CN1831132A - Method for expression of Aspergillus flavus urate oxidase and the special gene thereof - Google Patents
Method for expression of Aspergillus flavus urate oxidase and the special gene thereof Download PDFInfo
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Abstract
The invention discloses a method and special expression gene to express aspergillus flavus urate oxidase that contains one sequence from DNA sequence of SEQ ID No:3 in sequence table, and DNA sequence cross breeding nucleotide sequence of DNA sequence restricted by SEQ ID No:3 in sequence table. The invention has high expression level and could reach to 40% of solubility full mycoprotein, and is short cycle and low cost.
Description
Technical field
The present invention relates to a kind of method and special-purpose gene thereof of expressing Aspergillus flavus uricoxidase.
Background technology
Urico-oxidase (EC1.7.3.3, Uricase, Urate Oxidase, Uox) be a kind of enzyme in the purine degradation pathways metabolism in the organism, the oxidation of catalysis uric acid generates wallantoin, and wallantoin is that a kind of ratio is easier to the excretory metabolite, and its solubleness is 5～10 times of uric acid.Endogenous Uox is all arranged in most of mammalian bodies, but (ape and the mankind) lack this kind of enzyme and excrete with urine as end product with uric acid in the higher mammal body.
Uric acid in normal human's circulation of blood more than 99% exists with uric acid sodium salt (abbreviation urate) form, and serum uric acid salt fluctuates in narrower scope, according to domestic data, and male sex's average out to 5.7mg/dl, women 4.3mg/dl.If the concentration of uric acid is higher than normal value in the serum, just cause hyperuricemia.The propagation of malignant tumour causes nucleic acid katabolism to be strengthened, and purine metabolism produces a large amount of uric acid, and hyperuricemia appears in the patient.The rapid rising of serum uric acid is followed in a large amount of cracking of cell in the chemotherapy of tumors process, and patient a series of Physiology and biochemistries occur and changes, and comprises electrolyte disturbance, renal failure, spasm, arrhythmia, urinary stone disease, be called clinically tumor lysis syndrome (tumor lysissyndrome, TLS).
The gouty acute arthritis that hyperuricemia can cause is shown effect repeatedly, uratoma deposition, uratoma chronic arthritis and joint deformity, often involves kidney and causes that chronic interstitial nephritis and uric acid urinary stone disease form.This disease can be divided primary and Secondary cases two big classes, and primary person's cause of disease owing to enzyme defect causes, do not illustrate mostly, is often accompanied hyperlipidaemia, obesity, diabetes, essential hypertension, arteriosclerosis and coronary heart disease etc. except that minority, belongs to heredopathia.Secondary cases person can be caused by multiple reasons such as nephropathy, hemopathy and medicines.Have data to show the crowd of China more than 20 years old, the sickness rate of hyperuricemia is 2.4-5.7%, if be not careful in one's diet control and treatment, has 10% meeting to develop into gout approximately, infers that the sickness rate of the gout of China is 0.5%.
Suppress the uric acid synthetic drugs to having only Zyloric (allopurinol) so far, this medicine can suppress XOD, make xanthoglobulin and xanthine can not be converted into uric acid, the retardance uric acid forms, but can increase the load of renal excretion uric acid precursor (xanthoglobulin and xanthine).Different with xanthoglobulin, xanthine in urine than uric acid indissoluble.The patient of allopurinol treatment sometimes also xanthine ephrosis and calculus can occur.In addition, for the drainage of the uric acid that retains in the patient body, use Zyloric to fail to respond to any medical treatment.What uricosuric was at present commonly used has following three kinds: probenecid, sulfinpyrazone and benzbromarone, side effect comprise intestines and stomach reaction, renal colic and excite acute arthritis outbreak etc., because uricogenesis and discharge and morely easily increase the weight of the kidney burden and do not get.
Above data if the concentration of uric acid is higher than normal value in the serum, inevitably can cause the damage of joint and renal function as can be seen.With the hyperuricemia patient of the damage of renal function, suppress uric acid synthetic medicine Zyloric and also be to use uricosuric probenecid, sulfinpyrazone and benzbromarone no matter be to use, the side effect that their produce should be paid attention to.Recombined Aspergillus flavus Uox provides good selection for the hyperuricemia for the treatment of with renal dysfunction.
The non-reorganization Uox that France Sanofi-Synthelabo company produces is got by flavus nutrient solution purifying, and treatment hyperuricemia curative effect is remarkable than Zyloric.Yet, the non-recombinant product generation acute allergic reaction that extracts from flavus is (as bronchospasm, hypoxemia) person is about 5%, comprises the patient who does not in the past have allergies or suffers from methemoglobinemia and the hemolytic anemia patient of glucose-6-phosphate dehydrogenase shortage.The exploitation of France Sanofi-Synthelabo company, June calendar year 2001, the rasburicase (rasburicase) in Germany and Britain's Initial Public Offering was the recombined Aspergillus flavus Uox that the brewing yeast matrix reaches, can be used for treating and prevent to have the hematologic malignancies patient's of high-risk tumor lysis syndrome acute hyperuricemia, be particularly useful for the hyperuricemia patient that chemotherapy causes.After acute attack gout outbreak patient gave the Uox treatment, acute attack capable of blocking also reduced tophaceous volume.And former chemotherapeutics no matter be to suppress uric acid uric acid resisting medicine synthetic or the promotion uric acid excretion the suspicion that causes acute gouty arthritis is arranged, generally is not used in the treatment of gout acute attack.
The external flavus Uox cycle that reaches with the brewing yeast matrix is long, and price is costliness.The domestic investigator who has expresses the recombined Aspergillus flavus Uox merged the His label with prokaryotic expression system, although can remove its N end of His label for non-natural by proteolytic enzyme, remaining 4 amino-acid residues have increased the cost and the technology of purifying simultaneously.
Summary of the invention
The purpose of this invention is to provide a kind of method and special-purpose gene thereof of expressing Aspergillus flavus uricoxidase.
Aspergillus flavus uricoxidase gene provided by the present invention, name is called rUox, is the Aspergillus flavus uricoxidase gene of optimizing, and has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 3 dna sequence dna;
2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 3 dna sequence dnas hybridization that limit.
The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
The expression vector, engineering bacteria and the clone that contain said gene also belong to protection scope of the present invention.
The method of expression Aspergillus flavus uricoxidase provided by the present invention is that above-mentioned rUox is inserted procaryotic cell expression carrier, transforms the host bacterium, and screening obtains expressing the engineering bacteria of Aspergillus flavus uricoxidase, and the culturing engineering bacterium expresses obtaining Aspergillus flavus uricoxidase.
Described procaryotic cell expression carrier can be the pET-serial carrier.
Described rUox is inserted between the restriction enzyme site of the Nde I of pET-serial carrier and Sac I, or between the restriction enzyme site of Nde I and HindIII.
Described engineering bacteria can be intestinal bacteria, as e. coli bl21 (DE3).
In the aforesaid method, the described abduction delivering that is expressed as, used inductor are IPTG, and induced concentration 0.4-1.5mmol/L is preferably 1.0mmol/L.
The culture temperature of described engineering bacteria is 37 ℃, and the culture temperature of abduction delivering is 28-37 ℃, is preferably 37 ℃.
The step that also comprises the Aspergillus flavus uricoxidase that the purifying expression obtains in the aforesaid method; Described purifying comprises ion-exchange, hydrophobic chromatography and gel permeation chromatography.
The manually synthetic complete genome sequence of optimizing of the present invention adopts coli expression system, the expression level height, and the content of Aspergillus flavus uricoxidase reaches 40% of solubility whole bacterial protein, and the cycle is short, and cost is low.The method of express recombinant urico-oxidase of the present invention adopts the non-fusion expression strategy, the urico-oxidase that expression is obtained has natural N terminal sequence, not containing GST, 6 * His and Trx (Trx) etc. is used to increase solubleness, improves expression amount or makes things convenient for the sequence of purifying, avoid N end alpha-non-natural amino acid residue to exert an influence, and simplified purifying process active and immunogenicity.The reorganization urico-oxidase that the inventive method is expressed is complete soluble form in the buffer system of routine, help purifying, through ion-exchange series of strata, hydrophobic chromatography and gel permeation chromatography purifying, can reach 99% purity, determination of activity shows that the specific activity of the recombined Aspergillus flavus rUox that the present invention expresses reaches the 34IU/mg zymoprotein.
Description of drawings
Fig. 1 cuts the evaluation collection of illustrative plates for PCR and the enzyme of expression plasmid pET-rUox
Fig. 2 is that the SDS-PAGE of expressing protein-urico-oxidase analyzes
Fig. 3 is that the SDS-PAGE of the urico-oxidase behind the Phenyl-Sepharose FF chromatography purification analyzes
Fig. 4 is that the SDS-PAGE of the urico-oxidase behind Phenyl-Sepharose FF, DEAE-Sepharose FF and the Phenyl-Sepharose FF chromatography purification analyzes
Fig. 5 is that the SDS-PAGE of the urico-oxidase behind Phenyl-Sepharose FF, DEAE-Sepharose FF, Phenyl-Sepharose FF and HiloadTM 26/60 Superdex 75 chromatography purifications analyzes
Fig. 6 is a urico-oxidase determination of activity canonical plotting
Embodiment
Method among the embodiment is ordinary method if no special instructions.
The expression of the synthetic and urico-oxidase of the flavus UOX gene of embodiment 1, optimization
DNA restriction enzyme, T4 dna ligase, Pyrobest archaeal dna polymerase and pMD-T carrier are available from TaKaRa company.
The Omega test kit is adopted in the extraction of E.coli plasmid; The working method reference molecule of plasmid construction is cloned a book.
One, the structure of the synthetic and expression vector of rUox gene
The flavus UOX gene order of delivering with reference to GenBank (sequence number: X61766) and document, that analyzes existence may influence the complicated secondary structure that protein is translated in intestinal bacteria, this gene is optimized, guaranteeing to carry out rite-directed mutagenesis under the constant prerequisite of aminoacid sequence, remove complicated secondary structure, gene order after the sudden change is synthetic by Bo Ya biotech firm, and be cloned into the pMD-T carrier and carry out sequencing, determined dna sequence conclusive evidence synthetic gene is correct, the flavus Uox gene-rUox that is optimized, this rUox gene has the nucleotide sequence of sequence 1 in the preface sequence table, classifies encoding sequence (sequence 3 in the sequence table) as from the 7th-915 nucleotides sequences of 5 ' end.
RUox gene that determined dna sequence is correct with Nde I and Sac I from the pMD-T carrier under the double digestion, and gel reclaims purifying, the rUox gene of purifying is connected with the fragment of cutting the 5400bp that pET-32a (+) (Novagen) obtains through Nde I and Sac I enzyme, transformed into escherichia coli BL21 (DE3), on the LB flat board that contains penbritin (100 μ g/mL), cultivate, select positive colony special primer (upstream primer: 5-acc
CatatgTccgcagtaaaagcagcc-3 and downstream primer: 5-ggg
GagctcTtacaatttagacttcag3, line portion are respectively the restriction enzyme sites of Nde I and Sac I) carry out the PCR evaluation, screening obtains positive colony (band of swimming lane 1,1 treaty 900bp among Fig. 1).The PCR positive colony is extracted plasmid further carry out double digestion and evaluation, obtain 5400 and the band (swimming lane 2 among Fig. 1) of 900bp, the plasmid structure plasmid called after pET-rUox correct, that this structure is correct of structure is described with Nde I/Sac I; The PCR of pET-rUox and enzyme are cut qualification result as shown in Figure 1, and swimming lane M is dna molecular amount standard (DL-15 000) among Fig. 1; Swimming lane 1 is the PCR electrophoresis result of pET-rUox; Swimming lane 2 is the electrophoresis result of the product cutting the pET-rUox plasmid with Nde I and Sac I enzyme and obtain.
Two, the abduction delivering of recombined Aspergillus flavus Uox
With pET-rUox transformed into escherichia coli BL21 (DE3), on LB substratum (Pyocianil 100 μ g/ml) flat board, to cultivate, the single colony inoculation of picking is (Pyocianil 100 μ g/ml) in containing antibiotic LB substratum, cultivate 10 hours for 37 ℃; By 1: 100 volume ratio enlarged culturing, 37 ℃ are cultured to the nectar degree was A
600=0.8 o'clock, be divided into two parts, a add IPTG to final concentration be 1mmol/L, another part does not add IPTG, two parts are all continued to cultivate 5h at 37 ℃, the centrifugal collection thalline of 14000g, according to every gram thalline resuspended thalline of the ultrasonic damping fluid of 10ml, at ultrasonic (power 200w on ice, ultrasonic time 5s, 5s intermittently, ultrasonic total time is 8min), suspension after ultrasonic is at the centrifugal 20min of 14000rpm, precipitation is used with the ultrasonic damping fluid of the same volume of centrifugal back supernatant resuspended, and the centrifugal cleer and peaceful resuspended precipitation after ultrasonic is mixed with isopyknic 2 * SDS sample loading buffer respectively, boils 5min, behind the centrifugal 5min of 10000rpm, get 20 μ l respectively and carry out SDS-PAGE then and analyze, the SDS-PAGE analytical results as shown in Figure 2, the result shows, on the position of the about 34kD of molecular mass (arrow shows), the obvious expression band is arranged, conform to theory expectation; Expression amount through TotalLab software analysis target protein accounts for 40% of solubility whole bacterial protein, and the expression amount of urico-oxidase is 1090IU/g thalline (the same step 3 of the measuring method of enzymic activity).Wherein among Fig. 2, M is an albumen lower molecular weight standard, and swimming lane 1 is BL21 (DE3)/pET-rUox (changeing the e. coli bl21 (DE3) of pET-rUox), does not induce full bacterium; Swimming lane 2 is BL21 (DE3)/pET-uox, and IPTG induces full bacterium, ultrasonic supernatant; Swimming lane 3 is BL21 (DE3)/pET-uox, and IPTG induces, ultrasound precipitation.
Three, a large amount of abduction deliverings and the purifying of recombined Aspergillus flavus Uox
Inoculate single bacterium colony in 10ml LB substratum (containing Pyocianil 100 μ g/ml), cultivate 10h for 37 ℃.By the fresh LB substratum (containing Pyocianil 100 μ g/ml) of 1: 100 volume ratio inoculation, 37 ℃, 250rpm are cultivated 8h, are seeded to by 1: 50 volume ratio in the fermentor tank of 30L and are cultured to bacterium liquid A
600=1.4-1.6 adds IPTG to final concentration 1mmol/L, continues to cultivate 5h at 37 ℃, centrifugal collection thalline, and-20 ℃ are frozen.Frozen bacterium room temperature is melted, carries out following steps and handle:
1) the broken thalline of high-pressure homogenization: use 20mM, the resuspended thalline of PB damping fluid (20ml/g wet thallus) of pH 8.0, with high pressure homogenizer fragmentation 1 time under 900bar pressure, then at 4 ℃, the centrifugal 20min of 12000rpm (22000g), it is standby to get clarifying supernatant liquor.
2) hydrophobic chromatography of clarified supernatant: use 1M, (the NH of pH8.0
4)
2SO
4The balance chromatography column; With clarifying supernatant liquor and 3M, (the NH of pH8.0
4)
2SO
4, mix last sample according to 2: 1 volume ratios; 1M, (the NH of pH8.0
4)
2SO
4Wash non-specific combination albumen, last 20mM, the PB buffer solution for gradient elution sample of pH 8.0.Sample behind this wash-out is carried out SDS-PAGE analyzes, the result as shown in Figure 3, the arrow indication is the locational obvious expression band of each swimming lane at 34kD among Fig. 3.
3) desalting treatment: after the sample that step 2) obtains carries out SDS-PAGE, choose the good sample liquids of purity, use molecular weight cut-off 10kD, the ultrafiltration pipe of ultrafiltration volume 15ml carries out desalination, then the centrifugal 10-15min of 2000g (concentrating 1 times); Add 20mM, the PB damping fluid of pH 8.0 is to original volume ultrafiltration 2 times again.
4) ion exchange chromatography: the sample solution that obtains after the step 3) ultrafiltration desalination is diluted according to 1: 3 volume ratio with deionized water, electricity is led less than 2.5mS/cm, pH 8.0-8.4, last DEAE-Sepharose FF chromatography column, it is standby that stream is worn liquid, wherein level pad is 10mM, the PB damping fluid of pH 8.0-8.4.
5) stream is worn the hydrophobic chromatography of liquid: the stream that step 4) obtains is worn liquid and 3mol/L, (the NH of pH8.0
1)
2SO
4Mix according to 2: 1 volume ratios, cross Phenyl-Sepharose FF chromatography column; Use 1M, (the NH of pH8.0
4)
2SO
4, wash non-specific combinating substance; Use 20mM then, the PB buffer solution for gradient elution sample of pH 8.0 obtains the sample behind the purifying, with the sample filtering degerming.Sample behind this purifying is carried out SDS-PAGE analyzes, the result as shown in Figure 4, the arrow indication is the locational obvious expression band of each swimming lane at 34kD among Fig. 4.
6) gel permeation chromatography: used chromatography column is Hiload
TM26/60 Superdex 75 (Phamacia), 20mM, the PB damping fluid of pH 7.2 (NaCl that contains 0.15M) balance chromatography column, 0.5M NaOH washing pillar, 20mM, the PB damping fluid of pH 7.2 (NaCl that contains 0.15M) balance chromatography column, last sample (at every turn going up sample 12ml), collect the target protein peak, obtain purity greater than 99% urico-oxidase.Sample behind the purifying carries out preserving after the filtration sterilization.This urico-oxidase that obtains purifying is carried out SDS-PAGE analyzes, the result as shown in Figure 5, the arrow indication is the locational obvious expression band of each swimming lane at 34kD among Fig. 5.
Three, the external activity analysis of urico-oxidase (rUox)
Enzyme activity assay adopts reported method (J Biol Chem, 1992,267 (12): 8565-8570) such as Legoux.Per minute is oxidized to 1 international unit (IU) that the required enzyme amount of wallantoin is an enzymic activity with 1 μ mol uric acid.The specific activity of enzyme (IU/mg) is every milligram of unit of enzyme activity's number that albumen is contained.At first measure the absorbance (A of different uric acid concentrations under 292nm
292), the production standard curve, concrete grammar is as follows: according to the volume numerical value of 0-6 in the table 1 with 0.2 μ mol/ml uric acid stock solution and trolamine damping fluid (TEA buffer, 7.5g/L trolamine, 0.38/LEDTA, pH 8.9) mix, 30 ℃ of insulation 5min add stop buffer, the detection wavelength is 292nm, measure the absorbance value of each uric acid, each concentration repeats 3 times, averages.Utilize the numerical evaluation slope k of 0-6, formula y=kx, typical curve as shown in Figure 6, the result shows K=5.568/2.1=2.651.
Step 3 is expressed and the urico-oxidase of purification carries out active mensuration: in the reaction system of 5ml, add 3ml trolamine damping fluid (TEA buffer respectively, 7.5g/L trolamine, 0.38g/L EDTA, pH8.9), 1.5ml 0.2 μ mol/ml uric acid stock solution, the urico-oxidase of 1 μ g (10 μ l) purifying, 30 ℃ of insulation 5min, the KOH stop buffer reaction that adds 0.5ml 20% then finishes, and measures uric acid concentration, the detection wavelength is 292nm, the result carries out enzymic activity calculating, the activity of enzyme=[(0.3-OD according to numerical value in the table and the K value that obtains of aforementioned calculation shown in table 1 (sample)
292/ k)/5] * 1000; The result shows, the specific activity of enzyme=[(0.3-0.339/2.651)/5] * 1000=34IU/mg zymoprotein.
Table 1. urico-oxidase enzyme assay result
| Numbering |
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | Sample | |
| Uric acid stock solution (ml) | 0 | 0.5 | 1 | 1.5 | 2.0 | 2.5 | 3.0 | 1.5 |
| Trolamine damping fluid (ml) | 5 | 4.5 | 4 | 3 | 2.5 | 2 | 1.5 | 3 |
| Reaction conditions | Mix 30 ℃ of reaction | |||||||
| Stop buffer | ||||||||
| 20% KOH, 0.5ml | ||||||||
| OD 292-1 | 0 | 0.331 | 0.556 | 0.764 | 1.025 | 1.257 | 1.568 | 0.339 |
| OD 292-2 | 0 | 0.373 | 0.537 | 0.785 | 1.011 | 1.323 | 1.584 | 0.338 |
| OD 292-3 | 0 | 0.327 | 0.575 | 0.793 | 1.060 | 1.342 | 1.490 | 0.339 |
| OD 292Average | 0 | 0.344 | 0.556 | 0.781 | 1.032 | 1.308 | 1.547 | 0.339 |
Four, the N terminal amino acid sequence of rUox is measured
Get 10 μ g samples and reduce the SDS-PAGE electrophoresis, electrophoretic band is transferred on the pvdf membrane with the CAPS damping fluid, send consonance medical university N terminal amino acid sequence that fundamental research is carried out to measure, the sequencing result is SAVKAARYG KDNVR, and is consistent with natural aminoacid sequence (sequence 2 in the sequence table).
Sequence table
<160>3
<210>1
<211>924
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
acccatatgt ccgcagtaaa agcagcccgc tacggcaagg acaatgtccg cgtctacaag 60
gttcacaagg acgagaagac cggtgtccag acggtgtacg agatgaccgt ctgtgtgctt 120
ctggagggtg agattgagac ctcttacacc aaggccgaca acagcgtcat tgtcgcaacc 180
gactccatta agaacaccat ttacatcacc gccaagcaga accccgttac tcctcccgag 240
ctgttcggct ccatcctggg cacacacttc attgagaagt acaaccacat ccatgccgct 300
cacgtcaaca ttgtctgcca ccgctggacc cggatggaca ttgacggcaa gccacaccct 360
cactccttca tccgcgacag cgaggagaag cggaatgtgc aggtggacgt ggtcgagggc 420
aagggcatcg atatcaagtc gtctctgtcc ggcctgaccg tgctgaagag caccaactcg 480
cagttctggg gcttcctgcg tgacgagtac accacactta aggagacctg ggaccgtatc 540
ctgagcaccg acgtcgatgc cacttggcag tggaagaatt tcagtggact ccaggaggtc 600
cgctcgcacg tgcctaagtt cgatgctacc tgggccactg ctcgcgaggt cactctgaag 660
acttttgctg aagataacag tgccagcgtg caggccacta tgtacaagat ggcagagcaa 720
atcctagcgc gccagcagct gatcgagact gtcgagtact cgttgcctaa caagcactat 780
ttcgaaatcg acctgagctg gcacaagggc ctccaaaaca ccggcaagaa cgccgaggtc 840
ttcgctcctc agtcggaccc caacggtctg atcaagtgta ccgtcggccg gtcctctctg 900
aagtctaaat tgtaagagct cccc 924
<210>2
<211>301
<212>PRT
＜213〉flavus (Aspergillus flavus)
<400>2
Ser Ala Val Lys Ala Ala Arg Tyr Gly Lys Asp Asn Val Arg Val Tyr
1 5 10 15
Lys Val His Lys Asp Glu Lys Thr Gly Val Gln Thr Val Tyr Glu Met
20 25 30
Thr Val Cys Val Leu Leu Glu Gly Glu Ile Glu Thr Ser Tyr Thr Lys
35 40 45
Ala Asp Asn Ser Val Ile Val Ala Thr Asp Ser Ile Lys Asn Thr Ile
50 55 60
Tyr Ile Thr Ala Lys Gln Asn Pro Val Thr Pro Pro Glu Leu Phe Gly
65 70 75 80
Ser Ile Leu Gly Thr His Phe Ile Glu Lys Tyr Asn His Ile His Ala
85 90 95
Ala His Val Asn Ile Val Cys His Arg Trp Thr Arg Met Asp Ile Asp
100 105 110
Gly Lys Pro His Pro His Ser Phe Ile Arg Asp Ser Glu Glu Lys Arg
115 120 125
Asn Val Gln Val Asp Val Val Glu Gly Lys Gly Ile Asp Ile Lys Ser
130 135 140
Ser Leu Ser Gly Leu Thr Val Leu Lys Ser Thr Asn Ser Gln Phe Trp
145 150 155 160
Gly Phe Leu Arg Asp Glu Tyr Thr Thr Leu Lys Glu Thr Trp Asp Arg
165 170 175
Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Gln Trp Lys Ash Phe Ser
180 185 190
Gly Leu Gln Glu Val Arg Ser His Val Pro Lys Phe Asp Ala Thr Trp
195 200 205
Ala Thr Ala Arg Glu Val Thr Leu Lys Thr Phe Ala Glu Asp Asn Ser
210 215 220
Ala Ser Val Gln Ala Thr Met Tyr Lys Met Ala Glu Gln Ile Leu Ala
225 230 235 240
Arg Gln Gln Leu Ile Glu Thr Val Glu Tyr Ser Leu Pro Asn Lys His
245 250 255
Tyr Phe Glu Ile Asp Leu Ser Trp His Lys Gly Leu Gln Asn Thr Gly
260 265 270
Lys Asn Ala Glu Val Phe Ala Pro Gln Ser Asp Pro Asn Gly Leu Ile
275 280 285
Lys Cys Thr Val Gly Arg Ser Ser Leu Lys Ser Lys Leu
290 295 300
<210>3
<211>909
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atgtccgcag taaaagcagc ccgctacggc aaggacaatg tccgcgtcta caaggttcac 60
aaggacgaga agaccggtgt ccagacggtg tacgagatga ccgtctgtgt gcttctggag 120
ggtgagattg agacctctta caccaaggcc gacaacagcg tcattgtcgc aaccgactcc 180
attaagaaca ccatttacat caccgccaag cagaaccccg ttactcctcc cgagctgttc 240
ggctccatcc tgggcacaca cttcattgag aagtacaacc acatccatgc cgctcacgtc 300
aacattgtct gccaccgctg gacccggatg gacattgacg gcaagccaca ccctcactcc 360
ttcatccgcg acagcgagga gaagcggaat gtgcaggtgg acgtggtcga gggcaagggc 420
atcgatatca agtcgtctct gtccggcctg accgtgctga agagcaccaa ctcgcagttc 480
tggggcttcc tgcgtgacga gtacaccaca cttaaggaga cctgggaccg tatcctgagc 540
accgacgtcg atgccacttg gcagtggaag aatttcagtg gactccagga ggtccgctcg 600
cacgtgccta agttcgatgc tacctgggcc actgctcgcg aggtcactct gaagactttt 660
gctgaagata acagtgccag cgtgcaggcc actatgtaca agatggcaga gcaaatccta 720
gcgcgccagc agctgatcga gactgtcgag tactcgttgc ctaacaagca ctatttcgaa 780
atcgacctga gctggcacaa gggcctccaa aacaccggca agaacgccga ggtcttcgct 840
cctcagtcgg accccaacgg tctgatcaag tgtaccgtcg gccggtcctc tctgaagtct 900
aaattgtaa 909
Claims (10)
1, a kind of Aspergillus flavus uricoxidase gene has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 3 dna sequence dna;
2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 3 dna sequence dnas hybridization that limit.
2, contain the described expression carrier of claim 1, engineering bacteria and clone.
3, a kind of method of expressing Aspergillus flavus uricoxidase, be that the described Aspergillus flavus uricoxidase gene of claim 1 is inserted procaryotic cell expression carrier, transform the host bacterium, screening obtains expressing the engineering bacteria of Aspergillus flavus uricoxidase, the culturing engineering bacterium expresses obtaining Aspergillus flavus uricoxidase.
4, method according to claim 3 is characterized in that: described procaryotic cell expression carrier is the expression vector of pET series.
5, method according to claim 4 is characterized in that: described Aspergillus flavus uricoxidase gene is inserted between the restriction enzyme site of the Nde I of expression vector of pET series and Sac I, or between the restriction enzyme site of Nde I and HindIII; The expression vector of described pET series is preferably pET-32a (+).
6, method according to claim 3 is characterized in that: described engineering bacteria is intestinal bacteria.
7, method according to claim 6 is characterized in that: described engineering bacteria is e. coli bl21 (DE3).
8, method according to claim 7 is characterized in that: in the described method, the described abduction delivering that is expressed as, used inductor are IPTG, and induced concentration 0.4-1.5mmol/L is preferably 1.0m mol/L.
9, method according to claim 8 is characterized in that: the culture temperature of described engineering bacteria is 37 ℃, and the culture temperature of abduction delivering is 28-37 ℃, is preferably 37 ℃.
10, method according to claim 3 is characterized in that: the step that also comprises the Aspergillus flavus uricoxidase that the purifying expression obtains in the described method; Described purifying comprises ion-exchange, hydrophobic chromatography and gel permeation chromatography.
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|---|---|---|---|
| CNB2006100598364A CN100406560C (en) | 2006-03-15 | 2006-03-15 | Method for expression of Aspergillus flavus urate oxidase and the special gene thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102250848A (en) * | 2011-07-12 | 2011-11-23 | 湖北工业大学 | Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli |
| CN101255440B (en) * | 2008-03-12 | 2012-02-01 | 中国科学院微生物研究所 | Recombinant polymorphism hansenula as well as special recombinant expression vectors and uses thereof |
| CN103160522A (en) * | 2013-03-26 | 2013-06-19 | 湖北工业大学 | High-efficiency expression and purification method of aspergillus flavus uricase in Pichia pastoris |
| CN113956989A (en) * | 2021-12-08 | 2022-01-21 | 北京化工大学 | Gene engineering bacterium for separating urinary oxidase and construction method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382518A (en) * | 1989-07-13 | 1995-01-17 | Sanofi | Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells |
| CN100371439C (en) * | 2003-04-07 | 2008-02-27 | 北京双鹭药业股份有限公司 | Preparation method of recombinant can diad urate oxidase |
| CN100334207C (en) * | 2004-04-27 | 2007-08-29 | 杭州北斗生物技术有限公司 | Preparation method of aspergillus flavus urate oxidase |
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2006
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255440B (en) * | 2008-03-12 | 2012-02-01 | 中国科学院微生物研究所 | Recombinant polymorphism hansenula as well as special recombinant expression vectors and uses thereof |
| CN102250848A (en) * | 2011-07-12 | 2011-11-23 | 湖北工业大学 | Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli |
| CN102250848B (en) * | 2011-07-12 | 2012-10-17 | 湖北工业大学 | Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli |
| CN103160522A (en) * | 2013-03-26 | 2013-06-19 | 湖北工业大学 | High-efficiency expression and purification method of aspergillus flavus uricase in Pichia pastoris |
| CN113956989A (en) * | 2021-12-08 | 2022-01-21 | 北京化工大学 | Gene engineering bacterium for separating urinary oxidase and construction method and application thereof |
| CN113956989B (en) * | 2021-12-08 | 2023-12-15 | 北京化工大学 | Genetically engineered bacterium secreting urate oxidase, construction method and application thereof |
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| CN100406560C (en) | 2008-07-30 |
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