CN1446912A - Reformed lyophylization preparation of recombinant staphylokinase (r-Sak), its preparing method and application - Google Patents
Reformed lyophylization preparation of recombinant staphylokinase (r-Sak), its preparing method and application Download PDFInfo
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Abstract
A freeze-dried preparation of recombinant glucokinase in the form free dried powder, injection, lipoplasm, or microcapsule for thrombolytic purpose contains the glucokinase with mutation of amino acids at positions 7, 3 and 43, and one or more of mannitol, phosphate, EDTA and sodium chloride. Its preparing process is also disclosed.
Description
Technical field
The present invention relates to a kind of recombinant staphylokinase freeze-dried preparation, its preparation method and, belong to biological pharmacy technical field as the application of thrombolytic drug.
Background technology
Cardiovascular and cerebrovascular diseases is first killer who endangers people ' s health at present, wherein thrombotic diseases such as Acute Myocardial Infarction be the main diseases that causes patient death because of.Though still there are a lot of problem demanding prompt solutions at present existing clinically commercially available thrombolytic drugs at aspects such as the specificity of its thrombolysis effect, thrombolysis and drug prices.Staphylokinase (Staphylokinase, be called for short SAK) be a kind of protein that produces by streptococcus aureus, molecular weight is 15.5KD, it can combine specifically with Profibrinolysin, form mixture, plasminogen activation makes it to become plasmin, thereby fibrin degradation specifically makes thrombolysis.According to the foreign literature report, the thrombolysis effect of staphylokinase is better than streptokinase and urokinase etc., and is suitable with tissue-type plasminogen activator (t-PA) at least, and side effect is littler, is the most promising a kind of thrombolysis candidate medicine.
Summary of the invention
The object of the present invention is to provide a kind of recombinant staphylokinase of on 7,36,43 amino acids, finishing sudden change, and combine the preparation freeze-dried preparation, use clinically as thrombolytic drug with additive such as N.F,USP MANNITOL.
：,：TCA AGT TCA TTC GAC AAA GCA AAA TAT AAA AAA GGC GAT GAC GCG AGT TAT TTT GAA CCA ACA GGCSer Ser Ser Phe Asp Lys Ala Lys Tyr Lys Lys Gly Asp Asp Ala Ser Tyr Phe Glu Pro Thr GlyCCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GAT GGT AAA GGA AAT GAA TTG CTA TCC CCT CGT TATPro Tyr Leu Met Val Asn Val Thr Gly Val Asp Gly Lys Gly Asn Glu Leu Leu Ser Pro Arg TyrGTC GAG TTT CCT ATT AAA CCT GGG ACT ACA CTT ACA AAA GAA AAA ATT GAA TAC TAT GTC GAA TGG GCAVal Glu Phe Pro Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp AlaTTA GAT GCG ACA GCA TAT AAA GAG TTT AGA GTA GTT GAA TTA GAT CCA AGC GCA AAG ATC GAA GTC ACTLeu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu Leu Asp Pro Ser Ala Lys Ile Glu Val ThrTAT TAT GAT AAG AAT AAG AAA AAA GAA GAA ACG AAG TCT TTC CCT ATA ACA GAA AAA GGT TTT GTT GTCTyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Tnr Glu Lys Gly Phe Val ValCCA GAT TTA TCA GAG CAT ATT AAA AAC CCT GGA TTC AAC TTA ATT ACA AAG GTT GTT ATA GAA AAG AAAPro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile Thr Lys Val Val Ile Glu Lys LysTAA,
* *,
Recombination sequence length is 411 bases, and it is codon GCA, 115-118 base codon GGA that recombination sequence is characterized as 19-21 base; The 127-129 base is codon CGT, and corresponding amino acid is Ala, Gly and Arg.
Also comprise in the described recombinant staphylokinase freeze-dried preparation excipient N.F,USP MANNITOL, buffer reagent phosphoric acid salt, EDTA, permeate agent sodium-chlor wherein one or more.
The proportion of composing of described recombinant staphylokinase freeze-dried preparation is: recombinant staphylokinase 10mg, N.F,USP MANNITOL 15mg, Sodium phosphate dibasic 3.4mg, SODIUM PHOSPHATE, MONOBASIC 0.63mg, EDTA 1.0mg, sodium-chlor 8.5mg.
The preparation method of recombinant staphylokinase freeze-dried preparation of the present invention comprises
(1), the structure of recombinant staphylokinase engineering bacteria:
Select the streptococcus aureus strain to carry out the separation of chromosomal DNA, primer is:
F
SAK：5，GGAATTCATATGTCAAGTTCATTCGACAA 3’
R
SAK: 5 ' CGGGATCCTTATTTCTTTTCTATAACAACC 3 ' is that template is carried out pcr amplification with above-mentioned chromosomal DNA; Use pBV220 to be expression vector, the e. coli bl21 strain is the host bacterium, utilize the glucokinase gene of restriction enzyme EcoRI and BamHIPCR reaction amplification, simultaneously with identical enzymic digestion expression vector pBV220, electrophoresis reclaims, carrier is connected with gene, Transformed E .coliBL21 competent cell, the picking transformed clone is inoculated in the LB substratum that contains penbritin, the concussion overnight incubation, the plasmid DNA of extraction transformant utilizes EcoRI and BamHI enzyme to cut, enzyme is cut the product reaction product carry out the agarose gel electrophoresis analysis, the recombinant plasmid that filters out the glucokinase gene insertion is as engineering strain;
(2), fermentation
The clone strain that will contain recombinant plasmid is inoculated in the LB substratum that contains penbritin, 30 ℃ of concussion overnight incubation, as primary seed solution; Be inoculated in the concentration of 0.1-0.5% and contain in the penbritin LB substratum, 30 ℃ of concussion overnight incubation are as fermentation seed liquid; Again with LB as nutrient solution, inoculation 5-6% fermentation seed liquid carries out jar to be cultivated, mixing speed 300rpm, air flow 0.8-1.0vvm, oxygen dissolving value generally by the initial reduction gradually of fermentation, get OD
600Induce program to start heating up between the 3-3.5, wait to be warming up to 42 ℃ induce beginning after, portion-wise addition glucose by regulating stirring velocity, remains on more than 40% the DO value to supplement the nutrients, and induces after 5 hours and receives bacterium; The phosphate buffered saline buffer of zymophyte at pH7.6 suspended, under condition of ice bath, ultrasonication;
(3), purifying
The phosphate buffered saline buffer of zymophyte at pH7.6 suspended, under condition of ice bath, ultrasonication; Anion-exchange chromatography post damping fluid balance is passed the anion-exchange chromatography post with the supernatant liquor after the fragmentation, collects and passes the peak; Again the protein liquid of collecting being transferred pH with the 0.1N dilute phosphoric acid is 6.20, Zhiyang ion chromatography column purification, the recombinant staphylokinase protein molecular is adsorbed on the column packing, earlier with the flushing of pH6.20 damping fluid, remove unconjugated albumen, then, with the phosphoric acid salt EDTA pH6.20 buffer solution elution that contains NaCl, collect elution peak; Through S200 gel permeation chromatography purifying, collect elution peak again;
(4), preparation
With containing 0.1mol/L sodium-chlor, 20mmol/L phosphate buffered saline buffer (pH7.4), the solvent of the EDTA of 2mmol/L is diluted to 8.0mg/ml with recombinant staphylokinase, filtering with microporous membrane degerming with 0.22 μ m, adding stablizer and vehicle are 1.5% N.F,USP MANNITOL (weight percent), recombinant staphylokinase is sub-packed in the 10mg/ bottle in the cillin bottle of handling, carries out lyophilize then.
Streptococcus aureus described in preparation method's step (1) of described recombinant staphylokinase is selected good strains in the field for seed and is selected as 1697 strains.
Pcr amplification reaction condition described in preparation method's step (1) of described recombinant staphylokinase be g4 ℃ 40 seconds, 54 ℃ 40 seconds, 72 ℃ 2 minutes, carry out 30 circulations altogether, last circulation was extended 5 minutes for 72 ℃.
Ultrasonication bacterium described in preparation method's step (3) of described recombinant staphylokinase is adopted intermittently broken and remains in the ice bath and carries out, and each 30 seconds, 30 seconds at interval.
Recombinant staphylokinase freeze-dried preparation of the present invention can be made into freeze drying powder preparations form, vein and intramuscular injection pharmaceutical solutions form, Liposomal formulation form, the microcapsule formulation form of thrombolysis clinically as the application of thrombolytic drug.
Recombinant staphylokinase freeze-dried preparation clinical test results of the present invention is analyzed: the thrombolysis potentiality of staphylokinase are estimated in the patient who suffers from AMI and peripheral arterial infraction.In two small-sized pilot scale researches, 10 patients with the coronary artery infraction that vasography confirms give 10mg recombinant staphylokinase through vein, promptly injected 1mg medicine group earlier at 30 minutes, transfusion gives 9mg again, use with acetylsalicylic acid and combination with heparin simultaneously, it is logical fully again to obtain coronary artery in eight patients, and a personal sector is logical again, and perfusion postpones to be about 20 minutes mean time again.Fibrinogen, plasminogen and α
2The blood plasma level of-antiplasmin remains unchanged, and proves that staphylokinase has the fibrin-specific of height in vivo.In addition, in the patient of 13 carrying out property of trouble saturating wall (transmural) myocardial infarctions, carried out a bolus contrast agent staphylokinase, with the pilot scale research of acetylsalicylic acid and intravenous injection heparin.First patient has the big area anterior wall infarction, adopts two medicines to roll into a ball the 20mg staphylokinase twice, treatment in 15 minutes at interval, and the result has produced the intracerebral hemorrhage that the non-lethality moderate disables.Therefore, all the other 12 patients have given corresponding medicine and have rolled into a ball injection under the vasography monitoring.The 20mg staphylokinase was injected in 5 minutes and is finished during beginning, and in the expert vasography monitoring of 60 fens clock times thrombolysis process, as the logical again 10mg that then do not append entirely of infraction related arteries, 7 examples (58%) obtain fully again 60 minutes the time logical after administration as a result.All the other 5 examples give second dosage (10mg/5 minute), and the result is three people total reflux in the time of 90 minutes wherein, and total recurrence rate reached 83% in the time of 90 minutes.On the basis of these clinical trials, in 102 routine patients, carried out two medicine staphylokinase 15mg of group administration in 30 minutes (50 routine patient) at interval, to the comparative studies of preposition (front loaded) rt-PA (52 routine patient).As a result, after administration, obtained total reflux 90 minutes the time with staphylokinase treatment patient's 68% and rt-PA treatment patient's 57%.Staphylokinase be scleroproein optionally, and do not cause transformation reactions, but two week of most patient the back produce neutralizing antibodies.The clinical study of these pilot scales shows that it is optionally thrombolytics of the rapid also tool high fiber albumen of a kind of brute force the acute myocardial infarction patient that intravenously gives staphylokinase.Compare with the relevant thrombolysis medicine of present clinical application, staphylokinase has the following advantages: can avoid the activation of general Profibrinolysin; Can not cause that the general blood coagulation system is malfunctioning, reduces hemorrhage syndrome.
26S Proteasome Structure and Function specificity analysis from staphylokinase, because itself is exactly a kind of former nucleoprotein, there are not problems such as processing behind the protein expression and modification, and there is not disulfide linkage to exist with interchain in the molecular chain, this makes that utilizing genetic engineering technique to produce in enormous quantities becomes possibility, thereby can reduce production costs greatly.In recent years, the development of DNA reorganization equimolecular biology techniques utilizes genetically engineered to carry out the means that bio-pharmaceuticals has been ripe and widespread use at present rapidly.
Embodiment
Describe technology contents of the present invention in detail below in conjunction with embodiment:
(1), the structure of recombinant staphylokinase engineering bacteria:
The present invention selects to adopt staphylococcus aureus 1697 (taking from DSMZ of Microbe Inst., Chinese Academy of Sciences) to carry out the separation of chromosomal DNA, the preservation bacterium liquid of getting 1697 strains of 0.5ml streptococcus aureus is inoculated in the 100ml LB substratum, 37 ℃ of concussion overnight incubation, 7000rpm4 ℃ of centrifugal 8 minutes collection thalline, wash thalline once with 20ml TE, the resuspended thalline of 20ml TE, add 120 μ l Proteinase K mixings, add the SDS mixing of 2.2ml 10%, 37 ℃ of temperature were bathed 1 hour, once (put upside down mixing 1 minute) with isopyknic phenol extracting, with the 3M NaAc (pH5.2) of 0.1 volume, isopyknic Virahol precipitates 2 hours, 12000rpm4 ℃ centrifugal 10 minutes, get precipitation, wash once, be dissolved in the 200 μ l distilled waters with 75% ethanol, get 2 μ l samples and carry out 1% agarose gel electrophoresis, get its OD of an amount of detection simultaneously
260And OD
280, measure its purity and concentration.
Glucokinase gene separates: according to the primer design principle, designed a pair of Oligonucleolide primers, introduced the EcoRI restriction enzyme site, introduced the BamHI restriction enzyme site at 3 ' end at 5 of primer ' end, and regulated distance and based composition between SD sequence and the initiator codon, sequence is:
F
SAK：5’GGAATTCATATGTCAAGTTCATTCGACAA 3’
R
SAK：5’CGGGATCCTTATTTCTTTTCTATAACAACC 3’
Pcr amplification reaction: with above-mentioned chromosomal DNA is template, carries out pcr amplification, and reaction process is: g4 ℃ 40 seconds, 54 ℃ 40 seconds, 72 ℃ 2 minutes, carry out 30 circulations altogether, last circulation was extended 5 minutes for 72 ℃, got amplified production 5 μ l and carried out agarose gel electrophoresis.
The evaluation of glucokinase gene (dna sequence analysis): the distilled water that utilizes sterilization is with one times of PCR product dilution, utilize again that phenol/the chloroform extracting once, alcohol precipitation DNA, and be dissolved in the distilled water of sterilization, making up the order-checking plasmid, concrete grammar is: get an amount of PCR product and pBV220 carrier, respectively with EcoRI and BamHI double digestion, electrophoresis reclaims, and both mix according to a certain percentage, at T
4Connect under the effect of dna ligase, transformed into escherichia coli DH5 α competent cell, extract the plasmid DNA of transformant, analyze with EcoRI and BamHI double digestion, the row agarose gel electrophoresis of going forward side by side detects, and detected result shows have foreign DNA to insert in the recombinant clone, further extract its plasmid DNA, utilize pBV220 sequencing primer and automatic sequence analyser to carry out determined dna sequence.The nucleotide sequence of isolated genes and the aminoacid sequence of supposition are as mentioned above.Analytical results shows that the isolating gene order of the present invention is except that the discrete point mutation, and whole sequence is basic consistent with the SAK of bibliographical information, but because the sudden change of indivedual bases, thereby caused corresponding amino acid whose variation.
The structure of expression plasmid: the present invention uses pBV220 to be expression vector, the e. coli bl21 strain is the host bacterium, utilize the glucokinase gene of restriction enzyme EcoRI and BamHIPCR reaction amplification, simultaneously with identical enzymic digestion expression vector pBV220, electrophoresis reclaims, carrier is connected with gene, transformed into escherichia coli BL21 competent cell, the picking transformed clone is inoculated in the LB substratum that contains penbritin, the concussion overnight incubation, extract the plasmid DNA of transformant, utilize EcoRI and BamHI enzyme to cut, enzyme is cut the product reaction product carry out the agarose gel electrophoresis analysis, the recombinant plasmid that filters out the glucokinase gene insertion is as engineering strain;
(2), fermentative production
The seed preparation: the clone strain that will contain recombinant plasmid is inoculated in the LB substratum that contains penbritin, and 30 ℃ of concussion overnight incubation are as primary seed solution; Be inoculated in the concentration of 0.1-0.5% and contain in the penbritin LB substratum, 30 ℃ of concussion overnight incubation are as fermentation seed liquid; The cell of fermentation seed liquid should be in the logarithmic growth later stage, cell density OD
600Between 2.5-3.0, this moment, cell density was higher, energetic.
Fermentative production: as nutrient solution, inoculation 5-6% fermentation seed liquid carries out jar to be cultivated with LB, and mixing speed 300rpm, air flow 0.8-1.0vvm, oxygen dissolving value generally by 100% initial reduction gradually of fermentation, get OD
600Induce program to start heating up between the 3-3.5, wait to be warming up to 42 ℃ induce beginning after, portion-wise addition glucose is to supplement the nutrients, by regulating stirring velocity, the DO value is remained on more than 40%, induce the expression amount of target protein after 5 hours to reach the highest, therefore generally after inducing 5 hours, receive bacterium.Final OD ferments
600Value is generally between the 11.0-14.0, receives bacterium in centrifugal 15 minutes in 6000rpm below 4 ℃, and with 0.1mol/L NaCl, 20mmol/L phosphoric acid salt and edta buffer liquid (pH7.8) washing bacterial sediment twice claim wet bacterium weight with the zymophyte body and function, and-20 ℃ freezing standby;
(3), purifying
Excusing from death is broken: with zymophyte at phosphoric acid salt and edta buffer liquid (20mM phosphoric acid salt pH7.6,2mMEDTA) suspend, place ice bath, utilize the broken bacterium of Ultrasonic Cell Disruptor, in the shattering process, therefore owing to action of ultrasonic waves produces a large amount of heats, adopt intermittently broken method, and remain in the ice bath and carry out.Each 30 seconds, 30 seconds at interval, cause the albumen inactivation with the rising of anti-crushing liquid temp.Adding 10ml damping fluid ratio with the 1g bacterium is advisable.
Anion-exchange chromatography purifying: anion-exchange chromatography post (Q Sepharose FF) the phosphoric acid salt edta buffer liquid balance of pH7.6 20mM, utilize the negative adsorption method of ion-exchange, supernatant after the fragmentation is passed the anion-exchange chromatography post, recombinant staphylokinase albumen is passed, the peak is passed in collection, and most of tropina is adsorbed on the chromatography column.Through behind this step purifying, remove most of foreign protein, the purity of recombinant staphylokinase is improved, thereby reaches the effect of preliminary purification.
The cation-exchange chromatography purifying: will transfer pH with the 0.1N dilute phosphoric acid through the protein liquid that the anion-exchange chromatography purifying is collected is 6.20, the purifying of last sample Zhiyang ion chromatography post (SP Sepharose FF), the recombinant staphylokinase protein molecular is adsorbed on the column packing, phosphoric acid salt EDTA pH6.20 damping fluid with 20mM washes earlier, remove unconjugated albumen, then, phosphoric acid salt EDTApH6.20 buffer solution elution with the NaCl 20mM that contains 0.2M, collect elution peak, the SDS-PAGE detected result shows that the proteic purity of recombinant staphylokinase in the 0.2M NaCl elutriant further increases.
Gel permeation chromatography: the SephacrylS200 that selects Amersham-Pharmacia Biotech company to produce is a gel filter medium.At first with damping fluid (20mM phosphoric acid salt pH7.4,2mM EDTA, 0.1MNaCl) two column volumes of wash-out, with sample on the elutriant of cation seperation column 0.2M NaCl, carry out wash-out then, collect elution peak with 1.2ml/ minute flow velocity, elutriant is carried out SDS-PAGE to be detected, the result shows that recombinant staphylokinase albumen presents single band in the elutriant, and no foreign protein is taken out of existing.
(4), preparation
After the recombinant staphylokinase of chromatography column purifying passes through the SDS-PAGE electrophoresis, with the method that silver dyes its purity is determined, measure protein concentration and calculate total protein content with the Lowry method then.
According to the pharmacological experiment data, concentration is about 0.9～1.7 μ g/ml in the recombinant staphylokinase blood, and dosage also has certain interval range, generally in 0.25～0.4mg/kg body weight.From the angle of clinical application, generally select the at first quick administration 10m9 of vein, then according to the variation of the state of an illness and the further needs of treatment, slowly intravenous drip 10mg or no longer administration again.So the embodiment that the present invention provides determines that the packing dosage of recombinant staphylokinase is the 10mg/ bottle.Composition in every vial formulation is:
Recombinant staphylokinase 10mg
N.F,USP MANNITOL 15mg
Sodium phosphate dibasic 3.4mg
SODIUM PHOSPHATE, MONOBASIC 0.63mg
EDTA?1.0mg
Sodium-chlor 8.5mg
Formulation method comprises:
With containing 0.1mol/L sodium-chlor, 20mmol/L phosphate buffered saline buffer (pH7.4), the solvent of the EDTA of 2mmol/L is diluted to 8.0mg/ml with recombinant staphylokinase, and with the filtering with microporous membrane degerming of 0.22 μ m, adding stablizer and vehicle are 1.5% N.F,USP MANNITOL (weight percent).Recombinant staphylokinase is sub-packed in the 10mg/ bottle in the cillin bottle of handling, carries out lyophilize then.Adopt vacuum seal after the freeze-drying, add the plastic-aluminum enclosing cover ,-20 ℃ of preservations.
0.1mol/L the N.F,USP MANNITOL of sodium chloride solution and 1.5% is close with blood of human body osmotic pressure, phosphate buffered saline buffer is near the potential of hydrogen of blood of human body, and the EDTA of trace is suppressed at the remaining protease activity of possibility denier in the purge process.
Determining according to preparation freeze-drying aftershaping outward appearance of N.F,USP MANNITOL consumption utilizes 20% formula mannitol injection liquid to originate for auxiliary material, adds freeze-drying behind 2%, 4%, 6% and 10% 20% formula mannitol injection liquid respectively in recombinant staphylokinase stoste.The thrombolysis activity of preparation does not have significant difference after the freeze-drying, but mannitol content is when being lower than cumulative volume 1.2%, and preparations shaping is relatively poor, is higher than 1.2% and does not see difference, so select 1.5% mannitol concentration for use.
Human serum albumin is a stablizer main in finished product freeze-drying and the storage process, human serum albumin of too much planting different concns on probation reaches and does not add human serum albumin in the preparation manufacture, with thrombolysis activity after the freeze-drying and preparations shaping outward appearance is index, the tentative experiment observations is thought, do not add and add the human serum albumin of different concns, outward appearance after the preparation freeze-drying there is not influence, thrombolysis activity unknown significance difference.Because also there are some problems in blood products management at present, still there is risk in the biological products in blood source in quality control simultaneously.So comprehensive above consideration the present invention abandons end user's seralbumin, for the freeze-dried preparation that does not contain human serum albumin, has observed one-year age, its thrombolysis activity does not have to change substantially.
Recombinant glucokinase sequence table＜110〉Hebei Yiling Medicine Inst. Co., Ltd＜120〉a kind of recombinant glucokinase lyophilized formulations and application＜160〉2＜210〉1＜211〉411＜212〉DNA＜213〉recombinant glucokinase (recombinant stephylokinase)＜220〉＜221〉mutation; Old_sequence＜222〉(19) ... (21); (115) ... (118), (127) ... (129)＜223〉n=A or C or T or G＜400〉1TCA AGT TCA TTC GAC AAA GCA AAA TAT AAA AAA GGC GAT GAC GCG AGTSer Ser Ser Phe Asp Lys Ala Lys Tyr Lys Lys Gly Asp Asp Ala Ser1 5 10 15TAT TTT GAA CCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTTTyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val
20 25 30GAT?GGT?AAA?GGA?AAT?GAA?TTG?CTA?TCC?CCT?CGT?TAT?GTC?GAG?TTT?CCTAsp?Gly?Lys?Gly?Asn?Glu?Leu?Leu?Ser?Pro?Arg?Tyr?Val?Glu?Phe?Pro
35 40 45ATT?AAA?CCT?GGG?ACT?ACA?CTT?ACA?AAA?GAA?AAA?ATT?GAA?TAC?TAT?GTCIle?LyS?Pro?Gly?Thr?Thr?Leu?Thr?Lys?Glu?Lys?Ile?Glu?Tyr?Tyr?Val
50 55 60GAA?TGG?GCA?TTA?GAT?GCG?ACA?GCA?TAT?AAA?GAG?TTT?AGA?GTA?GTT?GAAGlu?Trp?Ala?Leu?ASp?Ala?Thr?Ala?Tyr?Lys?Glu?Phe?Arg?Val?Val?Glu65 70 75 80TTA?GAT?CCA?AGC?GCA?AAG?ATC?GAA?GTC?ACT?TAT?TAT?GAT?AAG?AAT?AAGLeu?ASp?Pro?Ser?Ala?Lys?Ile?Glu?Val?Thr?Tyr?Tyr?Asp?Lys?Asn?Lys
85 90 95AAA?AAA?GAA?GAA?ACG?AAG?TCT?TTC?CCT?ATA?ACA?GAA?AAA?GGT?TTT?GTTLys?Lys?Glu?Glu?Thr?Lys?Ser?Phe?Pro?Ile?Thr?Glu?Lys?Gly?Phe?Val
100 105 110GTC?CCA?GAT?TTA?TCA?GAG?CAT?ATT?AAA?AAC?CCT?GGA?TTC?AAC?TTA?ATTVal?Pro?Asp?Leu?Ser?Glu?His?Ile?Lys?Asn?Pro?Gly?Phe?Asn?Leu?Ile
115 120 125ACA?AAG?GTT?GTT?ATA?GAA?AAG?AAA?
TAAThr?Lys?Val?Val?Ile?Glu?Lys?Lys?***
130 135＜210〉2＜211〉136＜212〉PRT＜213〉recombinant staphylokinase (recombinant stephylokinase)＜220〉＜221〉CONFLICT, VARIANT, CHAIN, SITE＜222〉(7,36,43)＜400〉2Ser Ser Ser Phe Asp Lys Ala Lys Tyr Lys Lys Gly Asp Asp Ala Ser1 5 10 15Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val
20 25 30Asp?Gly?Lys?Gly?Asn?Glu?Leu?Leu?Ser?Pro?Arg?Tyr?Val?Glu?Phe?Pro
35 40 45Ile?Lys?Pro?Gly?Thr?Thr?Leu?Thr?Lys?Glu?Lys?Ile?Glu?Tyr?Tyr?Val
50 55 60Glu?Trp?Ala?Leu?Asp?Ala?Thr?Ala?Tyr?Lys?Glu?Phe?Arg?Val?Val?Glu65 70 75 80Leu?Asp?Pro?Ser?Ala?Lys?Ile?Glu?Val?Thr?Tyr?Tyr?Asp?Lys?Asn?Lys
85 90 95Val?Pro?Asp?Leu?Ser?Glu?His?Ile?Lys?Asn?Pro?Gly?Phe?Asn?Leu?Ile
115 120 125Thr?Lys?Val?Val?Ile?Glu?Lys?Lys?***
130 135
Recombinant staphylokinase of the present invention carries out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date on March 13rd, 2003, preserving number 0913, classification called after colon bacillus.
Claims (9)
1, a kind of recombinant staphylokinase freeze-dried preparation is characterized in that comprising in the preparation nucleotide sequence and the amino acid sequence coded of following recombinant staphylokinase isolated genes:
TCA?AGT?TCA?TTC?GAC?AAA?GCA?AAA?TAT?AAA?AAA?GGC?GAT?GAC?GCG?AGT?TAT?TTT?GAA?CCA?ACA?GGC
Ser?Ser?Ser?Phe?Asp?Lys?Ala?Lys?Tyr?Lys?Lys?Gly?Asp?Asp?Ala?Ser?Tyr?Phe?Glu?Pro?Thr?Gly
CCG?TAT?TTG?ATG?GTA?AAT?GTG?ACT?GGA?GTT?GAT?GGT?AAA?GGA?AAT?GAA?TTG?CTA?TCC?CCT?CGT?TAT
Pro?Tyr?Leu?Met?Val?Asn?Val?Thr?Gly?Val?Asp?Gly?Lys?Gly?Asn?Glu?Leu?Leu?Ser?Pro?Arg?Tyr
GTC?GAG?TTT?CCT?ATT?AAA?CCT?GGG?ACT?ACA?CTT?ACA?AAA?GAA?AAA?ATT?GAA?TAC?TAT?GTC?GAA?TGG?GCA
Val?Glu?Phe?Pro?Ile?Lys?Pro?Gly?Thr?Thr?Leu?Thr?Lys?Glu?Lys?Ile?Glu?Tyr?Tyr?Val?Glu?Trp?Ala
TTA?GAT?GCG?ACA?GCA?TAT?AAA?GAG?TTT?AGA?GTA?GTT?GAA?TTA?GAT?CCA?AGC?GCA?AAG?ATC?GAA?GTC?ACT
Leu?Asp?Ala?Thr?Ala?Tyr?Lys?Glu?Phe?Arg?Val?Val?Glu?Leu?Asp?Pro?Ser?Ala?Lys?Ile?Glu?Val?Thr
TAT?TAT?GAT?AAG?AAT?AAG?AAA?AAA?GAA?GAA?ACG?AAG?TCT?TTC?CCT?ATA?ACA?GAA?AAA?GGT?TTT?GTT?GTC
Tyr?Tyr?Asp?Lys?Asn?Lys?Lys?Lys?Glu?Glu?Thr?Lys?Ser?Phe?Pro?Ile?Thr?Glu?Lys?Gly?Phe?Val?Val
CCA?GAT?TTA?TCA?GAG?CAT?ATT?AAA?AAC?CCT?GGA?TTC?AAC?TTA?ATT?ACA?AAG?GTT?GTT?ATA?GAA?AAG?AAA
Pro?Asp?Leu?Ser?Glu?His?Ile?Lys?Asn?Pro?Gly?Phe?Asn?Leu?Ile?Thr?Lys?Val?Val?Ile?Glu?Lys?Lys
TAA
***?；
Recombination sequence length is 411 bases, and it is codon GCA, 115-118 base codon GGA that recombination sequence is characterized as 19-21 base; The 127-129 base is codon CGT, and corresponding amino acid is Ala, Gly and Arg.
2, recombinant staphylokinase freeze-dried preparation according to claim 1, it is characterized in that comprising in the preparation recombinant staphylokinase, excipient N.F,USP MANNITOL, buffer reagent phosphoric acid salt, EDTA, permeate agent sodium-chlor wherein one or more.
3, recombinant staphylokinase freeze-dried preparation according to claim 1 and 2 is characterized in that the preparation proportion of composing is: recombinant staphylokinase 10mg, N.F,USP MANNITOL 15mg, Sodium phosphate dibasic 3.4mg, SODIUM PHOSPHATE, MONOBASIC 0.63mg, EDTA 1.0mg, sodium-chlor 8.5mg.
4, the preparation method of recombinant staphylokinase freeze-dried preparation comprises:
(1), the structure of recombinant staphylokinase engineering bacteria:
Select the streptococcus aureus strain to carry out the separation of chromosomal DNA, glucokinase gene separates primer and is:
F
SAK：5’GGAATTCATATGTCAAGTTCATTCGACAA 3’
R
SAK：5’CGGGATCCTTATTTCTTTTCTATAACAACC 3’
With above-mentioned chromosomal DNA is that template is carried out pcr amplification; Use pBV220 to be expression vector, the e. coli bl21 strain is the host bacterium, utilize the glucokinase gene of restriction enzyme EcoRI and BamHIPCR reaction amplification, simultaneously with identical enzymic digestion expression vector pBV220, electrophoresis reclaims, carrier is connected with gene, transformed into escherichia coli BL21 competent cell, the picking transformed clone is inoculated in the LB substratum that contains penbritin, the concussion overnight incubation, the plasmid DNA of extraction transformant utilizes EcoRI and BamHI enzyme to cut, enzyme is cut the product reaction product carry out the agarose gel electrophoresis analysis, the recombinant plasmid that filters out the glucokinase gene insertion is as engineering strain;
(2), fermentation
The clone strain that will contain recombinant plasmid is inoculated in the LB substratum that contains penbritin, and 30 ℃ of concussion overnight incubation are as primary seed solution; Be inoculated in the concentration of 0.1-0.5% and contain in the penbritin LB substratum, 30 ℃ of concussion overnight incubation are as fermentation seed liquid; Again with LB as nutrient solution, inoculation 5-6% fermentation seed liquid carries out jar to be cultivated, mixing speed 300rpm, air flow 0.8-1.0vvm, oxygen dissolving value generally by the initial reduction gradually of fermentation, get OD
600Induce program to starting between the 3-3.5 to heat up, wait to be warming up to 42 ℃ induce beginning after, portion-wise addition glucose is to supplement the nutrients, by regulating stirring velocity, the DO value is remained on more than 40%, induce after 5 hours in centrifugal receipts bacterium below 4 ℃, with the washing precipitation of zymophyte body and function damping fluid ,-20 ℃ freezing standby;
(3), purifying
The phosphate buffered saline buffer of zymophyte at pH7.6 suspended, under condition of ice bath, ultrasonication; Anion-exchange chromatography post damping fluid balance is passed the anion-exchange chromatography post with the supernatant liquor after the fragmentation, collects and passes the peak; Again the protein liquid of collecting being transferred pH with the 0.1N dilute phosphoric acid is 6.20, Zhiyang ion chromatography column purification, the recombinant staphylokinase protein molecular is adsorbed on the column packing, earlier with the flushing of pH6.20 damping fluid, remove unconjugated albumen, then, with the phosphoric acid salt EDTA pH6.20 buffer solution elution that contains NaCl, collect elution peak; Through S200 gel permeation chromatography purifying, collect elution peak again;
(4), preparation
With containing 0.1mol/L sodium-chlor, 20mmol/L phosphate buffered saline buffer (pH7.4), the solvent of the EDTA of 2mmol/L is diluted to 8.0mg/ml with recombinant staphylokinase, filtering with microporous membrane degerming with 0.22 μ m, adding stablizer and vehicle are 1.5% N.F,USP MANNITOL (weight percent), recombinant staphylokinase is sub-packed in the 10mg/ bottle in the cillin bottle of handling, carries out lyophilize then.
5, the preparation method of recombinant staphylokinase according to claim 4 is characterized in that the streptococcus aureus described in the step (1) is selected good strains in the field for seed to be selected as 1697 strains.
6, the preparation method of recombinant staphylokinase according to claim 4, the pcr amplification reaction condition described in the step (1) that it is characterized in that be 94 ℃ 40 seconds, 54 ℃ 40 seconds, 72 ℃ 2 minutes, carry out 30 circulations altogether, last circulation was extended 5 minutes for 72 ℃.
7, the preparation method of recombinant staphylokinase according to claim 4 is characterized in that the ultrasonication bacterium described in the step (3), adopts intermittently broken and remains in the ice bath and carry out, and each 30 seconds, 30 seconds at interval.
8, the recombinant staphylokinase freeze-dried preparation is characterized in that can be made into freeze drying powder preparations form, vein and intramuscular injection pharmaceutical solutions form, Liposomal formulation form, the microcapsule formulation form of thrombolysis clinically as the application of thrombolytic drug.
9, recombinant staphylokinase freeze-dried preparation according to claim 8 is as the application of thrombolytic drug, it is characterized in that can be used as preparing the raw material of the oral preparations of thrombolysis clinically.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445389C (en) * | 2005-10-14 | 2008-12-24 | 中国人民解放军军事医学科学院基础医学研究所 | Low antigen recombination glucokinase mutant and its preparation method |
| CN100445390C (en) * | 2005-10-14 | 2008-12-24 | 中国人民解放军军事医学科学院基础医学研究所 | A highly active, low antigenic staphylokinase derivative and its preparation method |
| CN101112358B (en) * | 2006-07-26 | 2011-04-20 | 中国科学院上海生命科学研究院 | Staphylokinase intramuscular injection and method for preparing the same |
| CN105727267A (en) * | 2016-02-05 | 2016-07-06 | 苏州康聚生物科技有限公司 | Recombined human hyaluronidase freeze-drying preparation and preparing method and application thereof |
-
2003
- 2003-03-14 CN CN03119315A patent/CN100580082C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445389C (en) * | 2005-10-14 | 2008-12-24 | 中国人民解放军军事医学科学院基础医学研究所 | Low antigen recombination glucokinase mutant and its preparation method |
| CN100445390C (en) * | 2005-10-14 | 2008-12-24 | 中国人民解放军军事医学科学院基础医学研究所 | A highly active, low antigenic staphylokinase derivative and its preparation method |
| CN101112358B (en) * | 2006-07-26 | 2011-04-20 | 中国科学院上海生命科学研究院 | Staphylokinase intramuscular injection and method for preparing the same |
| CN105727267A (en) * | 2016-02-05 | 2016-07-06 | 苏州康聚生物科技有限公司 | Recombined human hyaluronidase freeze-drying preparation and preparing method and application thereof |
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|---|---|
| CN100580082C (en) | 2010-01-13 |
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