Ala Pro

65 70 75 80

Phe Gly Leu Asn Pro Pro His Ser His Pro Arg Ser Ser Glu Ile Leu

85 90 95

His Val Ala Glu Gly Thr Leu Tyr Ala Gly Phe Val Thr Ser Asn Thr

100 105 110

Glu Asn Gly Asn Leu Leu Phe Ala Lys Lys Leu Lys Lys Gly Asp Ala

115 120 125

Phe Val Phe Pro Arg Gly Leu Ile His Phe Gln Phe Asn Ile Gly Asp

130 135 140

Thr Asp Ala Val Ala Phe Ala Thr Phe Gly Ser Gln Ser Pro Gly Leu

145 150 155 160

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

165 170 175

Tyr Ile Leu Ala Gln Ala Val Gln Leu Ser Lys Thr Thr Val Gly Trp

180 185 190

Leu Gln Gln Gln Gln Trp Leu Asp Ile Ala Gln Glu Tyr Gly Gln Arg

195 200 205

Leu Val Gln Ala Asn

210

<210> 9

<211> 639

<212> DNA

<213> germin-like

<400> 9

atggctcccc tactctacct tgtagtattc ttgcttgctc cttttctctc ccatgctgcg 60

gatcccgatc ctttgctaga tttttgtgta gcggacctta atgcctctcc ctcatttgct 120

aatttccctt gcaaacaaac ctcaaatgtg acctctgaag atttcttctt tgatgggttt 180

atgaatgagg gaaacacatc aaactcgttt ggatcaaggg tcacacccgg aaacgtcctc 240

acatttcctg cccttaatat gctcgggatt tcaatgaatc gggttgatct tgctgtggat 300

gggatgaacc cgccccattc ccacccacga gcaagtgaga gcggtgtggt gatgaagggg 360

agagttctag tagggttcgt aaccacgggg aatgtgtact attcaaaggt gttggttcca 420

ggacagatgt ttgtaatccc aagggggttg gttcattttc aaaagaatgt tggacaaaat 480

aaggcactca tcattacagc tttcaatagt cagaatccag gagtagtgtt attatcctca 540

accctgtttg gtacaaaccc ttcaattcca gatgatgttt taagccaaac tttcctagtg 600

gaccagagca ttgtcgaagg aataaaatcc aacttttga 639

<210> 10

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<213> germin-like

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atggaagtcg tcgcagctgt atcttttctg gccgtgttat tggctctggt ttcccctgcc 60

ctcgccaatg atcctgatat cttttctggc cgtgttattg gctctggttt cccctgccct 120

cgccaatgat cctgatatgc tccaagatgt ttgtgtcgct gattccacct ctggagtgaa 180

attgaatgga tttgcatgca aggatgcagc aagcattaca ccagaagact tcttctttgc 240

tggaatatcc aaacccggaa tgacaaacaa tacaatgaaa tccctagtaa ccggagctaa 300

cgtcgaaaag ataccgggtt taaacacact cggagtgtcc atgggtcgta tcgacttcgg 360

cccaggtggt cttaacccac ctcacactca cccacgagcc acagaaatgg tctttgtgtt 420

atatggagaa ttggacgttg gtttcctaac tacttctaat aagctcattt ctaagcatat 480

taaaactggt gaaacttttg tttttcctag agggttagtc cactttcaga aaaataatgg 540

ggataaacct gctgctttag tcactgcttt taatagtcag ttgcctggca cccaatcaat 600

agctgccacg ttgtttacgt cgaccccacc tgttccagat aatgttttaa ctatgacttt 660

ccaagtcggt actaaacaag tccagaagat caaggctagg ctcgctccta agaagtaa 718

<210> 11

<211> 669

<212> DNA

<213> germin-like

<400> 11

atggcggctg tttgggtagt cttggtggtg ctagcggcgg cttttgctgt tggggtcttt 60

gccagcgatc ctgatatgct tcaagatgtt tgtgttgctg atcgtacatc tggaatatta 120

gtgaatggat tcacatgtaa aaatatgacc atgataaccc ctgaagactt cttcttcacc 180

ggaatttcac aaccaggcca aatcacaaat aaaatccttg gttctcgagt caccggagcg 240

aatgtgcagg acatccctgg tctcaacacc ttgggagtct cgatggctcg tgtcgacttt 300

actccctacg gtctaaaccc acctcacatt caccctagaa tcgtccaccc tcgtgccact 360

gaaatgatct atgttcttaa gggtgaattg tacgttggtt ttataacgac cgacaataag 420

ctcatttcca aggttgttaa agctggagaa gtatttgttt tccctagagg tttggctcac 480

tttcagaaaa acatgttgaa atatccagct gctgcattag ctgccttcaa cagccaactt 540

ccaggcactc aacaatttgc agctgctctc tttacttcca atcctcctgt gtctaatgat 600

gtgttggctc aggcttttaa cattgacgaa cacaatgtca aaaagattag ggctggcctt 660

actccatag 669

<210> 12

<211> 660

<212> DNA

<213> germin-like

<400> 12

atggagtcgc actacacgaa gagaccattc ctcctctttc tctccttcac cgtcctcctc 60

gtgttgatcc gcgctgaccc tgatcctctc caggacttct gcgtcgccga cctcggagct 120

actgtggtcg tcaatgggtt cccgtgcaag cccgcgtccg gagtcacgtc cgacgacttc 180

ttcttcgccg gactgtccag ggagggcaac accagcaata tcttcgggtc caacgtgacc 240

aacgccaaca tgctcagctt cccggggctc aacaccctcg gcgtctccat gaaccgcgtc 300

gacgtcgccc ccggcggcac caacccgccc cacagccacc cgagggctac cgagctcatc 360

atcctcctca agggccggct gctggtgggg ttcatcagca ccagtaacca gttcttctcc 420

aaggtcttga accccggcga gatgttcgtg gtgcccaagg ggctcatcca cttccagtac 480

aacgtcggca aggagaaggc gctcgccatc accaccttcg acagccagct ccccggagta 540

gtgatcgcct ccaccaccct gttcgcatcg aatccggcga ttcccgacga tgtgctggcc 600

aaagcttttc aggtggacgc gaaggtcgtc gctctcatca agtccaagtt tgagagataa 660

<210> 13

<211> 212

<212> PRT

<213> germin-like

<400> 13

Met Ala Pro Leu Leu Tyr Leu Val Val Phe Leu Leu Ala Pro Phe Leu

1 5 10 15

Ser His Ala Ala Asp Pro Asp Pro Leu Leu Asp Phe Cys Val Ala Asp

20 25 30

Leu Asn Ala Ser Pro Ser Phe Ala Asn Phe Pro Cys Lys Gln Thr Ser

35 40 45

Asn Val Thr Ser Glu Asp Phe Phe Phe Asp Gly Phe Met Asn Glu Gly

50 55 60

Asn Thr Ser Asn Ser Phe Gly Ser Arg Val Thr Pro Gly Asn Val Leu

65 70 75 80

Thr Phe Pro Ala Leu Asn Met Leu Gly Ile Ser Met Asn Arg Val Asp

85 90 95

Leu Ala Val Asp Gly Met Asn Pro Pro His Ser His Pro Arg Ala Ser

100 105 110

Glu Ser Gly Val Val Met Lys Gly Arg Val Leu Val Gly Phe Val Thr

115 120 125

Thr Gly Asn Val Tyr Tyr Ser Lys Val Leu Val Pro Gly Gln Met Phe

130 135 140

Val Ile Pro Arg Gly Leu Val His Phe Gln Lys Asn Val Gly Gln Asn

145 150 155 160

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

165 170 175

Leu Leu Ser Ser Thr Leu Phe Gly Thr Asn Pro Ser Ile Pro Asp Asp

180 185 190

Val Leu Ser Gln Thr Phe Leu Val Asp Gln Ser Ile Val Glu Gly Ile

195 200 205

Lys Ser Asn Phe

210

<210> 14

<211> 219

<212> PRT

<213> germin-like

<400> 14

Met Glu Val Val Ala Ala Val Ser Phe Leu Ala Val Leu Leu Ala Leu

1 5 10 15

Val Ser Pro Ala Leu Ala Asn Asp Pro Asp Met Leu Gln Asp Val Cys

20 25 30

Val Ala Asp Ser Thr Ser Gly Val Lys Leu Asn Gly Phe Ala Cys Lys

35 40 45

Asp Ala Ala Ser Ile Thr Pro Glu Asp Phe Phe Phe Ala Gly Ile Ser

50 55 60

Lys Pro Gly Met Thr Asn Asn Thr Met Lys Ser Leu Val Thr Gly Ala

65 70 75 80

Asn Val Glu Lys Ile Pro Gly Leu Asn Thr Leu Gly Val Ser Met Gly

85 90 95

Arg Ile Asp Phe Gly Pro Gly Gly Leu Asn Pro Pro His Thr His Pro

100 105 110

Arg Ala Thr Glu Met Val Phe Val Leu Tyr Gly Glu Leu Asp Val Gly

115 120 125

Phe Leu Thr Thr Ser Asn Lys Leu Ile Ser Lys His Ile Lys Thr Gly

130 135 140

Glu Thr Phe Val Phe Pro Arg Gly Leu Val His Phe Gln Lys Asn Asn

145 150 155 160

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

165 170 175

Gly Thr Gln Ser Ile Ala Ala Thr Leu Phe Thr Ser Thr Pro Pro Val

180 185 190

Pro Asp Asn Val Leu Thr Met Thr Phe Gln Val Gly Thr Lys Gln Val

195 200 205

Gln Lys Ile Lys Ala Arg Leu Ala Pro Lys Lys

210 215

<210> 15

<211> 217

<212> PRT

<213> germin-like

<400> 15

Met Ala Ala Val Trp Val Val Leu Val Val Leu Ala Ala Ala Phe Ala

1 5 10 15

Val Gly Val Phe Ala Ser Asp Pro Asp Met Leu Gln Asp Val Cys Val

20 25 30

Ala Asp Arg Thr Ser Gly Ile Leu Val Asn Gly Phe Thr Cys Lys Asn

35 40 45

Met Thr Met Ile Thr Pro Glu Asp Phe Phe Phe Thr Gly Ile Ser Gln

50 55 60

Pro Gly Gln Ile Thr Asn Lys Ile Leu Gly Ser Arg Val Thr Gly Ala

65 70 75 80

Asn Val Gln Asp Ile Pro Gly Leu Asn Thr Leu Gly Val Ser Met Ala

85 90 95

Arg Val Asp Phe Thr Pro Tyr Gly Leu Asn Pro Pro His Ile His Pro

100 105 110

Arg Ala Thr Glu Met Ile Tyr Val Leu Lys Gly Glu Leu Tyr Val Gly

115 120 125

Phe Ile Thr Thr Asp Asn Lys Leu Ile Ser Lys Val Val Lys Ala Gly

130 135 140

Glu Val Phe Val Phe Pro Arg Gly Leu Ala His Phe Gln Lys Asn Met

145 150 155 160

Leu Lys Tyr Pro Ala Ala Ala Leu Ala Ala Phe Asn Ser Gln Leu Pro

165 170 175

Gly Thr Gln Gln Phe Ala Ala Ala Leu Phe Thr Ser Asn Pro Pro Val

180 185 190

Ser Asn Asp Val Leu Ala Gln Ala Phe Asn Ile Asp Glu His Asn Val

195 200 205

Lys Lys Ile Arg Ala Gly Leu Thr Pro

210 215

<210> 16

<211> 219

<212> PRT

<213> germin-like

<400> 16

Met Glu Ser His Tyr Thr Lys Arg Pro Phe Leu Leu Phe Leu Ser Phe

1 5 10 15

Thr Val Leu Leu Val Leu Ile Arg Ala Asp Pro Asp Pro Leu Gln Asp

20 25 30

Phe Cys Val Ala Asp Leu Gly Ala Thr Val Val Val Asn Gly Phe Pro

35 40 45

Cys Lys Pro Ala Ser Gly Val Thr Ser Asp Asp Phe Phe Phe Ala Gly

50 55 60

Leu Ser Arg Glu Gly Asn Thr Ser Asn Ile Phe Gly Ser Asn Val Thr

65 70 75 80

Asn Ala Asn Met Leu Ser Phe Pro Gly Leu Asn Thr Leu Gly Val Ser

85 90 95

Met Asn Arg Val Asp Val Ala Pro Gly Gly Thr Asn Pro Pro His Ser

100 105 110

His Pro Arg Ala Thr Glu Leu Ile Ile Leu Leu Lys Gly Arg Leu Leu

115 120 125

Val Gly Phe Ile Ser Thr Ser Asn Gln Phe Phe Ser Lys Val Leu Asn

130 135 140

Pro Gly Glu Met Phe Val Val Pro Lys Gly Leu Ile His Phe Gln Tyr

145 150 155 160

Asn Val Gly Lys Glu Lys Ala Leu Ala Ile Thr Thr Phe Asp Ser Gln

165 170 175

Leu Pro Gly Val Val Ile Ala Ser Thr Thr Leu Phe Ala Ser Asn Pro

180 185 190

Ala Ile Pro Asp Asp Val Leu Ala Lys Ala Phe Gln Val Asp Ala Lys

195 200 205

Val Val Ala Leu Ile Lys Ser Lys Phe Glu Arg

210 215

<210> 17

<211> 25

<212> DNA

<213> Artificial

<223> B5100/5102F

<400> 17

atgtctgatc ctggtctcct acagg 25

<210> 18

<211> 24

<212> DNA

<213> Artificial

<223> B5100/5102R

<400> 18

agcaacatca gttaagagtt taag 24

<210> 19

<211> 25

<212> DNA

<213> Artificial

<223> B5601F

<400> 19

atgtccgatc ctgcacccct tcaag 25

<210> 20

<211> 20

<212> DNA

<213> Artificial

<223> B5601R

<400> 20

tcaataagtt tcagatagca 20

<210> 21

<211> 25

<212> DNA

<213> Artificial

<223> M30640F

<400> 21

atgtttgatc cgagtcctct ccaag 25

<210> 22

<211> 25

<212> DNA

<213> Artificial

<223> M30640R

<400> 22

tcaattagct tgaactaagc gttgt 25

<210> 23

<211> 36

<212> DNA

<213> Artificial

<223>Bichi yeast system B5102F

<400> 23

ccgctcgaga aaagatctga tcctggtctc ctacag 36

<210> 24

<211> 37

<212> DNA

<213> Artificial

<223>Bichi yeast system B5102R

<400> 24

aaatatgcgg ccgctcaagc aacatcagtt aagagtt 37

<210> 25

<211> 33

<212> DNA

<213> Artificial

<223>Bichi yeast system B5601F

<400> 25

ccgctcgaga aaagatccga tcctgcaccc ctt 33

<210> 26

<211> 39

<212> DNA

<213> Artificial

<223>Bichi yeast system B5601R

<400> 26

aaatatgcgg ccgctcaata agtttcagat agcaatttc 39

<210> 27

<211> 35

<212> DNA

<213> Artificial

<223>Bichi yeast system M30640f

<400> 27

ccgctcgaga aaagatttga tccgagtcct ctcca 35

<210> 28

<211> 38

<212> DNA

<213> Artificial

<223>Bichi yeast system M30640R

<400> 28

aaatatgcgg ccgctcaatt agcttgaact aagcgttg 38

<210> 29

<211> 33

<212> DNA

<213> Artificial

<223>plant expression system 5102-F

<400> 29

gctctagaat gtctgatcct ggtctcctac agg 33

<210> 30

<211> 34

<212> DNA

<213> Artificial

<223>plant expression system 5102-R

<400> 30

cggggtacct caagcaacat cagttaagag ttta 34

<210> 31

<211> 34

<212> DNA

<213> Artificial

<223>plant expression system 5601-R

<400> 31

gctctagaat gtccgatcct gcaccccttc aaga 34

<210> 32

<211> 34

<212> DNA

<213> Artificial

<223>plant expression system 5601-R

<400> 32

cggggtacct caataagttt cagatagcaa tttc 34

<210> 33

<211> 33

<212> DNA

<213> Artificial

<223>plant expression system 30640-F

<400> 33

gctctagaat gtttgatccg agtcctctcc aag 33

<210> 34

<211> 34

<212> DNA

<213> Artificial

<223>plant expression system 30640-R

<400> 34

cggggtacct caattagctt gaactaagcg ttgt 34

Claims

1. a kind of oxalate oxidase, can degrade oxalic acid or oxalates under physiological ph conditions, it is characterised in that: its gene order As shown in SEQ ID NO. 4.

2. oxalate oxidase described in claim 1, it is characterised in that: be the polyethyleneglycol modified oxalic acid oxygen of different molecular weight Change enzyme.

3. oxalate oxidase according to claim 2, it is characterised in that: the molecular weight of the polyethylene glycol is 2kD, Any one in 5kD, 10kD, 20kD, 4 arm -20kD, 30kD, 40kD or 4 arm -40kD.

4. a kind of expression vector includes the gene order in claim 1.

5. a kind of recombinant strains include expression vector as claimed in claim 4, can produce grass described in claim 1 Acid oxidase.

The oxalic acid of oxalic acid or oxalates 6. expression vector as claimed in claim 4 can degrade under the conditions of physiological pH is expressed in building Application in oxidizing ferment genetically modified plants.

7. oxalate oxidase described in any one of claim 1-3 is dense in preparation reduction oxalosis disease patient oxalic acid Application in the drug of degree.

8. application as claimed in claim 7, it is characterised in that: the oxalosis disease is primary hyperoxaluria, Two type hyperoxalurias, zellweger disease or chronic urinary system damageability disease.

9. a kind of pharmaceutical composition comprising recombinating oxalate oxidase described in claim 1-3 any one.

10. pharmaceutical composition as claimed in claim 9, it is characterised in that: injection is made or is embedded through enteric material.