CN1367176A - Novel recombinant human tissue factor pathway inhibitor active peptide and its preparation method - Google Patents

Abstract

The present invention relates to a recombinant human tissue factor pathway inhibitor active peptide with anticoagulant and antithrombotic activities, its preparation and application. According to the analysis of the structure of total-length tissue factor pathway inhibitor and its bio-chemical property, the said invention designs novel rh TFPI-API and rh TFPI-AP2 structures, and utilizes the processes of recombining the mutated tissue factor pathway inhibitor structure domain 1 and structure domain 2 genes with eucaryotic expression factor pPIC9K, converting methyl alcohol nutrient yeast GS 115, screening high-expression engineering bacterium, fermenting and amplifying, centrifugally collecting supernatant fluid and adopting a three-step method to purity rh TFPI-AP1 and rh TFPI-AP2. After having been freezed-dried, the obtained product possesses anticoagulant and antithrombotic activities, and can produce obvious effect for preventing thrombotic diseases.

Description

Novel recombinant human tissue factor pathway inhibitor active peptide and preparation method thereof

Technical field

The invention belongs to biological technical field, be specifically related to novel recombinant human institutional approach inhibitor active peptide.More specifically, the present invention relates to novel recombinant human institutional approach inhibitor active peptide 1 and peptide 2, compare with total length people institutional approach inhibition, bioactive peptide 1 keeps anti-tissue factor/factor VIIa activity, and peptide 2 keeps anti-factor xa activity.The invention still further relates to the preparation method and the application thereof of this recombinant human tissue factor pathway inhibitor active peptide.

Background technology

Tissue factor pathway inhibitor (tissue factor pathway inhibitor, TFPI) be a kind of endogenous anticoagulant substances, serine protease inhibitor for multivalence Kunitz type, its major function is to regulate tissue factor (TF) inductive coagulation process (Broze GJ.AnnuRev Med, 1995; 46:103-112).The structure of TFPI comprises the amino acid district, three Kunitz type proteolytic enzyme inhibitory areas and carboxyl petiolarea, and first Kunitz district is essential by supressor VIIa/TF mixture; The inhibition of second Kunitz district and FXa is closely related; The 3rd Kunitz district has the heparin combining site; The structure of carboxyl petiolarea helps (Enjyoji K et al.Biochemistry, 1995 of combining of TFPI and lipoprotein, sugared ammonia polysaccharide and heparin; 34:5725-5735).

In recent years, the researchist uses recombinant human TFPI to carry out a series of experimentation on animals and some clinical experiments, the result shows that TFPI all has better curative effect for Coronary thrombosis, pyemia, disseminated intravascular coagulation, apoplexy, cancer, adult respiratory distress syndrome and ischemical reperfusion injury etc., and danger of bleeding is starkly lower than Low molecular heparin, be that a kind of security is good, anti-freezing that curative effect is high and antithrombotic reagent (Jeske W, et al.Seminars in thrombosis and hemostasis, 1996; 22 (2): 213-219).

Summary of the invention

The purpose of this invention is to provide TFPI bioactive peptide with anti-freezing and anti-bolt function and preparation method thereof.The present invention transforms people TFPI, and the bioactive peptide 1 of acquisition has the active and antiplatelet function of tangible anti-TF/FVIIa, and the called after RECOMBINANT HUMAN TFPI ACTIVE PEPTIDE 1 (recombinant human TFPI active peptide 1, rhTFPI-AP1); The peptide 2 that obtains has tangible anti-FXa activity, and the called after RECOMBINANT HUMAN TFPI ACTIVE PEPTIDE 2 (recombinant human TFPI active peptide 2, rhTFPI-AP2).Both all have anti-thrombosis function, and the associating result of use is better.

TFPI is the strand glycoprotein of the about 40kd of molecular weight, is made up of 276 amino-acid residues.The structure of TFPI comprises amino petiolarea, three Kunitz type proteolytic enzyme inhibitory areas (structural domain).Each structural domain all is ring texturees that 51 amino-acid residues are formed, and three pairs of disulfide linkage are arranged.The avtive spot of structural domain 1 is positioned at Lys36-Ala37, and the avtive spot of structural domain 2 is positioned at Arg107-Gly108, and the avtive spot of structural domain 3 is positioned at Arg199-Ala200.TFPI has three glycosylation sites, lays respectively at Asn117, Asn167 and Asn228, but glycosylated activity (Nakahara Y, et al.Biochemistry, 1996 that influence TFPI are invariably arranged; 35:6450-6459; Burgering M, et al.J.Mol.Biol.1997; 269:395-407).According to the characteristic more than the TFPI, the present invention has designed the molecular structure of rhTFPI-AP1 and rhTFPI-AP2.RhTFPI-AP1 has kept TFPI 22-79, and Asp31 and Asp32 are replaced by Lys31 and Ala32.RhTFPI-AP2 has kept TFPI 93-150, and Asp100, Glu101, Glu102 and Asn117 are replaced by Arg100, Arg101, Gly102 and Gln103.Table 1 sequence table SEQ ID NO:1, the aminoacid sequence that shows rhTFPI-AP1 of the present invention, table 2 sequence table SEQ ID NO:2, the aminoacid sequence that shows rhTFPI-AP2 of the present invention, table 3 sequence table SEQ ID NO:3, the nucleotide sequence that shows rhTFPI-AP1 of the present invention, table 4 sequence table SEQ ID NO:4, the nucleotide sequence of demonstration rhTFPI-AP2 of the present invention.

The present invention also provides the method for preparing rhTFPI-AP1 of the present invention and rhTFPI-AP2, comprises that preparation comprises the cDNA that expresses bioactive rhTFPI-AP1 and rhTFPI-AP2 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, reclaim and required rhTFPI-AP1 of purifying and rhTFPI-AP2.

The preparation of rhTFPI-AP1 and rhTFPI-AP2 gene comprises through the gene after the point mutation among the present invention, and with plasmid PUC19 reorganization, DNA digestion with restriction enzyme evaluation and screening positive colony, whether nucleotide sequence analysis checking gene is correct.

With 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 carrier for expression of eukaryon, for example pPIC9K 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 methyl alcohol nutritional type yeast strain such as GS115, KM71 etc.

Expression product of the present invention is secreted into outside the born of the same parents, is present in the host cell nutrient solution supernatant.Only need centrifugal removal host cell, can separate and the purifying desired product the nutrient solution supernatant.

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 2 kinds of bioactive peptides, products obtained therefrom has anti-freezing efficiently and anti-bolt function, and preparation technology is easy, safety, comparison shows that with total length TFPI character, the anti-freezing of rhTFPI-AP1 and rhTFPI-AP2 and anti-bolt function and TFPI are similar, but molecular weight obviously reduces, and research slow-release injected for non-vein and non-injection provides raw material, can be used for prevention and treatment thrombotic diseases

The design of embodiment embodiment 1 rhTFPI-AP1 and rhTFPI-AP2, preparation and character are identified

(1) structure of the clone of rhTFPI-AP1 and rhTFPI-AP2 gene, transformation and eukaryon expression plasmid rhTFPI-AP1-pPIC9K and rhTFPI-AP2-pPIC9K

According to the design amino-acid sequence of rhTFPI-AP1 and rhTFPI-AP2, method of design is: rhTFPI-AP1 has kept TFPI 22-79, and Asp31 and Asp32 are replaced by Lys31 and Ala32.RhTFPI-AP2 has kept TFPI 93-150, and Asp100, Glu101, Glu102 and Asn117 are replaced by Arg100, Arg101, Gly102 and Gln103.According to yeast preference codon design nucleotide sequence.Aminoacid sequence and nucleotide sequence inspection accompanying drawing.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 rhTFPI-AP1 and rhTFPI-AP2 gene are cut out,, constitute eukaryon expression plasmid rhTFPI-AP1-pPIC9K and rhTFPI-AP2-pPIC9K with Yeast expression carrier pPIC9K reorganization.Transformed into escherichia coli JM109 extracts plasmid, identifies with respective limits restriction endonuclease enzymolysis, obtains the characteristic segment, confirms to obtain positive colony.

Restriction enzyme is available from BRL company, and e. coli jm109, plasmid pUC19, methyl alcohol nutritional type yeast GS115, yeast multiple copied expression vector pPIC9K are available from Invitrogen company.

(2) the high copy of screening efficient expression strain

Plasmid rhTFPI-AP1-pPIC9K and rhTFPI-AP2-pPIC9K electricity are transformed methyl alcohol nutritional type yeast GS115, itself and yeast chromosomal are recombinated, G418 screens efficient expression strain.

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 (250 RPM), 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 (pH 7.4) 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 pH 7.4) 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 Pharmacia Inagemaster  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: use the prothrombin time after diluting.

(9) anti-FXa measures: use the low thing of color development and send out mensuration.Embodiment 2 preparation rhTFPI-AP1 and rhTFPI-AP2

The structure of rhTFPI-AP1 and rhTFPI-AP2 eukaryon expression plasmid, the screening of high copy efficient expression strain, the fermentation expression of engineering bacteria is all with embodiment 1.Supernatant liquor is collected in the centrifugal back of fermented liquid, carries out purifying by laxative remedy and prepares pure product rhTFPI-AP1 and rhTFPI-AP2.

(1) Poros HQ column chromatography concentrates

With 50mmol/L PB (pH 7.4) balance Poros HQ (Pharmacia company) post of 10 times of volumes, the supernatant that collect the centrifugal back of fermented liquid is adsorbed onto it on Poros HQ post with the speed of 100ml/min.Wash post until OD with PB ₂₈₀Reach 0.00, use 0-1mol/L NaCl (50mmol/L PB pH 7.4) linear gradient elution then, collecting has anti-freezing vigor part.

(2) gel-filtration

Sephadex G-50 (Pharmacia company) post is with after 20mmol/L PB (pH 7.4) balance, with sample on the Poros HQ column chromatography concentrated solution, and disturbance SephadexG-50 glue face not when noting application of sample.Use the PB wash-out then, flow velocity 10ml/min collects active peak.

(3) Q-Sepharose Fast Flow column chromatography

With 50mmol/L PB (pH 7.4) 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 pH 7.4) linear gradient elution then, collecting has the anticoagulating active part, packing, lyophilize ,-40 ℃ of preservations.

(4) 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 Pharmacia Inagemaster  VDS sweep measuring purity, molecular weight.Products obtained therefrom purity is more than 97%, the about 6kD of molecular weight.

(5) anti-TF/FVIIa determination of activity

(6) anti-FXa measures embodiment 3 rhTFPI-AP1 and the application of rhTFPI-AP2 in thrombotic diseases

Use prepared rhTFPI-AP1 of the present invention and rhTFPI-AP2 and carry out the antithrombotic experiment, confirm that it has good effect in the control of thrombotic diseases, compare with Low molecular heparin, anti-bolt effect is obvious, and the influence of coagulation function is significantly less than Low molecular heparin.

(1) suppresses venous thrombosis

Damage rabbit internal jugular vein brings out venous thrombosis.Model forms 2 hours posterior vein drug administration by injection.Negative control group gives physiological saline, and positive controls gives Low molecular heparin 60 anti-Xa U/kg, and the treatment group gives rhTFPI-AP1 and rhTFPI-AP2 0.5 and 1.0mg/kg respectively.Removal of thromboses after 4 hours is weighed after the administration.The result shows that rhTFPI-AP1 and rhTFPI-AP2 more can suppress venous thrombosis than Low molecular heparin, and very little to the influence of coagulation function.Table 5 is that rhTFPI-AP1 and rhTFPI-AP2 suppress the venous thrombosis experimental result.Table 5

		Negative control physiological saline	Low molecular heparin 60 anti-Xa U/kg	???rhTFPI-AP1 ???0.5mg/kg	???rhTFPI-AP1 ???1.0mg/kg	???rhTFPI-AP2 ???0.5mg/kg	??rhTFPI-AP1 ??1.0mg/kg
								Thrombus weight		????11.8	????2.2	????2.3	????0.4	????2.5	????0.6
Anti-Xa activity	Before the administration	????0.04	????0.03	????0.03	????0.03	????0.03	????0.04
								During administration	????0.03	????0.85	????0.4	????0.5	????0.8	????1.2
	After the administration	????0.03	????0.3	????0.09	????0.11	????0.4	????0.6
								Dilution PT experiment	Before the administration	????22	????22	????21	????21	????22	????21
During administration	????21	????93	????22	????24	????23	????24
							After the administration		????21	????37	????21	????22	????22	????23
?APTT	Before the administration	????26	????24	????25	????25	????26									????25
							During administration	????23	????61	????27	????29	????29	????30
	After the administration	????23	????34	????26	????28	????27								????28

(2) suppress artery thrombosis

With balloon catheter damage rabbit femoral artery endotheliocyte, and the blood vessel of ligation damaged portion, make regional flow's retardance and bring out artery thrombosis.Negative control group gives physiological saline, and positive controls gives Low molecular heparin 60-120 anti-Xa U/kg, and the treatment group gives rhTFPI-AP1 and rhTFPI-AP2 0.5-2.0mg/kg respectively.Experimental result shows that rhTFPI-AP1 and rhTFPI-AP2 can make the artery thrombosis rate descend, and are dosage correlation.Table 6 is the effect that rhTFPI-AP1 and rhTFPI-AP2 suppress artery thrombosis.Table 6

		Negative control physiological saline	Low molecular heparin 60 anti-Xa U/kg	??rhTFPI-AP1 ??0.5mg/kg	??rhTFPI-AP1 ??1.0mg/kg	???rhTFPI-AP2 ???0.5mg/kg	???rhTFPI-AP1 ???1.0mg/kg
								Thrombus weight		????12.5	????3.8	????2.9	????0.5	????2.8	????0.7
Anti-Xa activity	Before the administration	????0.05	????0.04	????0.03	????0.03	????0.03	????0.04
								During administration	????0.04	????0.91	????0.48	????0.54	????0.82	????1.22
	After the administration	????0.04	????0.36	????0.08	????0.14	????0.42	????0.66
								Dilution PT experiment	Before the administration	????22	????23	????22	????21	????22	????21
During administration	????22	????91	????23	????23	????23	????24
							After the administration		????23	????38	????21	????22	????22	????22
??APTT	Before the administration	????25	????24	????25	????24	????26									????25
							During administration	????23	????64	????26	????28	????28	????29
	After the administration	????24	????36	????25	????27	????26								????27

(3) prevent and treat disseminated intravascular coagulation (DIC)

DIC forms to rabbit injection endotoxin induced.Compare with Low molecular heparin, intravenous injection rhTFPI-AP1 and rhTFPI-AP2 can obviously correct dysfunction of blood coagulation and the thrombotic degree due to the DIC.Table 7 is the effect of rhTFPI-AP1 and rhTFPI-AP2 treatment DIC.

Table 7

		Negative control physiological saline	Low molecular heparin 60 anti-Xa U/kg	???rhTFPI-AP1 ???0.5mg/kg	??rhTFPI-AP1 ??1.0mg/kg	???rhTFPI-AP2 ???0.5mg/kg	???rhTFPI-AP1 ???1.0mg/kg
								Anti-Xa activity	Before the administration	????0.02	????0.02	????0.03	????0.03	????0.03	????0.02
During administration	????0.02	????0.66	????0.42	????0.51	????0.81	????1.18
							After the administration		????0.01	????0.43	????0.07	????0.13	????0.40	????0.61
Dilution PT experiment	Before the administration	????18	????18	????17	????18	????17									????18
							During administration	????18	????76	????24	????22	????21	????23
	After the administration	????16	????34	????20	????21	????20								????21
							??APTT	Before the administration	????20	????21	????21	????20	????20		????20
During administration	????19	????54	????25	????27	????26	????28
								After the administration	????17	????33	????24	????25	????25	????26
Mortality ratio		????5/5 ????100％	????3/5 ????60％	????2/5 ????40％	????1/5 ????20％	????3/5 ????60％									????1/5 ????20％

(4) control that thrombus forms again behind the coronary angioplasty (PTCA)

The ramus descendens anterior arteriae coronariae sinistrae endotheliocyte of damage dog brings out the obstructive coronary artery thrombus and forms.Use rhTFPI-AP1 and rhTFPI-AP2 additional medication, can promote coronary artery logical again, suppress thrombus and form again, reduce the weight of remaining thrombus, and be the dependency of dosage as recombined streptokinase.Compare with Low molecular heparin, the time that generation is led to again is shorter, and the dabbling again time length prolongs, and residual thrombus weight is lighter.Table 8 is that rhTFPI-AP1 and rhTFPI-AP2 suppress the effect that arterial thrombus forms again.

Table 8

		Negative control physiological saline	Low molecular heparin 60 anti-Xa U/kg	??rhTFPI-AP1 ??0.5mg/kg	???rhTFPI-AP1 ???1.0mg/kg	???rhTFPI-AP2 ???0.5mg/kg	????rhTFPI-AP1 ????1.0mg/kg
								Thrombus weight		????10.2	????3.2	????2.7	????0.4	????2.4	????0.5
Anti-Xa activity	Before the administration	????0.05	????0.05	????0.04	????0.05	????0.04	????0.05
								During administration	????0.05	????0.93	????0.46	????0.51	????0.78	????1.26
	After the administration	????0.04	????0.43	????0.06	????0.15	????0.34	????0.68
								Dilution PT experiment	Before the administration	????22	????23	????22	????22	????23	????23
During administration	????23	????94	????24	????24	????23	????25
							After the administration		????24	????36	????22	????23	????22	????23
??APTT	Before the administration	????25	????24	????25	????24	????25									????25
							During administration	????24	????61	????27	????27	????27	????29
	After the administration	????24	????38	????25	????26	????26								????26

Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front 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.Table 1 SEQ ID NO:11 11 21 31MHSFCEAFKA AKCPCKAIMK RFFFNIFTRQ CEEFIYGGCEG41 51NQNRFESLEE CKKNCTRD table 2 SEQ ID NO:21 11 21 31KPDFCFL GRRPGICRGYITR YFYN QQTKQC ERFKYGGCLG41 51NMNNFETLEE CKNICEDG table 3 SEQ ID NO:3ATG CAT TCA TTT TGT GCA TTC AAG GCG GCA AAGGGC CCA TGT AAA GCAATC ATG AAA AGA TTT TTC TTC AAT ATT TTC ACT CGA CAG TGC GAA GAATTT ATA TAT GGG GGA TGT GAA GGA AAT CAG AAT CGA TTT GAA AGT CTGGAA GAG TGC AAA AAA ATG TGT ACA AGA GAT table 4 SEQ ID NO:4AAG CCA GAT TTC TGC TTT TTG GGA CGA AGGCCT GGA ATA TGT CGA GGTTAT ATT ACC AGG TAT TTT TAT AAC AAT CGAACA AAA CAG TGT GAA CGTTTC AAG TAT GGT GGA TGC CTG GGC AAT ATG AAC AAT TTT GAG ACA CTGGAA GAA TGC AAG AAC ATT TGT GAA GAT GGT

Claims (11)

1. novel recombinant human tissue factor pathway inhibitor active peptide, it is characterized in that changing the structure of human tissue factor pathway inhibitor structural domain 1 and structural domain 2, obtain to keep bioactive peptide 1 and the active peptide 2 of anti-FXa of special, the efficient anti-tissue factor of hTFPI (TF)/FVIIa, bioactive peptide 1 rhTFPI-AP1 has the aminoacid sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ IDNO:3, and peptide 2 rhTFPI-AP2 has the aminoacid sequence of SEQ ID NO:2 and the nucleotide sequence of SEQ ID NO:4.

2. novel recombinant human tissue factor pathway inhibitor active peptide according to claim 1, it is characterized in that described bioactive peptide rhTFPI-AP1 has kept TFPI 22-79, D59 and D60 are replaced by K59 and A60, rhTFPI-AP2 has kept TFPI 93-150, and D128, E129 and E130 are replaced by R128, R129 and G130.

3. method for preparing novel recombinant human tissue factor pathway inhibitor active peptide is characterized in that adopting the following step: (1) according to the structure of total length hTFPI and the analysis of biochemical characteristic thereof, design rhTFPI-AP1 and rhTFPI-AP2 molecular structure; (2) clone rhTFPI-AP1 and rhTFPI-AP2 gene in the carrier that is suitable for expressing; (3) host cell that is suitable for expressing with this recombinant vectors transfection; (4) cultivate host cell being suitable for expressing under the segmental condition of this cDNA, and therefrom reclaim and the required product of purifying.

4. method according to claim 3, wherein employed carrier is not limited to specific expression vector, as long as it can be recombinated with described cDNA fragment, forms suitable plasmid of expressing.

5. according to right 4 described methods, wherein employed carrier is a carrier for expression of eukaryon.

6. according to right 5 described methods, wherein employed carrier is pPIC9K.

7. method according to claim 3, wherein employed host cell is not limited to any specific host cell, as long as it can express described recombinant expression vector.

8. according to right 7 described methods, wherein employed host cell is methyl alcohol nutritional type yeast strain GS115.

9. method according to claim 3, wherein cultivating the employed method of host cell is fermentation process, fermentation parameter is: 30 ℃ of temperature, oxygen capacity is controlled at 35 ± 5%, pH=5, stirring velocity and DO interlock.

10. method according to claim 3, wherein rhTFPI-AP1 and rhTFPI-AP2 end product are by centrifugal collection supernatant behind the engineering bacterium fermentation, through ultrafiltration, gel-filtration, ion-exchange three-step approach purifying and obtain.

11. the application in the medicine of preparation control thrombotic diseases according to described rhTFPI-AP1 of claim 1-3 and rhTFPI-AP2.SEQ?ID?NO：?11???????????11??????????21?????????31MHSFCEAFKA? AKCPCKAIMK?RFFFNIFTRQ?CEEFIYGGCEG41??????????51NQNRFESLEE?CKKNCTRD???SEQ?ID?NO：?21????????????11?????????21???????????31KPDFCFL GRR?PGICRGYITR?YFYN QQTKQC?ERFKYGGCLG41???????????51NMNNFETLEE?CKNICEDG???SEQ?ID?NO：?3ATG?CAT?TCA?TTT?TGT?GCA?TTC?AAG?GCG? GCA?AAG?GGC?CCA?TGT?AAA?GCAATC?ATG?AAA?AGA?TTT?TTC?TTC?AAT?ATT??TTC?ACT??CGA?CAG?TGC?GAA?GAATTT?ATA?TAT?GGG?GGA?TGT?GAA?GGA?AAT??CAG?AAT??CGA?TTT?GAA?AGT?CTGGAA?GAG?TGC?AAA?AAA?ATG?TGT?ACA?AGA??GAT???SEQ?ID?NO：?4AAG?CCA?GAT?TTC?TGC?TTT?TTG? GGA?CGA?AGG?CCT?GGA?ATA?TGT?CGA?GGTTAT?ATT?ACC?AGG?TAT?TTT?TAT??AAC?AAT CGA?ACA?AAA?CAG?TGT?GAA?CGTTTC?AAG?TAT?GGT?GGA?TGC?CTG??GGC?AAT?ATG??AAC?AAT?TTT?GAG?ACA?CTGGAA?GAA?TGC?AAG?AAC?ATT?TGT??GAA?GAT?GGT