CN100334207C - Preparation method of aspergillus flavus urate oxidase - Google Patents
Preparation method of aspergillus flavus urate oxidase Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a preparation method of aspergillus flavus urate oxidase, which comprises the following steps: (1) obtaining aspergillus flavus uricoxidase gene; (2) constructing an expression vector and transforming an engineering bacterium of escherichia coli; (3) screening, culturing and inducing expression of positive clones; (4) separating and purifying the fusion protein; (5) and (5) carrying out enzyme digestion and purification on the fusion protein. The expression vector plasmid is pET series vector plasmid, the preparation method of the recombinant aspergillus flavus urate oxidase has the advantages of simple expression scheme, high expression quantity, simple purification process, high yield, soluble expression product and no need of renaturation, and the purification is directly carried out by a chromatography method, so that the purification yield is greatly improved, and the preparation method is suitable for large-scale preparation.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of preparation method of Aspergillus flavus uricoxidase.
Background technology
(Urate Oxidase Uricase) is a kind of proteolytic enzyme that uric acid can be oxidized to wallantoin to urico-oxidase for uric acid oxydo-reductase, EC 1.7.3.3.The body purine nucleotides decomposes the product xanthine that produces and produce uric acid under the effect of XOD, and uric acid further is decomposed into CO under urico-oxidase, allantoinase, allantoicase, urease effect
2And NH
3The uricolytic degree difference of different plant species, most of xanthoglobulin that produces in mammalian body and guanine are to change inosine list phosphoric acid and guanosine monophosphate into by phosphoribosyltransferase, but still have 10% will be decomposed, and get rid of external.In the human body, uric acid is the final product of purine degradation, and gets rid of external with urine.
Normal male adult twenty-four-hour urine uric acid total amount is less than 600mg, and a large amount of generations of the uric acid that pathology causes often are higher than this value and many clinical symptom occur.The propagation of malignant tumour causes nucleic acid katabolism to be strengthened, and purine metabolism produces a large amount of uric acid, and hyperuricemia (Hyperuricemia) appears in patient.Tumor regression syndromes (the tumor lysissyndrome of hyperuricemia also appears following because of a large amount of cracking of cell in the tumor chemoradiotherapy process, TLS), a series of physiological functions change in the patient body: dielectric disorder, purine metabolism produce a large amount of uric acid, polarity renal failure, weakness, spasm, arrhythmia, urinary stone disease or death.Easier appearance in lymphoma, leukemia, bulk tumor treatment.TLS has increased the concentration of intracellular organic matter such as potassium, phosphorus, calcium, uric acid etc., has substantially exceeded the discharge capacity of human kidney, causes diseases such as high lithemia, high potassium, high phosphorus, high calcium, causes the formation of renal failure at last.
Gout is another kind of and the uric acid level diseases associated, causes because the heredity or the acquired cause of disease cause purine metabolism obstacle and serum uric acid to continue rising institute.The serum uric acid level that raises causes showing effect in the joint when forming uric acid sodium hydrate micro-crystallization, painful urarthritis just takes place, and can show effect repeatedly.The crystalline deposit thing in the joint, the deposition of bone, soft tissue and cartilage is called uratoma.Urate deposition causes inflammation in uriniferous tubules, pathologies such as renal glomerulus fibrosis.Gout patient accounts for 95% based on the male sex, and onset peak is at 30-40 between year.It is too much that the major cause that gout takes place is that endogenous produces uric acid, and exogenous food intake of being rich in purine is too much, and uric acid excretion reduces.
China cancer morbidity patient 2000 was 180～2,000,000 people, accounted for 1/5th of the world, estimated the year two thousand twenty, and global annual New Development cancer patient is 1,500 ten thousand people.China's composition of nearly 20 years malignant tumours in the cause of the death rises to 17.9% by 12.6%, annual neopathy number about 1,700,000, major part adopts means treatments such as chemotherapy and radiation after morbidity, cell fission is crossed and is contained and sharply destroy when chemotherapy and radiotherapy, the a large amount of uric acid of nucleic acid decomposition increasing suddenly generation, cause hyperuricemia, blood uric acid concentration is up to 2380 μ mol/L～3570 μ mol/L (40～60mg/dl).Cause calculus and renal failure, patient is in an extremity of pain.
The way of control TLS has dietary control, hydration, alkalization acidismus at present, with means such as Zyloric and urico-oxidase diuresis.But the activity of Zyloric inhibition XOD has blocked xanthine and xanthoglobulin is converted into uric acid, disturbs the metabolism of Ismipur, influences tumor treatment.Zyloric has increased the load of renal excretion uric acid precursor (xanthoglobulin and xanthine) because suppressing the yellow fast oxydase retardance uric acid of holding in the mouth or the eyes forms.Different with xanthoglobulin, xanthine in urine than uric acid indissoluble.The patient of Zyloric 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 allopurinol to fail to respond to any medical treatment.With the patient of Zyloric treatment, 2% produces anaphylaxis, even serious allergy syndromes occurs in addition.For the acute hyperuricemia patient, Zyloric can not in time obtain medical treatment patient because onset time is long.And urico-oxidase can be oxidized to uric acid wallantoin, is easy to be excreted.Greatly alleviate syndromes that high lithemia causes to the misery that patient produces, improved the quality of life of cancer, tumour patient.
The natural urico-oxidase Uricozyme that extracts from flavus (Aspergillus flavus) has the history in 20 years in Europe listing use, and administrated by injection is used for the treatment of the serious hyperuricemia relevant with leukemia chemotherapy.The U.S. also began this product is used for the leukaemic in 1997.Compare with Zyloric, it is rapid and single-minded that urico-oxidase reduces serum uric acid level.Gout patient gives urico-oxidase acute attack capable of blocking and reduces tophaceous volume.
Not only culture cycle is long, productive rate is low to extract urico-oxidase from the flavus of cultivating, and is subjected to pathogen contamination easily.Genetic engineering technique has been opened up new road for a large amount of preparation urico-oxidases.1992, Legoux etc. cloned the Aspergillus flavus uricoxidase gene, and attempted expressing in intestinal bacteria, product is expressed in born of the same parents with soluble form, and has enzymic activity, but expression amount is low, have only 4% of thalline whole protein, do not have industrialization development and be worth.The acetylize of natural Aspergillus flavus uricoxidase N end helps the stable of enzyme and opposing proteasome degradation, and this is very important to the long expression system of culture cycle, but is not necessary to activity.In the same year, Chevalet expresses with the aspergillus flavus strain of urico-oxidase defective, and it can not be only nitrogen source with the uric acid that the result transforms bacterial strain, and reverts to wild-type easily.And obtained remarkable progress hoc annos such as Leplatois, and they express the Aspergillus flavus uricoxidase gene in yeast saccharomyces cerevisiae, and expression amount reaches 13%.Other 2000, Hongoh etc. carried out amalgamation and expression with Nilaparvata Iugens urate oxidase gene at the large intestine bar, and 90% target protein exists with the inclusion body form, and expression amount estimates at 20%.Domestic Zhu offer army equal calendar year 2001 in intestinal bacteria clonal expression the urate oxidase gene of Candida utilis, product is expressed with soluble form, accounts for 30% of soluble proteins.
The subject matter that present Aspergillus flavus uricoxidase coli expression system exists is that expression amount is not high, can't scale operation.The product expression amount of yeast expression reaches 13%, and culture cycle is long, the cost height.Therefore be necessary to adopt the preparation system that more efficiently expresses urico-oxidase, to satisfy clinical needs.
Summary of the invention
The object of the present invention is to provide the preparation method of the high Aspergillus flavus uricoxidase of a kind of expression amount.
Aspergillus flavus uricoxidase cDNA sequence is known, 301 amino acid whose protein of the total length of encoding.The encoding sequence of Aspergillus flavus uricoxidase can derive from gene library, or synthetic according to the full gene of Aspergillus flavus uricoxidase cDNA sequence of bibliographical information, can also design primer, obtains by pcr amplification.
The preparation method of a kind of recombined Aspergillus flavus uricoxidase of the present invention comprises the steps: the acquisition of (1), Aspergillus flavus uricoxidase gene; (2) structure of expression vector and transformed into escherichia coli engineering bacteria: synthetic Aspergillus flavus uricoxidase cDNA fragment is inserted into the restriction enzyme site of expression vector plasmid, then direct transformed into escherichia coli host bacterium; (3), the screening of positive colony, cultivation, abduction delivering: filter out positive colony and on substratum, cultivate, target protein is expressed through inducing; (4), the separation of fusion rotein, purifying: at first with colibacillus engineering microorganism collection, broken bacterium, through the centrifugal bacterium liquid supernatant that obtains brokenly, then to broken bacterium liquid supernatant chromatographic technique purifying; (5), the enzyme of fusion rotein cuts, wherein said expression vector plasmid is pET-15b, pET-19b, pET-30a-c, pET-32a-c; The acquisition of Aspergillus flavus uricoxidase gene is synthetic or by the design primer, PCR obtains again by full gene; Described restriction enzyme site is Nde I/BamH I or Nco I/BamH I site; Step is to do resistance screening with penbritin in (3); The temperature of culture medium culturing is controlled at 28 ℃～40 ℃ in the step (3), preferably at 30 ℃～35 ℃; The IPTG that the middle abduction delivering working concentration of step (3) is 0.1～1.5mmol/L is as inductor, and the IPTG of preferred 0.4～1.0mmol/L is as inductor; Chromatographic technique is one or more methods in ion exchange chromatography, sieve chromatography, hydrophobic chromatography and the affinity chromatography; It is zymoplasm or Enteropeptidase that the middle enzyme of step (5) is cut used enzyme.
The preparation method of a kind of Aspergillus flavus uricoxidase of the present invention, its expressional scheme is simple, the expression amount height, purifying process is simple, the yield height.Expression product is solvable, does not need renaturation, directly carries out purifying with chromatography method, and purification yield improves greatly, is fit to mass preparation.
Description of drawings
Fig. 1: expression vector establishment synoptic diagram of the present invention;
Fig. 2: the SDS-PAGE electrophoretic analysis of fusion rotein is figure as a result;
Wherein: 1 and 8: protein molecular weight standard is respectively 97.4kd, 66.2kd, 43kd, 31kd, 20.1kd, 14.4kd from top to bottom; 2: do not induce full bacterium; 3: do not induce thalline to break the bacterium supernatant; 4: do not induce the broken bacterium precipitation of thalline; 5: induce full bacterium; 6: induce thalline to break the bacterium supernatant; 7: induce the broken bacterium precipitation of thalline.
Fig. 3: ion exchange chromatography collection of illustrative plates;
Fig. 4: the SDS-PAGE electrophoretic analysis after the enzyme of fusion rotein is cut is figure as a result;
Wherein: 1: not enzyme-added contrast; 2: enzyme 2000 diluting effect results: 3: enzyme 200 diluting effect results; 4: enzyme 20 diluting effect results; 5: the enzyme contrast.
Fig. 5: hydrophobic chromatography collection of illustrative plates;
Fig. 6: activation analysis is figure as a result.
Embodiment
The acquisition of embodiment 1:1, Aspergillus flavus uricoxidase gene
Table 1: Aspergillus flavus uricoxidase cDNA sequence
| ?001 ?061 ?121 ?181 ?241 ?301 ?361 ?421 ?481 ?541 ?601 ?661 ?721 ?781 ?841 ?901 | ?gag catatgt?ccgcggtTaa?agcagcccgc?tacggcaaGg?acaacgttcg?cgtgtacaag ?gttcacaaag?acgagaagac?cggtgtccag?acggtgtacg?agatgaccgt?ctgtgtgctt ?ctggagggtg?agattgagac?cAGttacacc?aaggccgaca?acagcgtcat?tgtcgcaacc ?gacAGcatta?agaacaccat?ttacatcacc?gccaagcaga?accccgttac?tcctcccgag ?ctgttcggct?ccatcctggg?cacacacttc?attgagaagt?acaaccacat?ccatgccgct ?cacgtcaaca?ttgtctgcca?ccgctggacc?cggatggaca?ttgacggcaa?gccacacccG ?cacAGcttca?tccgcgacag?cgaggagaag?cggaatgtgc?aggtggacgt?ggtcgagggc ?aagggcatcg?atatcaagtc?gtctctgtcc?ggcctgaccg?tgctgaagag?caccaactcg ?cagttctggg?gcttcctgcg?tgacgagtac?accacactta?aggagacctg?ggaccgtatc ?ctgagcaccg?acgtcgatgc?cacttggcag?tggaagaatt?tcagtggact?ccaggaggtc ?cgctcgcacg?tgcctaagtt?cgatgctacc?tgggccactg?ctcgcgaggt?cactctgaag ?acttttgctg?aagataacag?tgccagcgtg?caggccacta?tgtacaagat?ggcagagcaa ?atcctggcgc?gccagcagct?gatcgagact?gtcgagtact?cgttgcctaa?caagcactat ?ttcgaaatcg?acctgagctg?gcacaagggc?ctccaaaaca?ccggcaagaa?cgccgaggtc ?ttcgctcctc?agtcggaccc?Gaacggtctg?atcaagtgta?ccgtcggccg?gtcctctctg ?aagtctaaat?tgTAACTGA g?gatccAga (restriction enzyme site of line place) for introducing |
Expression vector adopts pET-15b (Invitrogen), and the host bacterium is e. coli bl21 (DE3).Aspergillus flavus uricoxidase cDNA sequence according to bibliographical information, see Table 1, synthesize 30 sections complementary oligonucleotide, and draw respectively as restriction enzyme site Nde I, BamH I, the ordinary method of pressing molecular cloning at 5 ' end and 3 ' end, at first handle 30min for 37 ℃ with T4 phage polynucleotide kinase, each oligonucleotide fragment of phosphorylation mixes to wait mole, 94 ℃ of sex change 5min, 65 ℃ of annealing 10min immediately, add the T4 ligase enzyme then, 16 ℃ of connections are spent the night.
2, the structure of expression vector pET-15b-202 and conversion host bacterium
The pET-15b plasmid reclaims big fragment with Nde I, BamH I double digestion, is connected with synthetic Aspergillus flavus uricoxidase cDNA fragment, and 20 μ L reaction system gene fragments and the big segmental ratio of carrier are 10: 1, add T
4Dna ligase 300 units, 15 ℃ of connections are spent the night, and get 10 μ L and connect product direct transformed into escherichia coli host bacterium BL21 (DE3) competent cell, coat the amicillin resistance flat board, 37 ℃ of overnight incubation obtain engineering bacteria through further screening, the engineering bacteria building process is seen Fig. 1
3, the screening of positive colony, cultivation, abduction delivering
Penbritin is done resistance screening, obtains positive colony pET-15b-Uox.Extract plasmid, identify with restriction enzyme.Positive transformant carries out sequential analysis with universal primer, and cloned sequence and implementation sequence are in full accord as a result.
The inoculation positive colony is cultivated, and induces through the IPTG of 0.8mmol/L.
(4), the separation of fusion rotein, purifying
Collect thalline, carry out ultrasonication, collect then and go up cleer and peaceful precipitation, use the 12%SDS-PAGE electrophoretic analysis, terminal objective albumen mainly is present in brokenly in the supernatant liquor behind the bacterium, electrophoresis result is seen Fig. 2, according to the strip analysis of electrophoresis result, induces the expression amount that No. 5 of full bacterium runs the target protein in the adhesive tapes should be more than 30%.With the broken bacterium supernatant liquor ion exchange chromatography separation and purification of collecting, its process makes its specific conductivity less than 5mS/cm for being that 8.5 20mmol/L Tris.CL damping fluid dilutes with pH value.Damping fluid balance CM post with same is washed till baseline with level pad behind the last sample, with 0.01～0.5mol/LD NaCL gradient elution, collects the target protein peak, and the chromatography collection of illustrative plates is seen Fig. 3.
(5), the enzyme of fusion rotein cut, purifying
To make the concentration of target protein reach 1mg/mL after the dilution of ion-exchange target protein peak, sample cuts damping fluid (50mmol/L Tris.CL, 150mmol/L NaCL, 2.5mmol/L CaCL to zymoplasm
2, pH7.5) buffer-exchanged is carried out in dialysis, adds the zymoplasm of 20mg/mL (0.5U/mL) then, Dilution ratio is respectively 20,200,2000 times, and 25 ℃ of incubated overnight enzymes are cut, the 12%SDS-PAGE electrophoretic analysis, the zymoplasm cutting effect that the results are shown in Figure 4,20,200 times of dilutions is fine.Target protein after enzyme is cut is added (NH
4)
2SO
4Making final concentration is 1.0-1.5mol/L, uses 1.2mol/L (NH on the sample
4)
2SO
4, 20mmol/L PB, the damping fluid equilibrated Phenyl-Sepharose Fast Flow post of pH7.4 is washed and is dragged by reducing the salt concn gradient, collects the target protein peak, sees the hydrophobic chromatography collection of illustrative plates of Fig. 5.
The hydrophobic chromatography peak that obtains is through Sephadex G-25 desalination, and balance liquid is 20mmol/L PB, pH7.4.Be the Aspergillus flavus uricoxidase of polishing purification after the sample desalination.
Embodiment 2: the Aspergillus flavus uricoxidase activation analysis
Aspergillus flavus uricoxidase behind purifying method (Legoux R.et al., J.Biol.Chem., 1992,267, (12), 8565-8570) enzyme analysis activity, 3ml buffer solution system (TEA buffer, the 7.5g trolamine of bibliographical information; 0.38g contain uric acid 0.18 μ mol EDTA), pH8.9,30 ℃ of insulations detect wavelength 292nm, and it is a unit of enzyme activity that per minute is oxidized to the required enzyme amount of allantoic acid with 1 μ mol uric acid.Enzyme 1 μ L, the 2 μ L, 5 μ L, the 10 μ L that add 1mg/mL, reaction finishes the back and surveys absorption value, the results are shown in Figure 6.
Claims (9)
1. the preparation method of an Aspergillus flavus uricoxidase is characterized in that comprising the steps:
(1) acquisition of Aspergillus flavus uricoxidase gene;
(2) structure of expression vector and transformed into escherichia coli engineering bacteria: synthetic Aspergillus flavus uricoxidase cDNA fragment is inserted into the restriction enzyme site of expression vector plasmid, then direct transformed into escherichia coli host bacterium;
(3) screening of positive colony, cultivation, abduction delivering: filter out positive colony and on substratum, cultivate, target protein is expressed through inducing;
(4), the separation of fusion rotein, purifying: at first with colibacillus engineering microorganism collection, broken bacterium, through the centrifugal bacterium liquid supernatant that obtains brokenly, then to broken bacterium liquid supernatant chromatographic technique purifying;
(5), the enzyme of fusion rotein cut, purifying: partially purified fusion rotein is obtained Aspergillus flavus uricoxidase with the chromatographic technique purifying after enzyme is cut, wherein said expression vector plasmid is a pET serial carrier plasmid, described pET serial carrier plasmid is pET-15b, pET-19b, pET-30a-c or pET-32a-c, and it is zymoplasm or Enteropeptidase that described enzyme is cut used enzyme.
2. the preparation method of a kind of recombined Aspergillus flavus uricoxidase according to claim 1, the acquisition that it is characterized in that the Aspergillus flavus uricoxidase gene is synthetic or by the design primer, PCR obtains again by full gene.
3. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 1 is characterized in that described restriction enzyme site is Nde I/BamH I or Nco I/BamH I site.
4. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 1 is characterized in that in the step (3) be to do resistance screening with penbritin.
5. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 1 is characterized in that the temperature of cultivating in the step (3) is controlled at 28 ℃～40 ℃.
6. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 5 is characterized in that the temperature of cultivating in the step (3) is controlled at 30 ℃～35 ℃.
7. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 1 is characterized in that abduction delivering working concentration in the step (3) is that the IPTG of 0.1～1.5mmol/L is as inductor.
8. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 7 is characterized in that abduction delivering working concentration in the step (3) is that the IPTG of 0.4～1.0mmol/L is as inductor.
9. the preparation method of a kind of Aspergillus flavus uricoxidase according to claim 1 is characterized in that chromatographic technique is one or more methods in ion exchange chromatography, sieve chromatography, hydrophobic chromatography and the affinity chromatography.
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