CN1180106A - Glucokinase gene and its high expression engineering strain - Google Patents
Glucokinase gene and its high expression engineering strain Download PDFInfo
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Abstract
The present invention discloses a kind of glucokinase (Sak) gene and its high expression engineering strain. It uses staphylococcus aureus chromosome DNA producing Sak as template, and passes through the process of PCR amplification treatment to obtain coded Sak gene. The amino acids coded at 34 th and 42 th locuses in said gene order are different from the reported Sak gene. The primer of specially-designed nucleotide order is adopted to make amplification to obtain recombined DNA fragment, and passes through purification, identification, enzymolysis and linkage, then transforming into host-cell to form the Sak high expression engineering strain. Said expression product possesses the action of plasminogen activator, so that it has the high hydrolytic activity for human fibre protein, therefore it can be used for curing embolic diseases of cardiac infarction, cerebral thrombosis and pulmonary thromboembolism.
Description
The present invention relates to the DNA genetically engineered, is a kind of staphylokinase (Sak) gene and high expression engineering strain thereof specifically, and this expression product has the plasminogen activator effect, and human fiber's albumen is had very high hydrolytic activity.
Pertinent data has reported that golden Portugal bacterium produces a kind of non-zymoprotein, can be converted into plasmin by plasminogen activation.This non-zymoprotein is staphylokinase (Sak).The gene of this enzyme is cloned by the Duo Jia laboratory.Have in the publication document CN1109508A specification sheets, disclose and obtained glucokinase gene and cloning from the S.aureus phage DNA.Document is arranged: Collen, Det al. (1992) Fibrinolysis discloses in the 6:226-231 literary composition and has cloned glucokinase gene from S.aureus bacterium chromosomal DNA.These two kinds of gene fragments all adopt enzyme to cut large fragment DNA and clone.Its gene fragment is bigger, and the copy number that is assembled into vector plasmid is lower, is difficult to obtain higher expression effect.The expression level of this S.aureus bacterium chromosomal DNA is generally at 10-15%, as disclosing in " biotechnology " 1994.2 the 12nd volumes " the high yield production and the purifying of treatment thrombus medication recombinant staphylokinase " literary composition: " with fermentor cultivation transfection Escherichia coli TG1 cell; produce staphylokinase in the cell, account for the 10-15% of total protein of cell." this is in the present open source information report, the generally acknowledged level that staphylokinase is expressed.
The objective of the invention is to improve staphylokinase (Sak) expression of gene level, obtain high expression level staphylokinase (Sak) product and be used for thrombus treatment as plasminogen activator.Screen the Sak gene that makes new advances, construct the engineering strain of high-expression plasmid.
The objective of the invention is to have following technical scheme to realize, concrete screening, the clone, identify, building process is as follows: at first filter out a kind of glucokinase gene, this gene is the chromosomal DNA by the golden bacterium SL1.063 of Portugal bacterial strain, obtain the following sequence of the codon of the complete sequence of this gene and coded amino acid thereof: TCA AGT TCA TTC GAC AAA GGA AAA TAT AAASer Ser Ser Phe Asp Lys Gly Lys Tyr Lys through pcr amplification
10AAA GGC GAT GAC GCG AGT TAT TTT GAA CCALys Gly Asp Asp Ala Ser Tyr Phe Glu Pro
20ACA GGC CCG TAT TTG ATG GTA AAT GTG ACTThr Gly Pro Tyr Leu Met Val Asn Val Thr
30GGA GTT GAT GGT AAG GGA AAT GAA TTG CTAGly Val Asp
Gly 34Lys Gly Asn Glu Leu Leu
40TCC CCT CGT TAT GTC GAG TTT CCT ATT AAASer Pro
Arg 43Tyr Val Glu Phe Pro Ile Lys
50CCT GGG ACT ACA CTT ACA AAA GAA AAA ATTPro Gly Thr Thr Leu Thr Lys Glu Lys Ile
60GAA TAC TAT GTC GAA TGG GCA TTA GAT GCGGlu Tyr Tyr Val Glu Trp Ala Leu Asp Ala
70ACA GCA TAT AAA GAG TTT AGA GTA GTT GAAThr Ala Tyr Lys Glu Phe Arg Val Val Glu
80TTA GAT CCA AGC GCA AAG ATC GAA GTC ACTLeu Asp Pro Ser Ala Lys Ile Glu Val Thr
90TAT TAT GAT AAG AAT AAG AAA AAA GAA GAATyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu
100ACG AAG TCT TTC CCT ATA ACA GAA AAA GGTThr Lys Ser Phe Pro Ile Thr Glu Lys Gly
110TTT GTT GTC CCA GAT TTA TCA GAG CAT ATTPhe Val Val Pro Asp Leu Ser Glu His Ie
120AAA AAC CCT GGA TTC AAC TTA ATT ACA AAGLys Asn Pro Gly Phe Asn Leu Ile Thr Lys
130GTT GTT ATT GAA AAG AAAVal Val Ile Glu Lys Lys
136
The high expression engineering strain of this glucokinase gene by the karyomit(e) template DNA that produces the high bacterial strain (SL1.063) of staphylokinase activity, adopts primer P-Nml and primer P-C through pcr amplification, obtains complete glucokinase gene DNA small segment; This gene DNA fragment and pUC 19 vector plasmids are cut with EcoRI and BamH1 enzyme respectively; Above-mentioned plasmid of again enzyme being cut and glucokinase gene DNA small segment are used T
4Dna ligase connects acquisition pUK408 plasmid DNA, is transformed in DH5 α or the JM109 Bacillus coli cells again, is built into the engineering strain DH5 α (pUK408) or the JM109 (pUK408) of staphylokinase high expression level.
The high expression engineering strain of this glucokinase gene, described primer P-Nm
1, its nucleotide sequence: 5 '-CGCGAATTCATGTCAAGTTCATTCGACAAAGG-3 ', bond distance: 32;
Described primer P-C, its nucleotide sequence: 5 '-CGCGGATCCTATTTCTTTTCTATAACAACCTTTG-3 ', bond distance: 34.
The high expression engineering strain of this glucokinase gene, described pUK408 recombinant plasmid dna also can with another kind of pUV606 vector plasmid, cut the small pieces segment DNA of pUK408 recombinant plasmid generation and the large fragment DNA that enzyme is cut pUV606 through EcoRI and BamH1 enzyme respectively, pass through T
4Dna ligase connects into the pUK408 plasmid DNA, is transformed in DH5 α or TG1 or the TG2 Bacillus coli cells, is built into the engineering strain DH5 α (pHK408) of the staphylokinase high expression level that contains recombinant plasmid pHK408.
Sak gene of the present invention and high expression engineering strain thereof, its advantage is: Sak gene of the present invention is made up of 408 Nucleotide, derives 136 aminoacid sequences of corresponding staphylokinase.Sak gene the 34th of the present invention and 43 amino acid are not only different with Sak gene in the phage DNA of external report, and the Sak gene of delivering with Collen etc. by golden Portugal bacterium chromosomal DNA clone is also incomplete same.Obviously, the present invention is to provide a new Sak gene.With the dna fragmentation that obtains, be cloned into respectively on the suitable carriers plasmid, all obtained higher expression, its corresponding expression product all has the plasminogen activator effect, compare with similar Sak expression of gene level (10-15%) and to have improved nearly 1.7 times, reach 40% efficiently express by golden Portugal bacterium chromosomal DNA clone.This expression product recombinant staphylokinase, compare with t-PA with clinical streptokinase commonly used, not only security is better for it, and it is fast to have a thrombolysis, effective, advantages such as dosage is few, it can be used for treating myocardial infarction, degree of depth phlebothrombosis, pulmonary infarction, acute or subacute peripheral arterial thrombosis and chronic arterial embolism and diseases such as central retina artery or venous thrombosis.
The embodiments of the invention accompanying drawings describes in further detail, and protection domain of the present invention not only is confined among the embodiment.
Fig. 1 is: the colony characteristics of the golden bacterium SL1.063 of Portugal bacterial strain on the manitol salt agar plate.
Fig. 2 is: the golden bacterium SL of Portugal 1.063 bacterial strains colony characteristics on the high salt agar plate of yolk.
Fig. 3 is: the golden bacterium SL of Portugal 1.063 bacterial strains haemolysis on blood agar plate.
Fig. 4 is: the golden bacterium SL of Portugal 1.063 bacterial strain heat stable nucleases test.
Fig. 5-1 is: the Sak in the golden bacterium SL of Portugal 1.063 bacterial strains, and with human fibrin standard plate detected result.
Fig. 5-2 is: the Sak in the golden bacterium SL of Portugal 1.063 bacterial strains, and with human fibrin heating plate detected result.
Fig. 6 is: the structure schema of recombinant plasmid pUK408 high-expression plasmid.
Fig. 7 is: the structure schema of recombinant plasmid PHK408 high-expression plasmid.
Fig. 8 is: engineering bacteria TG2 (pHK408) efficiently expresses SDS-PAGE (12%) the electrophoretic analysis figure of product.
Coli strain and genotype thereof that the present invention uses are as follows: the coli strain genotype
JM109 endA1，recA1，gyrA96，thi，hsdR17(rk-，mk+)，
relA1，supE44，D(lac-proAB)，[F′，traD36，
proAB，lacφZDM 15]
DH5α f80dlacZDM15，recA1，endA1，gyrA96，thi-1，
hsdR17(rk-，mk+)，supE44，relA1，deoR，D(lacZ
YA-argF)U169
TG1 F′traD36laclqD(lacZ)M 15proA+B+/supED
(hsdM-mcrB)5(rk-mk-McrB-)thiD(lac-proAB)
TG2 supEhsdD5thiD(lac-proAB)D(srl-recA)
306：：Tn?10(tetr)F′[traD36?proAB+lacIq
lacZDM15]
The embodiment of the invention is following 1, separation, evaluation and the product staphylokinase of golden Portugal bacterium (Staphylococcus aureus) (Staphylokinase, Sak) bacterial strain screening:
Be SEPARATION OF GOLD Portugal bacterium, gathered more than 90 sample from sections such as hospital surgery, ophthalmology and otorhinolaryngology, with dilution method or method of scoring, be seeded on the yolk sodium-chlor nutrient agar, cultivated 24-48 hour for 36 ℃, picking is golden yellow or lemon yellow, substratum is the bacterium colony of pearliness white precipitate on every side, insert in the common nutritional medium, 36 ℃ of overnight incubation, gramstaining, microscopy are selected G
+Thalline is spherical in shape and be piled into botryoidalis, no gemma bacterial strain, is further purified on the yolk substratum.The purifying bacterial strain is connected on respectively on the high salt mannite agar substratum, cultivates 24-48 hour for 36 ℃, and the yellow annulus of light appears in periphery of bacterial colonies.Through separation, purifying, evaluation, obtain 167 strains gold Portugal bacterium altogether, its form and cultural characteristic are as follows:
Gramstaining is positive, and thalline is sphere, is piled into botryoidalis, no gemma, and the thalline size is generally 0.8-0.9 μ m.
Cultural characteristic:
On the manitol salt agar substratum, to cultivate 24-48 hour for 36 ℃, bacterium colony is golden yellow, circular protrusions, the periphery is little yellow ring, as Fig. 1.
On the high salt nutrient agar of yolk, to cultivate 24-48 hour for 36 ℃, bacterium colony is golden yellow or lemon yellow, and lunar halo shape oil film that is white in color around it as Fig. 2, shows that this bacterial strain has the ability of the Phospholipid hydrolase of laying eggs, and this is one of golden Portugal bacterium feature.
Rule on blood agar plate, cultivated 18-24 hour for 36 ℃, periphery of bacterial colonies presents transparent zone of hemolysis, and promptly β type zone of hemolysis is seen Fig. 3.
Whether produce heat stable nuclease in order to measure, with strains tested in common nutrient medium 37 ℃ cultivated 18-24 hour, bacterium liquid boiled 10 minutes, click and enter in the aperture of toluidine blue DNA agar plate, 37 ℃ are incubated 3-5 hour, and positive person all produces pink annulus, sees Fig. 4.Among Fig. 4: 1,4 is physiological saline; 2,6 is SL1.02 strain fermentation supernatant liquor; 3,5,7, be SL1.063 strain fermentation supernatant liquor.
Coagulase test of blood plasma: get rabbit plasma: physiological saline (1: 1) mixes with the common nutrient medium of each bacterial strain of equal-volume.Contrast: be isopyknic physiological saline.37 ℃ of incubated overnight, positive control and test strain blood plasma all solidify, and control tube blood plasma flows freely.
The substratum that the present invention uses:
The high salt nutrient agar of yolk
Peptone 10g OX-heart immersion liquid 10g
N.F,USP MANNITOL 10g yolk suspension 100g
NaCl 70g agar 15g
LiCl 5g distilled water 890g
The manitol salt agar substratum
Peptone 10g
N.F,USP MANNITOL 10g
Extractum carnis 1g
NaCl 75g
Phenol red 0.025g
Agar 15g
Distilled water 1000ml
The ordinary nutrient agar substratum
Peptone 10g
Extractum carnis 3g
NaCl 5g
Agar 15g
The above keynote pH7.2 that cultivates of distilled water 1000ml sterilized 20 minutes for 121 ℃.Toluidine blue (Toluidine blue) DNA agar plate
Milt DNA 0.03g
NaCl 1.00g
Agar 1.00g
2% toluidine blue 0.3ml
0.01M?CaCl 0.1ml
0.05M 100ml121 ℃ of sterilization of Tris-HCl (pH9.0) 15 minutes.
In order to screen the golden Portugal bacterium that produces staphylokinase, with 167 strains, getting its single bacterium colony is connected in the 3mlLB liquid nutrient medium test tube, 37 ℃ of shaking culture are spent the night, centrifugal 4000rpm 5 minutes, supernatant liquor get 10 μ l points in the human fibrin plate as crude enzyme liquid, 37 ℃ the insulation 16 hours, now examine whether have transparent round-formed.Through repeatedly repeating, prove that the golden bacterium SL1.063 of Portugal bacterial strain product Sak level is the highest.In order to confirm that this is staphylokinase activity or protease activity, by manufactured two identical human fibrin plates with quadrat method, one was heated 85 ℃, 40 minutes.Another sees Fig. 5-1 routinely as the standard plate, after the punching, puts 10 μ l fermented supernatant fluids respectively, and 37 ℃ are incubated 16 hours.The result is: on the standard plate, 3, No. 5 is the SL1.063 crude enzyme liquid; Be respectively urokinase and t-PA 1, No. 2; No. 4 is Lumbrukinase, all produces transparent circle.On the heating plate, see shown in Fig. 5-2, except that No. 4 Lumbrukinases produced transparent circle, 3, No. 5 SL 1.063 crude enzyme liquids were the same with urokinase and t-PA, all there is not transparent circle, the result shows: the enzyme of SL 1.063 is to produce the plasmin hydrolysis of fibrin by plasminogen activation, handles in 40 minutes through 85 ℃ of heating, makes the whole inactivations of Profibrinolysin, thereby the enzyme that SL 1.063 produces no longer produces transparent circle as urokinase and t-PA adding on the thermal fiber plate.2, the clone of SL 1.063 Sak genes: (1) golden Portugal bacterium chromosomal DNA preparation:
In 25ml LB liquid nutrient medium, 37 ℃ of vibration trainings are spent the night with bacterial strain list colony inoculation, centrifugal 4000rpm, collect thalline in 8 minutes, thalline be suspended in 2.5mlGTE solution (50mM Glucose-50mM Tris-HCl-10mM EDTA, pH8.0) in.Adding the N,O-Diacetylmuramidase final concentration is 10mg/ml, and 37 ℃ are incubated 60 minutes, add the 10%SDS of 300 μ l, RNase A (10mg/ml) and RNase T
1(2000U/ml) each 40 μ l, 37 ℃ are incubated 60 minutes, add equal-volume phenol-chloroform-primary isoamyl alcohol (12: 12: 1) extracting three times, (20mM Tris-HCl (pH8.0)-1mMEDTA) back suction is carried twice with isopyknic TE solution to the white films on the interface, after water merges, add 95% ethanol of 1/10 volume 3M NaCl and 2 times of volumes, stir out fibrous DNA with glass rod, with the flushing of 70% ethanol and dehydrated alcohol each twice, catch up with ethanol.DNA is dissolved in an amount of TE solution, the about 0.2 μ g/ μ l of concentration.Check do not have RNA to exist through SDS-PAGE.(2) design of primers:
Structure with reference to known Sak gene has designed several primers, and has introduced suitable restriction site.
Primer restriction enzyme site nucleotide sequence (5 ' → 3 ') bond distance
P-Nm
1 EcoRI CGCGAATTCATGTCAAGTTC 32
ATTCGACAAAGG
P-C BamH1 CGCGGATCCTATTTCTTTTCT 34
ATAACAACCTTTG (3) polymerase chain reaction (PCR) system:
Anabolic reaction liquid (μ l) 1.SL 1.063 masterplate DNA (0.2 μ g/ μ l) 3.0 2. primer P-N
Ml(33pmol/ μ l) 3.0 3. primer P-C (33pmol/ μ l) 3.0 4.10 * PCR damping fluid, 5.0 5.4 * dNTP (2mM) 4.0 6.ddH
2O 31.0 7.Taq archaeal dna polymerases (2.5U/ μ l) 1.0
The 1-6 mixing, denaturing treatment 95 ℃, 7 minutes adds Taq archaeal dna polymerase mixing then, in Progene PCR circulation instrument, increases.
Extension 10 minutes.(4) separation of dna fragmentation, purifying, enzyme are cut, are connected and transform:
1. purifying:, under the 360mn wavelength, downcut amplification of DNA fragments through 1.5%Agarose electrophoretic examinations.Adopt the dna fragmentation of Genelute Agarose Spin Columns method purifying pcr amplification.DNA is dissolved in 20 μ lTE (PH8.0) solution.
2. enzyme is cut:
Endonuclease reaction liquid (μ l)
Dna fragmentation 20
10 * damping fluid 5
EcoRI (20U/μl) 2
BamH
1?(12U/μl) 3
ddH
2O 20
37 ℃, 2 hours.
With Genelute Agarose Spin Columns method purifying, dna fragmentation is dissolved among the 20 μ l TE again.
3. connect: pUC 19 plasmids extract the method with reference to people such as Sambrook.As stated above, with EcoRI and BamH
1Enzyme is cut the pUC19 plasmid, through Genelute AgaroseSpin Columns method purifying.With through EcoRI and BamH
1The Sak gene that enzyme is cut connects, and 16 ℃ are incubated 12 hours.
4. transform: according to people's such as Sambrook method, the competence bacterial strain is DH5 α or the JM109 that calcium ion is handled, and is coated in overnight incubation on the plate.Detect with the x_gal test, choose white colony, insert in the LB-AP liquid nutrient medium, 37 ℃ of shaking culture are spent the night, and extract plasmid DNA, with EcoRI and BamH
1Enzyme is cut, and through 1.5%Agarose electrophoretic examinations, 431bp dna fragmentation district band is arranged, and proves to obtain the pUK408 recombinant plasmid, sees flow process shown in Figure 6.(5) determined dna sequence:
Alkaline SDS lysing cell PEG NaCl method is taked in the extraction of recombinant plasmid dna, checks through the 7%Agarose gel electrophoresis, is substantially free of RNA, can use for determined dna sequence.What determined dna sequence adopted is T7 polysaccharase sequencing system (Promega company product), and the result is sequence as previously mentioned.
The Sak gene order and the zymoprotein amino acid identity thereof of the different sources of existing report are all very high.Yet, often different at the 34th and 43 codon and amino acids coding thereof.By table one as can be seen, codon and the amino acids coding thereof of Sak63 of the present invention on these sites not only is different from the Sak s -c and the Sak42D gene in phage DNA source, nor is same as the Sak star gene of golden Portugal bacterium chromosomal DNA.The 34th coding of Sak63 of the present invention be glycine, Sak star is a Serine; The 43rd coding of Sak63 of the present invention be arginine, and Sak star is a Histidine.Obviously, Sak63 provided by the invention is a new glucokinase gene.(6) structure of efficient expression plasmid pHK408:
The flow process of pressing Fig. 7 makes up efficient expression plasmid pHK408.The preparation of plasmid DNA is with reference to people's such as Sambrook method.Get DH5 α (pUK408) plasmid list bacterium colony, be connected in the LB-AP liquid nutrient medium, 37 ℃ of shaking culture are spent the night.Handle the cracking Bacillus coli cells with alkaline SDS, precipitate RNA, obtain plasmid DNA with PEG-NaCl.Check through SDS-PAGE, be substantially free of RNA.Cut pUK408 DNA with EcoRI and BamH1 enzyme,, downcut the small pieces segment DNA, press Genelute Agarose Spin Columns method, obtain purify DNA through the 1.5%Agarose electrophoretic separation.
PUV606 vector plasmid, separation and purification as stated above.With EcoRI and BamH
1Double digestion.
ddH
2O 36μl
10×buffer 5μl
Plasmid DNA (0.5 μ g/ μ l) 5 μ l
EcoRI (20U/μl) 2?μl
BamH1 (12U/μl) 2μl
37 ℃ are incubated 2 hours.
Endonuclease reaction liquid is walked the 0.7%Agarose electrophoresis, downcuts the big fragment of DNA under long wavelength ultraviolet lamp.Press Genelute Agarose Spin Columns method purifying.The said gene fragment is connected with the pUV606 plasmid DNA, and 16 ℃ were reacted 12 hours, and changed DH5 α recipient bacterium over to.Extract recombinant plasmid dna,, walk the 1.2%Agarose gel electrophoresis, confirm the insertion of DNA gene fragment through EcoRI and BamH1 double digestion.DH5 α (pHK408) bacterial strain that will contain recombinant plasmid inserts in the LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night, the centrifuging and taking supernatant liquor, point 37 ℃ of insulations 16 hours on the human fibrin plate produce transparent circle, confirm that this bacterial strain can express the generation staphylokinase.(7) engineering bacteria TG
2Efficiently expressing (pHK408):
The pHK408 plasmid changes different coli strains over to, as JM109, DH5 α, TG
1Or TG
2Higher expression level is all arranged.
Engineering bacteria TG
2(pHK408) be inoculated in the 50ml PY substratum (1000ml, 16g Tryptones, the 5g yeast powder, 5g NaCl, PH7.2) 30 ℃ of shaking culture are 6 hours, are warming up to 42 ℃ immediately and continue shaking culture 5-6 hour, centrifugal collection thalline.Thalline is suspended in (20mM Tris-HCl-50mM NaCl-10mM EDTApH8.0) in the 5mlTSE solution, carries out supersound process with JY92-II type ultrasonic cell disruptor, and centrifugal collection supernatant liquor carries out the SDS-PAGE electrophoretic analysis.Electrophoresis finishes, and the running gel plate is taken off, and is attached on the ready-made fiber flat board that contains human plasminogen, copy about 10-20 minute after, take out running gel and carry out Coomassie brilliant blue dyeing.The dull and stereotyped room temperature of fiber is placed or about 20 minutes of 37 ℃ of insulations, promptly the clear area band occurs at the staphylokinase place.
In order to investigate engineering bacteria TG
2(pHK408) expression product Sak is in the ratio of intestinal bacteria soluble protein, and the ultrasonic cell-break supernatant liquor is walked the SDS-PAGE electrophoresis.Coomassie brilliant blue dyeing, fade to background clear after, carry out gel with Pharmacia Image Master Software and Sharp J * 330 Scanner scanners and scan, as shown in Figure 8.Among Fig. 8: 1,9 is: cytochrome C (W.M 13370) 2,10 is: RNase A (W.M.15000); 3 are: do not induce the full cell pyrolysis liquid of DH5 α (pHK408); 4 are: do not induce DH5 α (pHK408) cell ultrasonication supernatant liquor; 5 are: do not induce DH5 α (pHK408) cell ultrasonication precipitation; 6 are: the full cell pyrolysis liquid of inductive DH5 α (pHK408); 7 are: induce DH5 α (pHK408) cell ultrasonication supernatant liquor; 8 are: induce DH5 α (pHK408) cell ultrasonication precipitation.Staphylokinase district band accounts for 40% in each district's band of cytoclasis supernatant liquor total protein.As seen, this expression system is very effective.
Claims (4)
1, a kind of glucokinase gene is characterized in that: this gene is the chromosomal DNA by the golden bacterium SL1.063 of Portugal bacterial strain, obtains the following sequence of the complete nucleotide sequence of this gene and amino acids coding thereof through pcr amplification:
TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA
Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys
10
AAA GGC GAT GAC GCG AGT TAT TTT GAA CCA
Lys Gly Asp Asp Ala Ser Tyr Phe Glu Pro
20
ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT
Thr Gly Pro Tyr Leu Met Val Asn Val Thr
30
GGA GTT GAT GGT AAG GGA AAT GAA TTG CTA
Gly Val Asp
Gly 34 Lys Gly Asn Glu Leu Leu
40
TCC CCT CGT TAT GTC GAG TTT CCT ATT AAA
Ser Pro?
Arg 43 Tyr Val Glu Phe Pro Ile Lys
50
CCT GGG ACT ACA CTT ACA AAA GAA AAA ATT
Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile
60
GAA TAC TAT GTC GAA TGG GCA TTA GAT GCG
Glu Tyr Tyr Val Glu Trp Ala Leu Asp Ala
70
ACA GCA TAT AAA GAG TTT AGA GTA GTT GAA
Thr Ala Tyr Lys Glu Phe Arg Val Val Glu
80
TTA GAT CCA AGC GCA AAG ATC GAA GTC ACT
Leu Asp Pro Ser Ala Lys Ile Glu Val Thr
90
TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA
Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu
100
ACG AAG TCT TTC CCT ATA ACA GAA AAA GGT
Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly
110
TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT
Phe Val Val Pro Asp Leu Ser Glu His Ile
120
AAA AAC CCT GGA TTC AAC TTA ATT ACA AAG
Lys Asn Pro Gly Phe Asn Leu Ile Thr Lys
130
GTT GTT ATT GAA AAG AAA
Val Val Ile Glu Lys Lys
136
2, according to the high expression engineering strain of the described glucokinase gene of claim 1, it is characterized in that: the karyomit(e) template DNA by producing the high bacterial strain (SL1.063) of staphylokinase activity, adopt primer P-Nm
1With primer P-C through pcr amplification, obtain complete glucokinase gene DNA small segment; This gene DNA fragment and pUC19 vector plasmid are cut with EcoR I and BamH1 enzyme respectively; Above-mentioned plasmid of again enzyme being cut and glucokinase gene DNA small segment are used T
4Dna ligase connects acquisition pUK408 plasmid DNA, is transformed in DH5 α or the JM109 Bacillus coli cells again, is built into the engineering strain DH5 α (pUK408) or the JM109 (pUK408) of staphylokinase high expression level.
3, according to the high expression engineering strain of the described glucokinase gene of claim 2, it is characterized in that: described primer P-Nm
1, its nucleotide sequence: 5 '-CGCGAATTCATGTCAAGTTCATTCGACAAAGG-3 ', bond distance: 32; Described primer P-C, its nucleotide sequence: 5 '-CGCGGATCGTATTTCTTTTCTATAACAACCTTTG-3 ', bond distance: 34.
4, according to the high expression engineering strain of the described glucokinase gene of claim 2, it is characterized in that: described pUK408 recombinant plasmid dna, also can with another kind of pUV606 vector plasmid, cut the small pieces segment DNA that the pUK408 recombinant plasmid produces through EcoRI and BamH1 enzyme respectively, cut the large fragment DNA of pUV606 with enzyme, pass through T
4Dna ligase connects into the pHK408 plasmid DNA, is transformed in DH5 α or TG1 or the TG2 Bacillus coli cells, is built into the engineering strain DH5 α (pHK408) of the staphylokinase high expression level that contains recombinant plasmid pHK408.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97105988A CN1059926C (en) | 1997-07-19 | 1997-07-19 | Glucokinase gene and its high expression engineering strain |
| PCT/CN1998/000129 WO1999004017A1 (en) | 1997-07-19 | 1998-07-17 | Recombinant staphylokinase and their high expression engineered strain |
| AU84297/98A AU8429798A (en) | 1997-07-19 | 1998-07-17 | Recombinant staphylokinase and their high expression engineered strain |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97105988A CN1059926C (en) | 1997-07-19 | 1997-07-19 | Glucokinase gene and its high expression engineering strain |
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| Publication Number | Publication Date |
|---|---|
| CN1180106A true CN1180106A (en) | 1998-04-29 |
| CN1059926C CN1059926C (en) | 2000-12-27 |
Family
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|---|---|---|---|
| CN97105988A Expired - Fee Related CN1059926C (en) | 1997-07-19 | 1997-07-19 | Glucokinase gene and its high expression engineering strain |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1059926C (en) |
| AU (1) | AU8429798A (en) |
| WO (1) | WO1999004017A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064406C (en) * | 1998-05-11 | 2001-04-11 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for producing glucokinase by gene engrg. technique |
| CN101240284B (en) * | 2007-09-20 | 2012-05-30 | 三峡大学 | Recombination staphylokinase and highly effective secretion expression method thereof |
| CN114480353A (en) * | 2022-01-17 | 2022-05-13 | 景泽生物医药(合肥)有限公司 | Method for preparing recombinant human akkerplasmin |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0398595A (en) * | 1989-09-11 | 1991-04-24 | Taisho Pharmaceut Co Ltd | Production of peptides in gene engineering |
| ES2089784T3 (en) * | 1991-12-30 | 1996-10-01 | Medac Klinische Spezialpraep | EXPRESSION OF STAPHYLOCINASES WITHOUT SIGNAL PEPTIDE. |
| CN1035192C (en) * | 1994-04-04 | 1997-06-18 | 上海医科大学 | Preparing method for recomposing staphylokinase |
| CN1109508A (en) * | 1995-01-23 | 1995-10-04 | 成都世新药物技术开发中心 | Full DNA sequence of recombinative glucokinase |
-
1997
- 1997-07-19 CN CN97105988A patent/CN1059926C/en not_active Expired - Fee Related
-
1998
- 1998-07-17 WO PCT/CN1998/000129 patent/WO1999004017A1/en active Application Filing
- 1998-07-17 AU AU84297/98A patent/AU8429798A/en not_active Abandoned
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064406C (en) * | 1998-05-11 | 2001-04-11 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for producing glucokinase by gene engrg. technique |
| CN101240284B (en) * | 2007-09-20 | 2012-05-30 | 三峡大学 | Recombination staphylokinase and highly effective secretion expression method thereof |
| CN114480353A (en) * | 2022-01-17 | 2022-05-13 | 景泽生物医药(合肥)有限公司 | Method for preparing recombinant human akkerplasmin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1059926C (en) | 2000-12-27 |
| AU8429798A (en) | 1999-02-10 |
| WO1999004017A1 (en) | 1999-01-28 |
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