CN1528894A - Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof - Google Patents
Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof Download PDFInfo
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Abstract
The invention belongs to biology technology field, which concretely refers to controlled release tissue factor path inhibitor (LTFPI) and the manufacturing method, and the application. The invention analyzes and experiments the biology information science and structure molecular biology science of tissue factor path inhibitor (TFPI) and its acceptor low density lipoprotein acceptor correspondent protein (LRP), it ascertains the part where the TFPI carboxy end combines with LRP and is eliminated. It designs TFPI carboxy end mutant, which is recombined with primary nucleus or eukarya expressing carrier after constructing LTFPI gene through PCR location, it converts bacillus coli or bici yeast, sifts the high expression project fungus. The primary project fungus are yeasted and expanded, crushed, then they are centrifugated and collects the inclusion body, purifies the LTFPI through molecular sift and ion interchanging two-step method; the eukarya project fungus are carried on with two-step purifying directly. The half-life are prolonged, it has good pour-depressant function.
Description
Technical field
The invention belongs to biological technical field, be specifically related to long-acting recombinant tissue factor pathway inhibitor (LTFPI).More specifically, the present invention relates to determine LTFPI mutational site and sudden change, gene clone, genetic expression, protein purification and renaturation, and functional examination.
Background technology
Tissue factor pathway inhibitor (tissue factor pathway inhibitor, TFPI) be serine protease inhibitor, the about 40kd of molecular weight, precursor has 304 amino acid, maturation protein then has 276 amino acid, and the latter is made up of N-terminal, three placed in-line Kunitz type structural domains and C-terminal, and major function has: (1) structural domain 1 suppresses tissue factor (Tissue Factor, TF) and proconvertin/VIIa, and by suppressing TF produce antiplatelet aggregative activity; (2) structural domain 2 anticoagulant factor X/Xa; (Broze GJ.Annu RevMed, 1995; 46:103-112); (3) structural domain 3 suppresses combining of intracellular toxins and CD14, produces significant anti-inflammatory effect; Structural domain 3 still is the binding site of heparin simultaneously, and heparin is by being incorporated into TFPI structural domain 3 performance anticoagulations; (4) C-terminal can combine with acceptor LDH receptor related protein (LRP), is the key position that influences the TFPI transformation period.Animal and human's body experiment confirm TFPI can effectively treat severe infections, dense toxicaemia, disseminated intravascular coagulation (DIC), MOFE (MOF), hypercoagulative state, thrombotic disease and tumour etc. and have good efficacy, is a kind of protein with applications well prospect.
Most of TFPI are synthetic and secretion by vascular endothelial cell, and smooth muscle cell, myocardial cell and fibroblast also can produce TFPI under certain condition.No matter be the TFPI of human body self, the reorganization wild-type TFPI that still gives, its transformation period is all very short, has only about 2 minutes, can remove from blood very soon.After TFPI entered blood circulation, LRP rapid and on the liver plasma membrane combined, and is degraded then and metabolism.The transformation period weak point is the shortcoming of TFPI maximum when the performance therapeutic action, and administration in 72-96 hour could keep curative effect continuously, needs 160-320mg each course of treatment approximately, and so big dosage has brought huge economical load to patient.Therefore, prolonging the transformation period of TFPI, reduce its dosage, is urgent problem.
Summary of the invention
The purpose of this invention is to provide long half time, have good anti-freezing function long-acting tissue factor pathway inhibitor (long half-life TFPI, LTFPI) and preparation method thereof.
The present invention is according to the analytical results of information biology and structural molecular biology, using gene engineering and protein engineering are suddenlyd change to the TFPI C-terminal, obtain the long-acting mutant of recombinant tissue factor pathway inhibitor (TFPI) that the transformation period obviously prolongs, show that with experimentation on animals LTFPI has good anti-freezing function in the long-acting tissue factor pathway inhibitor of called after (LTFPI), body.
The C-terminal of TFPI can combine with LRP, is degraded rapidly and metabolism then.The present invention has at first built TFPI and LRP molecule at the computer workstation patrix, then both is docked, and finds the interaction performance keying action of some key amino acid of TFPI C-terminal at TFPI and LRP.The present invention with some amino acid in the TFPI C-terminal 267-304 zone carry out in various degree substitute and deletion mutantion after, decline has in various degree appearred in both binding abilities, the transformation period of described LTFPI prolongs more than 10 times than TFPI, and possesses anti-tissue factor, proconvertin/VIIa and factor X/Xa function.The mutation effect that wherein 269,282,288 and 289 Methionins is all replaced with L-Ala is good.
The aminoacid sequence that has Sequence 1 behind the amino acid mutation of the present invention.
The present invention also provides the method for preparing LTFPI, comprise that preparation comprises the cDNA that expresses bioactive LTFPI encoding sequence part at least, clone cDNA segment in being fit to the carrier of expressing, with this carrier transfection host cell, be suitable for expressing this host cell of cultivation under the pulsating condition of this cDNA, and from culture, reclaiming and the required LTFPI of purifying.
The preparation of LTFPI gene comprises through the gene after the point mutation among the present invention, and with relevant plasmid reorganization, DNA digestion with restriction enzyme evaluation and screening positive colony, whether nucleotide sequence analysis checking gene is correct.
With LTFPI cDNA segment of the present invention and expression vector reorganization, form recombinant expression plasmid.The invention is not restricted to specific expression plasmid.In a preferred embodiment, the present invention uses prokaryotic expression carrier, for example pET28 etc.
Above-mentioned recombinant expression vector can import suitable host cell according to a conventional method.The present invention is not limited to any specific host cell, as long as it can express described recombinant expression vector.In a preferred embodiment, the present invention uses e. coli bl21 etc.
Expression product of the present invention is present in the inclusion body form in the cell space of host cell, and broken bacterium separates inclusion body, high concentration urea or guanidine hydrochloride dissolution inclusion body, and separation and purification LTFPI is through suitably promptly obtaining activated LTFPI after the renaturation.
All basic molecular biology operations are all with reference to＜molecular cloning experiment guide in the above technical scheme 〉.
Using gene engineering method of the present invention is produced LTFPI, and products obtained therefrom has anti-freezing efficiently, antiplatelet and anti-inflammatory properties, and preparation technology is easy, safety.Comparison shows that with wild TFPI character the LTFPI transformation period obviously prolongs, all the other function classes seemingly.LTFPI can be used for prevention and treatment severe infections disease and thrombotic disease etc. for the research of injection and non-injection provides raw material.
Description of drawings
Fig. 1 is that TFPI docks the interactional amino acid sites in back with LRP.
Fig. 2 is that the dPT method detects TFPI1-276 and TFPI1-161 anticoagulating active.
Embodiment
Embodiment 1 TFPI and LRP butt joint and the analysis on computer workstation
Use Insight II software, on computer workstation TFPI and LRP are docked, having searched out 269,282,288 and 289 Methionins of TFPI C-terminal is both interactional critical sites.
The design of embodiment 2 LTFPI, preparation and character are identified
(1) structure of the clone of LTFPI gene, transformation and prokaryotic expression plasmid rLTFPI-pET28
The method of design of LTFPI is: 269,282,288 and 289 Methionins of TFPI C-terminal are replaced with L-Ala.Sequence uses the PCR point mutation to obtain gene, and with the pUC19 reorganization, enzymolysis screening positive clone, nucleotide sequence analysis confirm that gene order is correct.Then the LTFPI gene is cut out,, constitute prokaryotic expression plasmid rLTFPI-pET28 with prokaryotic expression carrier pET28 reorganization.Transformed into escherichia coli JM109 extracts plasmid, identifies with respective limits restriction endonuclease enzymolysis, obtains the characteristic segment, confirms to obtain positive colony.
LTFPI gene acquisition methods is as follows:
Adopt overlapping extension, select the one section sequences Design downstream primer of 400bp behind the pET28 multiple clone site NotI for use:
5’＞CCA?CTA?CGT?GAA?CCA?TCA?CCC?TAA?TCA?AGT?＜3’
Overlapping extension primer 1: wherein the italic password from left to right is followed successively by K254A, K241A.
5’＞CCC?AAT?TAG?GCC?TCC?TTT?TGA?TAT?TCT?TTG?GAT?GAA?ACC?CGC?TTT?ACATGC?CCT?CAG?ACA＜3’
Overlapping extension primer 2: the italic password from left to right is followed successively by K254A, K260A, K261A.
5’＞AAA?GGA?GGC?CTA?ATT?GCG?ACC?AAA?AGA?AAA?AGA?GCG?GCG?CAG?AGA?GTGAAA?ATA?GCA?TAT?＜3’
With plasmid pET28-TFPI is template, upstream primer and 1 pairing of overlapping extension primer, downstream primer and the pairing of overlapping extension primer 2, PCR is used as intermediate behind the recovery product respectively, add upstream primer and downstream primer and be PCR, NcoI+NotI double digestion PCR product promptly gets K241A, K254A, K260A, the LTFPI of K261A sudden change.
Restriction enzyme is available from BRL company, e. coli jm109, plasmid pUC19 Invitrogen company.The structure of pLy4 is undertaken by currently known methods.
(2) the high copy of screening efficient expression strain
With plasmid rLTFPI-pLy4 electricity transformed into escherichia coli BL21, itself and escherichia coli chromosome are recombinated, G418 screens efficient expression strain.
Host bacterium: e. coli bl21
Nutrient solution: LBK
Kan working concentration: 30ug/ml
IPTG working concentration: 1mM
1. choose the mono-clonal colony inoculation to 5ml LBK medium, 37 ℃ * 250rpm to OD
600=1～2.
2. be seeded to 500ml LBK medium, 37 ℃ * 250rpm to OD
600=1～2.
3. be seeded to 25L LBK medium, 37 ℃ * 600rpm, dissolved oxygen 50% is to OD
600=0.6～0.8.
4. add 1M IPTG to final concentration 1mM, abduction delivering 3h.
5. 4 ℃ * 4000rpm * 35min collects thalline.
6. 50mM Tris.HCl pH8.0 washing thalline is 1 time, collects thalline, weighs.
The above-mentioned positive colony of picking is inoculated in 10ml low nutritive medium BMG " 1.34%YNB, (4 * 10
-5) vitamin H, 1% glycerine " in, 30 ℃ shake fast distant (250RPM), overnight incubation.Measure optical density(OD) OD next day
600, centrifugal reject BMG training liquid is trained liquid " 1.34%YNB, (4 * 10 with sterilized water washing back with BMM
-5) vitamin H, 0.5% methyl alcohol " be diluted to OD
600=1.Add percent by volume every day and be 0.5% methyl alcohol.Methanol induction was cultivated 7 days, every 24 hours sampling 1ml, measured anti-TF/FVIIa (rhTFPI-AP1) or anti-Fxa (rhTFPI-AP2) activity.The centrifugal precipitation of abandoning, supernatant is in-20 ℃ of preservations.Get 20ul after directly getting training liquid supernatant and 2x sample-loading buffer equal-volume mixing and go up sample, make reductibility SDS-PAGE electrophoresis.After the Xylene Brilliant Cyanine G R-250 dyeing, purity, molecular weight are decided in Pharmacia Imagenaster VDS scanning.As seen induce the back supernatant liquor at the about 6kd of molecular weight place the band that concentrates to be arranged, through scanning, target protein accounts for 84% of supernatant total protein; The result shows that expression product rhTFPI-AP1 has tangible anti-TF/FVIIa effect, and rhTFPI-AP2 has tangible anti-FXa effect.Above-mentioned reductibility SDS-PAGE is undertaken by the Laemmli method.
(3) fermentation expression engineering bacteria
As engineering bacteria, carry out high density fermentation with the 5L fermentor tank by above-mentioned screening high expression level bacterial strain then.Take out kind of a daughter bacteria from-70 ℃ of profound hypothermia refrigerators, thaw under the room temperature, at following stroke of YPD flat board of 100 grades of cleanliness factor conditions of kind of daughter bacteria culturing room, 30 ℃ of incubators were cultivated 2-3 days.Picking list bacterium colony from the flat board is inoculated under 100 grades of cleanliness factor conditions in the 10ml BMG nutrient solution equally, 30 ℃ of overnight incubation, and this is a primary seed solution.Again primary seed solution is added in the 140ml nutrient solution, cultivated 6-8 hour for 30 ℃, until OD
600=6, this is a secondary seed solution.After kind of daughter bacteria inserts, unearned increment, treat that the glycerine that adds in advance in the basic medium is depleted after, begin to replenish glycerine, replenish speed 16ml/L/h, treat OD
600Reach about 120, stop to add glycerine.After treating that glycerine all exhausts in the nutrient solution, the beginning methanol induction.Methanol feeding speed is increased to 12ml/L/h gradually from 1ml/L/h, keeps this speed later on always.Fermentation technique of the present invention is with low salt culture medium amplification engineering bacteria, carries out feed supplement with the glycerine solution that contains trace element before inducing, and carries out abduction delivering with the methanol solution that contains trace element after arriving certain cell concentration.
After the methanol induction 40h, stop fermentation, emit bacterium liquid immediately and carry out centrifugally from fermentor tank, the precipitation separation thalline is collected supernatant and is carried out purifying.RhTFPI-AP1 reaches Ki=0.12uM to the inhibition constant of TF/FVIIa in the supernatant, and rhTFPI-AP2 reaches Ki=0.09uM to the inhibition constant of FXa.Fermentation parameter is: 30 ℃ of temperature, oxygen capacity is controlled at 35 ± 5%, pH=5, stirring velocity and DO interlock.
(4) ultrafiltration and concentration desalination
The centrifugal institute supernatant that obtains with dilution in 1: 10, to 1.0L, is sloughed inorganic salt through Millipore ultra-filtration equipment (NMWL:3000, Millipore company) ultrafiltration and concentration.
(5) gel-filtration
Sephadex G-50 (Pharmacia company) post with sample on the ultrafiltration and concentration liquid, notes not stirring Sephadex G-50 glue face after using 20mmol/L PB (pH7.4) balance during application of sample.Use the PB wash-out then, flow velocity 10ml/min collects active peak.
(6) Q-Sepharose Fast Flow column chromatography
With 50mmol/L PB (pH7.4) balance Q-Sepharose F.F. (Pharmacia company) post of 10 times of volumes, the anti-freezing vigor of collecting after gel-filtration part is adsorbed onto it on Q-Sepharose F.F. post with the speed of 50ml/min.Wash post until OD with PB
280Reach 0.00, use 0-1mol/L NaCl (50mmol/L PB pH7.4) linear gradient elution then, collecting has the anticoagulating active part, packing, lyophilize ,-40 ℃ of preservations.
Chromatographic run of the present invention is routine operation.
(7) purity is identified and molecular weight determination
Sample carries out the 16.5%SDS-PAGE electrophoresis, after the Xylene Brilliant Cyanine G R-250 dyeing, and PharmaciaInagemaster VDS sweep measuring purity, molecular weight.Products obtained therefrom purity is more than 97%, the about 6kD of molecular weight.
(8) anti-TF/FVIIa determination of activity: (dilutedprothrombin time dPT) measures the prothrombin time after the use dilution, and method is as follows.
The thrombokinase powdered reagent is added 2mL physiological saline be diluted to final concentration 5000U/L, this is a thrombokinase stoste, dilutes 10,100 and 500 times respectively with physiological saline again, put 37 ℃ standby.Get 100 μ L normal humanplasmas, add 10 μ L protein solutions, add the thromboplastin solution of 100 μ L different concns respectively, hatch 1min for 37 ℃, add 100 μ L 30mmol/L CaCl
2Solution, PCT picks up counting.TFPI1-276 and TFPI1-161 final concentration are 0.2 μ mol/L in the reaction system, get 3 empirical average values and are designated as experimental result, and all reagent all need 37 ℃ of preheatings, all operate in the 2h and finish.The result shows that LTFPI obviously prolongs dPT.
(9) anti-FXa measures: use the low thing of color development and send out mensuration, method is as follows.
Adopt normal people's whole blood, the anti-freezing in 1: 9 of 109mM Sodium Citrate, 4 ℃ of centrifugal 15min, 4000r/min, equal-volume mix 30 portions of normal people's fresh plasmas, hatch 15min for 56 ℃, put 37 ℃ standby, this is with reference to blood plasma.Every milliliter this be defined as 1 activity unit (1U) with reference to the TFPI in the blood plasma.
Wild-type TFPI and LTFPI are dissolved in TS (20mM Tris/HCl pH7.8+150mM NaCl) 96 orifice plates respectively, every hole adds 100 μ L solution to be determined, add 100 μ L reaction solutions (100ng/mL FXa+0.2%BSA) again, incubated at room 30min, add chromophoric substrate Spectrozyme Xa to final concentration 0.25mM, survey OD
405The result shows that the anti-xa activity of LTFPI and wild-type TFPI are similar.Table 1 is wild-type TFPI and the anti-xa activity of LTFPI.
Table 1
| Sample | Anti-xa activity (U/mgx10 3) |
| ????TFPI | ????41 |
| ????LTFPI | ????39 |
The 3 LTFPI transformation period of embodiment are measured
The dPT method is surveyed the TFPI transformation period in the rat body: rats by intraperitoneal injection vetanarcol (40mg/kg) anesthesia, femoral vein administration (1mg/kg), the arteria carotis communis intubate, different time is got blood respectively after the administration, the anti-freezing in 1: 9 of 109mM Sodium Citrate, 4 ℃ of centrifugal 15min, 4000r/min, draw upper plasma, measure dPT.
With 500 times of thrombokinase standardized solution dilutions, this is the thrombokinase working fluid with physiological saline.Get 100 μ L blood plasma, add 100 μ L thrombokinase working fluids, hatch 1min for 37 ℃, add 10 μ L 250mmol/LCaCl
2Solution, PCT picks up counting.Get 3 empirical average values and be designated as experimental result.
With wild-type TFPI is control group, observes the transformation period of LTFPI (K282A) and LTFPI (K269A, K282A, K288A and K289A), the T of visible LTFPI of result (K282A) and LTFPI (K269A, K282A, K288A and K289A)
1/2 α5.1 times and 14.5 times have been prolonged than TFPI respectively.Transformation period calculates and uses the Shanghai grand energy PKS of softcom limited statistics software.Table 2 is the variations of dPT before and after the rat administration.Table 3 is secondary fitting parameter table---two-compartment models.
Table 2
| Time after the administration (minute) | PCT (second) | ||
| ?????TFPI | ????LTFPI ????(K282A) | LTFPI (K269A, K282A, K288A and K289A) | |
| ????0 | ?????77.6 | ????78 | ???78.8 |
| ????1 | ?????106 | ????104 | ???106.2 |
| ????2 | ?????103 | ????104 | ???105.8 |
| ????3 | ?????95.3 | ????104 | ???104.6 |
| ????4 | ?????93.8 | ????99.7 | ???104 |
| ????5 | ?????93.2 | ????99.5 | ???95.6 |
| ????7 | ?????92.5 | ????99.2 | ???93.2 |
| ????9 | ?????91.8 | ????98.0 | ???92 |
| ????12 | ?????91.5 | ????96.6 | ???91.4 |
| ????15 | ?????89.6 | ????95.1 | ???91.4 |
| ????20 | ?????86.6 | ????94.1 | ???89.6 |
| ????25 | ?????84.2 | ????92.5 | ???89 |
| ????30 | ?????84.2 | ????92.9 | ???86 |
Table 3.
| The medication object | Dosage | ?T 1/2α | ?T 14/2β | ??CL | ?V d | ??V 1 | ??V 2 | ??AUC ∞ | ??K 10 | ??K 21 | ??K 12 |
| ????1 | ??1 | ?1.70 | ?46.21 | ??0.062168 | ?4.1445 | ??1.6406 | ??2.5039 | ?16.085 | ??0.037893 | ??0.16133 | ??0.22332 |
| ????2 | ??1 | ?8.66 | ?187.2 | ??0.013921 | ?3.7598 | ??2.2779 | ??1.4820 | ?71.832 | ??0.006112 | ??0.04849 | ??0.02914 |
| ????3 | ??1 | ?24.72 | ?25.16 | ??0.076884 | ?2.7908 | ??2.7612 | ??0.0296 | ?13.007 | ??0.027845 | ??0.02774 | ??0.00000 |
| Mean | ?11.70 | ?86.19 | ??0.05099 | ?3.5650 | ??2.2266 | ??1.338 | ??33.64 | ??0.02395 | ??0.07919 | ??0.0842 | |
| ????SD | ?11.81 | ?88.11 | ??0.03294 | ?0.6976 | ??0.5620 | ??1.243 | ?33.11 | ??0.01624 | ??0.07189 | ??0.1214 |
Wherein, 1 in the medication object represented TFPI,
2 represent LTFPI (K282A),
3 represent LTFPI (K269A, K282A, K288A and K289A).
Need not further to elaborate, according to above-mentioned disclosed content, those skilled in the art can use the present invention to greatest extent.Therefore, above-mentioned preferred specific embodiments should be understood that only to illustrate, but not limits the scope of the invention by any way.
From the above description, those skilled in the art can understand essential characteristic of the present invention at an easy rate, and under the situation that does not depart from its purport and scope, can carry out various changes and improvement to the present invention.
SEQUENCE?LISTING
Organization?Applicant
Street: No. 138, medical college road
City: Shanghai
State: Shanghai
Country: China
PostalCode：200032
PhoneNumber：8621-54237045
FaxNumber：8621-64037324
＜110〉Fudan University
＜120〉long-acting recombinant tissue factor pathway inhibitor and preparation method thereof
<160>1
<170>PatentIn?version?3.2
<210>1
<211>304
<212>PRT
<213>human
<400>1
Met?Ile?Tyr?Thr?Met?Lys?Lys?Val?His?Ala?Leu?Trp?Ala?Ser?Val?Cys
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Asn?Leu?Ala?Pro?Ala?Pro?Leu?Asn?Ala?Asp?Ser?Glu?Glu
20??????????????????25??????????????????30
Asp?Glu?Glu?His?Thr?Ile?Ile?Thr?Asp?Thr?Glu?Leu?Pro?Pro?Leu?Lys
35??????????????????40??????????????????45
Leu?Met?His?Ser?Phe?Cys?Ala?Phe?Lys?Ala?Asp?Asp?Gly?Pro?Cys?Lys
50??????????????????55??????????????????60
Ala?Ile?Met?Lys?Arg?Phe?Phe?Phe?Asn?Ile?Phe?Thr?Arg?Gln?Cys?Glu
65??????????????????70??????????????????75??????????????????80
Glu?Phe?Ile?Tyr?Gly?Gly?Cys?Glu?Gly?Asn?Gln?Asn?Arg?Phe?Glu?Ser
85??????????????????90??????????????????95
Leu?Glu?Glu?Cys?Lys?Lys?Met?Cys?Thr?Arg?Asp?Asn?Ala?Asn?Arg?Ile
100?????????????????105?????????????????110
Ile?Lys?Thr?Thr?Leu?Gln?Gln?Glu?Lys?Pro?Asp?Phe?Cys?Phe?Leu?Glu
115?????????????????120?????????????????125
Glu?Asp?Pro?Gly?Ile?Cys?Arg?Gly?Tyr?Ile?Thr?Arg?Tyr?Phe?Tyr?Asn
130?????????????????135?????????????????140
Asn?Gln?Thr?Lys?Gln?Cys?Glu?Arg?Phe?Lys?Tyr?Gly?Gly?Cys?Leu?Gly
145?????????????????150?????????????????155?????????????????160
Asn?Met?Asn?Asn?Phe?Glu?Thr?Leu?Glu?Glu?Cys?Lys?Asn?Ile?Cys?Glu
165?????????????????170?????????????????175
Asp?Gly?Pro?Asn?Gly?Phe?Gln?Val?Asp?Asn?Tyr?Gly?Thr?Gln?Leu?Asn
180?????????????????185?????????????????190
Ala?Val?Asn?Asn?Ser?Leu?Thr?Pro?Gln?Ser?Thr?Lys?Val?Pro?Ser?Leu
195?????????????????200?????????????????205
Phe?Glu?Phe?His?Gly?Pro?Ser?Trp?Cys?Leu?Thr?Pro?Ala?Asp?Arg?Gly
210?????????????????215?????????????????220
Leu?Cys?Arg?Ala?Asn?Glu?Asn?Arg?Phe?Tyr?Tyr?Asn?Ser?Val?Ile?Gly
225?????????????????230?????????????????235?????????????????240
Lys?Cys?Arg?Pro?Phe?Lys?Tyr?Ser?Gly?Cys?Gly?Gly?Asn?Glu?Asn?Asn
245?????????????????250?????????????????255
Phe?Thr?Ser?Lys?Gln?Glu?Cys?Leu?Arg?Ala?Cys?Lys?Ala?Gly?Phe?Ile
260?????????????????265?????????????????270
Gln?Arg?Ile?Ser?Lys?Gly?Gly?Leu?Ile?Ala?Thr?Lys?Arg?Lys?Arg?Ala
275?????????????????280?????????????????285
Ala?Gln?Arg?Val?Lys?Ile?Ala?Tyr?Glu?Glu?Ile?Phe?Val?Lys?Asn?Met
290?????????????????295?????????????????300
Claims (8)
1. the long-acting mutant of a class recombinant tissue factor pathway inhibitor (TFPI), the long-acting tissue factor pathway inhibitor of called after (LTFPI), it is characterized in that the aminoacid sequence that has sequence 1 behind some amino acid mutation in the TFPI C-terminal 267-304 zone, the transformation period of described LTFPI prolongs more than 10 times than TFPI, has anti-tissue factor, proconvertin/VIIa and factor X/Xa function.
2. based on the described LTFPI of claim 1, the sudden change in the C-terminal 267-304 zone can be to replace or excision.
3. based on claim 2, amino acid whose replacement can be any amino acid in the C-terminal 267-304 zone.
4. based on claim 2, the excision in the C-terminal 267-304 zone can be one, a plurality of or whole.
5. based on claim 3,269,282,288 and 289 Methionins are replaced with L-Ala.
6. based on the preparation method of the LTFPI of claim 1, obtain by following step:
(1) gene of acquisition encoding sequence 1;
(2) gene after will suddenling change and protokaryon or carrier for expression of eukaryon reorganization;
(3) recombinant expression vector is transduceed protokaryon or eukaryotic expression system;
(4) LTFPI after expressing is behind molecular sieve and ion exchange chromatography, obtains purity greater than 95% LTFPI;
(5) LTFPI behind the prokaryotic system expression and purification obtains active through renaturation.
7. based on claim 6, prokaryotic expression carrier can be pET-28 or pLY-4; Carrier for expression of eukaryon can be pPIC9K.
8. based on claim 6, prokaryotic expression system can be BL21 or JF1125; Eukaryotic expression system can be GS115.
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100451116C (en) * | 2006-09-14 | 2009-01-14 | 中国医学科学院生物医学工程研究所 | Human tissue factor pathway inhibitory factor mutation gene mTFPI, recombination carrier including the same and host cell thereof |
| CN100460507C (en) * | 2006-09-14 | 2009-02-11 | 中国医学科学院生物医学工程研究所 | Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same |
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