CN1706943A - Prepn, activity detection method ad disease treating application of recombinant human prourokinase amino terminal fragment - Google Patents

Abstract

The present invention relates to the preparation process and activity identification of one kind of recombinant protein of mutual action of prourokinase (proUK) and prourokinase receptor (uPAR). Owing to that uPA/uPAR takes important role in many pathologic physiology processes, blocking off this passage will inhibit the occurrence and development of diseases. The extracorporeal activity identification shows that the human prourokinase amino terminal fragment (ATF) of the present invention can inhibit the combination between prourokinase and prourokinase receptor on the surface of cell, and inhibit the activation of UK on plasminogen and the activation of plasmin on proUK. This shows that rhATF may take important role in inhibiting the uPA/uPAR participated pathologic physiology processes.

Description

RhATF's preparation, activity determination method and the application in disease treatment thereof

Technical field

The present invention relates to suppress proUK/UK and interactional protein fragments of uPAR and the application in disease treatment with gene engineering method production is a kind of.

Background technology

Cell migration is cell adhesion and goes the adherent periodic process.In organism growing development, organize in multiple physiology, the pathologic processes such as formation, differentiation, inflammatory reaction, trauma repair, atherosclerosis, tumor vascular generation, invasion by tumor cells and all have cell migration.In recent years some studies show that uPA/uPAR has brought into play very important effect [1] in the cell migration process.The maincenter [2] that the cell surface protein cracking of uPAR mediation and non-protein cleavage are cell migration and chemotactic.The zygotic induction uPAR of uPA and uPAR and glass protein binding are regulated the interaction between uPAR and the integration element or are promoted the intracellular signal transduction cascade, promote that leader sticks, thus irritation cell migration [3-4]; At the cell afterbody, uPA promotes Profibrinolysin to transform the generation plasmin, promotes the degraded of migrating cell afterbody extracellular matrix and cell adhesion molecule, causes the retraction of cell afterbody simultaneously.Major part stick and the epithelial cell and tumour cell that move in, uPA can plainly interact together with being rich in the various protein moleculars in caveolin or kinase whose cytolemma zone by integrating with β 1 β 3, regulates and control to integrate plain characteristic and the signal transmissibility thereof of sticking.UPAR is the limitation distribution at the guiding surface of migrating cell, thereby by promote the conversion of Profibrinolysin to plasmin in conjunction with uPA.Plasmin is a kind of fibronectin of can directly degrading, the serine proteinase enzyme of extracellular matrix components such as ln and glass Fibronectin.Simultaneously, plasmin makes it to become great-hearted matrix metalloprotease lytic enzyme by hydrolysing substrate metalloprotein prohydrolase, can play the purpose of indirect degradation of cell epimatrix.Thereby for cell migration is removed the obstacles.

Humanized uPA is the protein that molecular weight is 54kD, and holding the C end from N is respectively Urogastron structural domain (EGF domain), handle structural domain (kringle domain) and serine protease structural domain.Wherein handle structural domain and kringle structural domain and be called together urokinase the N-terminal fragment (amino-terminal fragment, ATF).In recent years some studies show that uPA is by its ATF structural domain and uPAR bonded.This combination is irrelevant with serine proteinase enzymatic structure territory.The Urogastron structural domain that ATF comprised has participated in the combination with urokinase receptor, and the sticking and transporting action [5,6,7] of inducing cell; The function [9] that the handle structural domain of ATF then has chemical chemotactic activity [8] and suppresses the tumor vascular endothelial cell growth.ATF can suppress the propagation and the migration [10～15] of tumour cell by the interaction of blocking-up uPA and uPAR.Studies show that in a large number the uPAR expression amount of tumor cell surface is significantly higher than normal cell [16], and its back poor, weak point lifetime [17] of the tumour patient of high expression level uPA, uPAR.Because uPA, the vital role of uPAR system in tumor invasion shifts generally believe that uPA, uPAR are the important targets of antineoplastic invasion, transfer.Suppress the tumor cell invasion transfer by interaction and the inhibition uPA activity of disturbing uPA, uPAR, become a kind of new approaches [18,19] that antineoplastic invasion shifts treatment.Because binding specificity between ATF and the uPAR and affinity (there is the people Kd=4 * 10-10mol/L) [20] at present with ATF and biotoxin coupling, targeted inactivation tumour cell [21], so ATF receives publicity as a kind of potential anti-tumor medicine.Discoveries such as Wada M and Alfano M recently, ATF can suppress the release [22,23,24] with virus particle of duplicating of HIV virus, thereby provides a new way for the treatment of acquired immune deficiency syndrome (AIDS).

Summary of the invention

The objective of the invention is to propose a kind of rhATF's preparation, activity determination method and the application in disease treatment thereof, the invention provides the proteic method of a kind of preparation, and its activity measured, find that it can suppress the combination of proUK and uPAR.

Technical solution of the present invention:

A kind of rhATF's preparation method is characterized by the structure of expression plasmid, the screening of positive colony, rhATF's abduction delivering and purifying.

A kind of method of measuring rhATF's determination of activity, after it is characterized by rhATF and treated U937 cell and proUK insulation, infer the active method of rhATF thereby measure the dissociated proUK content of cell surface with the zymograph method.

A kind of method of measuring the rhATF is characterized by with the enzyme kinetics method and measures the rhATF activates proUK to UK plasminogen activation (plasminogen) and plasmin (plasmin) influence.

The ATF that the present invention produced suppress HIV virus duplicate and virus particle release aspect effect and in the application aspect the treatment acquired immune deficiency syndrome (AIDS).

Effect and the effect inhibition growth of tumour cell and migration aspect of the ATF that the present invention produced aspect inhibition of endothelial cell proliferation and migration.

The ATF that the present invention produced is used for and some biotoxins or medicine coupling join to come the effect of targeted inactivation tumour cell aspect.

The ATF that the present invention produced is used for suppressing the application of the enzymatic reaction aspect that proUK or UK participate in.

The invention provides and a kind ofly prepare rhATF's method, and the recombinant human urokinase zymogen of producing by this method in one embodiment can suppress the combination of the urokinase receptor and the uPA of cell surface by gene engineering method.In another embodiment, the rhATF can suppress urokinase to the activation to uPA of the activation of Profibrinolysin and plasmin.Wherein human pro-urokinase's N-terminal fragment is the part between 135 residues of the 1st residue to the of human urokinase.

Broadly, any cell all can be used to deliver in an embodiment, and is not limited only to hereinafter listed examples as long as there is urokinase receptor on the surface.Among another embodiment, the method for measuring the ATF vigor by the enzyme kinetics method is not limited only to that two kinds of reactions.As long as the reaction that has urokinase or uPA to participate in the enzymatic reaction process all can be used to measure its vigor.

The inventive method is the prelude of subsequent applications research, for follow-up study provides a kind of great-hearted pharmaceutical protein.Follow-up study is meant that the ATF that all available the present invention of pathophysiological process of every uPA of having and uPAR participation produces blocks research.As, growth of tumor and invasive procedure, tumor-blood-vessel growth, wound healing etc.

Description of drawings

Fig. 1 is the structure schema of recombinant expression plasmid pET-29a (+)/ATF.

Fig. 2 cuts the evaluation collection of illustrative plates for the enzyme of recombinant plasmid pET-29a (+)/ATF.

Wherein, first swimming lane (Lane1) is a dna molecular amount standard, and second swimming lane (Lane2) is a PCR product band, and the 3rd swimming lane (Lane3) is XhoI and NdeI double digestion product.

Fig. 3 is a rhATF IPTG abduction delivering collection of illustrative plates.

Fig. 4 is a rhATF CM-cellulose ion exchange chromatography collection of illustrative plates, wash-out collection of illustrative plates behind the CM-cellulose post on promptly sample Dichlorodiphenyl Acetate solution is dialysed afterwards after the renaturation.

Fig. 5 is the sieve chromatography collection of illustrative plates of rhATF, and promptly the rhATF sample is further gone up the sieve chromatography collection of illustrative plates behind the Superdex G-75 gel-filtration column.

Fig. 6 is the SDS-PAGE electrophoretogram of rhATF, the rhATF sample electrophoresis collection of illustrative plates that final purifying obtains.Wherein, first swimming lane is middle molecular weight protein standard, and second swimming lane is the sample behind the CM-cellulose column purification, and the 3rd swimming lane is the sample behind Superdex G-75 sieve chromatography purifying.

Fig. 7 is the influence collection of illustrative plates of rhATF to proUK and uPAR keying action.Zymograph measures and relatively from the dissociated proUK of cell surface.Wherein, first swimming lane is that ATF handles with the cell insulation with proUK, and second swimming lane only is a cell culture fluid, and the 3rd swimming lane only is that proUK and cell insulation are handled.

Fig. 8 is rhATF activates plasminogen to UK influence curve figure.

Fig. 9 is rhATF activates proUK to Lys-plasmin influence curve figure.

Embodiment

The invention provides a kind of rhATF's of preparation method.At first extract the total RNA of human umbilical vein's endotheliocyte, amplify human pro-urokinase's N-terminal fragment gene, join with the carrier coupling behind the double digestion through RT-PCR.Transform and identify that positive colony and order-checking back give expression to the rhATF with e. coli bl21 (DE3).Afterwards its vigor is measured.The rhATF that discovery is produced by this method can suppress combining of uPA and its cell surface receptor, and can suppress plasmin to the activation to Profibrinolysin of the activation of uPA and urokinase.

Embodiments of the invention:

1.1 material

1.1.1 bacterial strain and plasmid human umbilical vein endothelial cell strain (HUVEC) are available from Clonetic company, coli strain BL21 (DE3) and expression vector pET-29a (+) are available from Novagen company.

(Abbott Laboratories, Abbott Park IL) is so kind as to give 1.1.2 the reorganization proUK of reagent C HO cell expressing (MW 55000) is by Dr.Jack Henkin.Trayslol is available from Calbiochem company.The RNA purification kit is available from Qigen company.The RT-PCR test kit is available from Invitrogen company.DNA glue reclaims test kit available from magnificent company.The T4DNA ligase enzyme, NdeI, XhoI is available from Takara company.CM-cellulose is available from Sigma company.Other chemical reagent is analytical pure.

1.1.3PCR primer is synthetic by Takara company, the upstream primer sequence is GGT GGT CAT ATGAGC AAT GAA CTT CAT CAA GTT, the downstream primer sequence is GGT GGT CTC GAGTTA TTT TCC ATC TGC GCA GTC ATG CAC C, wherein CATATG and CTCGAG are respectively the restriction enzyme site of NdeI and XhoI.

1.2 method

1.2.1 the preparation reference literature [25] of Profibrinolysin (plasminogen) and plasmin (plasmin) carries out.

1.2.2 the extraction reference literature [26] of the total RNA of human umbilical vein's endotheliocyte carries out.

1.2.3RT-PCR amplified reaction is got the total RNA 0.8 μ g of HUVEC, adds oligodT primer 1 μ L, adds water and supplies volume to 12.5 μ L, behind 65 ℃ of insulation 10min, room temperature is placed 3min.Add 1.0 μ L RNAase inhibitor, 4.0 μ L, 5 * reaction buffer, 1.0 μ L dNTP, 1.0 μ L80mmol/L trisodium phosphates and 0.5 μ L AMV reversed transcriptive enzyme then successively, at 42 ℃ of reaction 60min.Get above-mentioned reaction solution 5 μ L, add each 1 μ L of PCR primer, 10 * damping fluid, 5 μ L, the 25mmol/LMgCl of 50 μ mol/L ₂5 μ L, 10mmol/L dNTP 1.0 μ L, Taq enzyme 0.3 μ L add water and supply volume to 50 μ L.The pcr amplification condition is: 95 ℃ of pre-sex change, 2min; 95 ℃ then, 30s, 52 ℃, 1min, 72 ℃, 1min, totally 26 circulations; Last 72 ℃ are extended 8min.

1.2.4 the structure of recombinant expression plasmid pET-29a (+)/ATF reclaims the pcr amplification product of ATF gene with 1% Agarose gel electrophoresis purifying.With PCR product behind the purifying and expression vector plasmid pET-29a (+), use the NdeI/XhoI double digestion respectively.After Agarose gel electrophoresis recovery enzyme is cut product, spend the night 16 ℃ of connections with the T4DNA ligase enzyme.

Transform DH5 α competent cell 1.2.5 the conversion of recombinant plasmid, the screening of positive colony and sequencing connect product, transformant is coated on the LB flat board that contains 50 μ g/mL kantlex, 37 ℃ of overnight incubation.To the LB overnight incubation that contains 50 μ g/mL kantlex,, and carry out determined dna sequence from picking list colony inoculation on the culture plate with NdeI and XhoI double digestion screening positive clone.

1.2.6rhATF abduction delivering and purifying transform the BL21 competent cell with the right-on pET-29a of gene order (+)/ATF recombinant expression plasmid, transformant is coated on the LB flat board that contains 50 μ g/mL kantlex, put 37 ℃ of overnight incubation.The picking positive colony is seeded to the LB substratum incubated overnight that contains 50 μ g/mL kantlex, is seeded to fresh LB substratum by 2% inoculum size again.Nectar degree OD in nutrient solution ₆₀₀Reach at 0.8 o'clock, adding IPTG is 0.5mmol/L to final concentration, and 3h, 4 ℃ of centrifugal collection thalline are cultivated in the continuation jolting.

By the ultrasonic damping fluid of every gram thalline 10mL (50mmol/L Tris-HCl, 0.15mol/L NaCl, 1mmol/L EDTA, 0.5mmol/L PMSF, 0.5% Triton X-100, ratio pH8.0) suspends thalline, the broken ultrasonic 5min of bacterium in ice bath.It is 1000W that ultrasonic frequency is set.At 4 ℃ of centrifugal 25min of 6000g, remove supernatant, precipitation is the rhATF135 inclusion body.Ratio in the ultrasonic damping fluid of every gram inclusion body 10mL suspends inclusion body and supersound washing 5min centrifugal collection inclusion body.Inclusion body is pressed this step repeated washing 2 times.

In the ratio of every gram inclusion body 40mL with solubilization of inclusion bodies in sex change damping fluid (6mol/L Guanidinium hydrochloride, 50mmol/L Tris-HCl, 5mmol/L EDTA, the 100mmol/L beta-mercaptoethanol pH8.6), is put 37 ℃ and is stirred 6h, the centrifugal 25min of 8000r/min keeps supernatant liquor, and precipitation discards.

With ratio renaturation buffer (50mmol/L Tris-HCl, 1.5mol/L urea, the 0.1mol/L NaCl of supernatant liquor in 1: 30,1mmol/L EDTA, pH8.0) 4 ℃ of renaturation 24h are put in dilution, Dichlorodiphenyl Acetate damping fluid (5mmol/L HAc-NaAc, pH4.8) fully dialysis.

At 4 ℃, the centrifugal 25min of 8000r/min gets on the supernatant liquor sample in advance through 5mmol/L HAc-NaAc pH4.8 damping fluid equilibrated CM-cellulose post (1.6cm * 4cm) with the renaturation solution after the dialysis.Last sample finishes, and (5mmol/L HAc-NaAc, pH4.8) (5mmol/L HAc-NaAc, 0.5mol/L NaCl pH4.8) carry out gradient elution with the ultimate damping fluid of 200mL with 200mL initial buffer liquid.Merge the rhATF elution peak, 4 ℃ of the dialysis of HAc-NaAc damping fluid, freeze-drying to 5mmol/L.

With the rhATF sample dissolution in 3mL 50mmol/L Tris-HCl, 0.1mol/L NaCl, in the pH7.4 damping fluid, and (gel chromatography column of 1.6cm * 60cm), elution flow rate is 15mL/h to using above-mentioned damping fluid equilibrated Superdex G-75 in advance to go up sample.Collect rhATF sample peak, the sds gel electrophoresis with 15% detects its purity.

1.2.7rhATF activity identification

1.2.7.1rhATF proUK and cell surface uPAR bonded are influenced the mensuration reference literature [13] of rhATF to proUK and the influence of uPAR keying action.With RPMI RPMI-1640 suspension culture U937 cell to the density that contains 10% foetal calf serum is 2 * 10 ⁷/ mL, the centrifugal 10min of 800r/min removes supernatant.The RPMI 1640 that adding 40mL contains 100kIU/mL trayslol washs the U937 cells twice.At 5mL 0.1mol/L glycine-hydrochloric acid, 0.1mol/L NaCl is in the damping fluid of pH3.0 with cell suspension, room temperature is placed 3min (make with cell surface uPAR bonded UK and dissociate), the centrifugal 10min of 800r/min abandons supernatant, adds 3mL RPMI 1640 suspension cells.In the 1.5mL centrifuge tube, respectively get 285 μ L cell suspensions, add 15ul PRMI 1640 respectively; 7.5 μ L 0.8 μ mol/L proUK and 7.5 μ L RPMI 1640; 7.5 μ L 20 μ mol/L rhATF and 7.5 μ L, 0.8 μ mol/L proUK, 37 ℃ of insulation 30min, the centrifugal supernatant that goes.With cell 1.5mL 50mmol/L Na ₂HPO ₄-NaH ₂PO ₄, 0.1mol/L NaCl, pH7.4 damping fluid washing 3 times is to remove not and the free proUK of U937 cell bonded.Cell is resuspended in 150 μ L0.1mol/L glycine-hydrochloric acid, 0.1mol/L NaCl, in the pH3.0 damping fluid, room temperature is placed 3min, makes with U937 cell bonded proUK and dissociates centrifugal collection supernatant liquor from cell surface.

With zymograph mensuration and relatively from the dissociated proUK of cell surface ^[27]Getting 40 μ L supernatant liquors, to carry out gum concentration be 12% non-reduced SDS-PAGE, electrophoresis finishes, polyacrylamide gel is immersed in 100mL 50mmol/L Tris-HCl, 10% Triton X-100, in the pH8.0 damping fluid,, use 100mL 50mmol/L Tris-HCl again room temperature jolting 30min (removing the SDS in the gel), the pH8.0 damping fluid washs 3 times at room temperature jolting 20min.

Get the 0.5g low melting-point agarose, add 30mL 50mmol/L Tris-HCl, the pH8.0 damping fluid, heating makes the agarose fusing, when treating that its temperature is cooled to 40 ℃, adds skim-milk (being preheated to 37 ℃) and the 0.1mg plasminogen of 5mL 5%, mixing is poured over agarose in the culture dish.After treating that agarose solidifies, polyacrylamide gel is covered the agarose surface, remove the bubble in the two interface, put 37 ℃ of insulation 15h.

1.2.7.2 rhATF activates the influence of plasminogen at 50mmol/L Tris-HCl to UK, 100mmol/L NaCl, 0.1% Tween80, in the pH7.4 reaction buffer, with Glu-plasminogen (0.2 μ mol/L) respectively with S2251 (1.2mmol/L), UK (0.15nmol/L), and rhATF (0～10.0 μ mol/L) mixing, working sample is at OD _410/490The variation of absorbance value.

1.2.7.3 rhATF activates the influence of proUK at 50mmol/L Tris-HCl to plasmin, 100mmol/L NaCl, 0.1% Tween80, in the reaction buffer of pH7.4, with proUK (0.2 μ mol/L) respectively with S2444 (1.2mmol/L), Lys-plasmin (0.1nmol/L), and rhATF (0-10.0 μ mol/L) mixing, working sample is at OD _410/490The variation of absorbance value.

The result:

1, the building process of pET-29a (+)/ATF such as Fig. 1.From human umbilical vein's endotheliocyte, extract total RNA earlier, obtain cDNA by reverse transcription, amplify the ATF gene fragment by special PCR reaction again, 1% agarose glue reclaims back XhoI and NdeI double digestion, and glue reclaims and obtains ATF gene fragment to be connected again.Carrier pET-29a (+) cuts with these two kinds of enzyme enzymes equally, and 1% glue obtains carrier segments to be connected after reclaiming.The ATF gene fragment with obtained recombinant plasmid pET-29a (+)/ATF after carrier segments is connected.

Introduced NdeI and XhoI restriction enzyme site respectively at 5 ' of PCR primer-and 3 '-end, increasing from the total RNA of HUVEC with RT-PCR obtains the ATF gene and sees Fig. 2, and Lane 2.By NdeI and XhoI restriction enzyme site the PCR product is inserted among the expression vector pET-29a (+), obtained recombinant plasmid pET-29a (+)/ATF.The NdeI/XhoI enzyme is cut evaluation and is seen Fig. 2, and Lane 3.Connect after the product transformed competence colibacillus cell 37 ℃ of overnight incubation of coated plate.Picking mono-clonal bacterium colony, 37 ℃ of 250rpm shake bacterium and spend the night, and take out plasmid, and with XhoI and NdeI double digestion, enzyme is cut product and is run 1% agarose glue afterwards.Sequencing is the result show, the ATF gene order is entirely true.

2, with correct recombinant plasmid pET-29a (+)/ATF transformed into escherichia coli BL21 (DE3) of order-checking, after IPTG induced, the expression level of rhATF accounted for 20% of full bacterium total protein, sees Fig. 3.Among Fig. 3, correct recombinant plasmid pET-29a (+)/ATF is transformed into and expresses among the bacterium BL21, runs SDS-PAGE behind the IPTG abduction delivering.Wherein first road is middle molecular weight protein standard, and second road is the full bacterium band behind the IPTG abduction delivering, and the 3rd road is the full bacterium band of inductive not.The rhATF of escherichia coli expression exists with the inclusion body form of non-activity, must could obtain activated product after external renaturation.The renaturation sample is directly gone up sample to CM-cel lulose post after the acetate buffer solution of 5mmol/L is fully dialysed, through gradient elution (Fig. 4), the purity of rhATF sample reach 85% (Fig. 6, Lane2).With the rhATF sample further use the SuperdexG-75 gel-filtration column (purifying (Fig. 5) of 1.6cm * 60cm), the rhATF sample purity of acquisition surpass 95% (Fig. 6, Lane3).Through above-mentioned purification step, can obtain 15mg rhATF from every liter of fermented liquid.

3, in order to identify whether rhATF has uPAR in conjunction with activity, we have studied the influence of rhATF to the uPAR keying action on proUK and the U973 cell.In the U937 cell culture fluid, add proUK and rhATF (Fig. 7 simultaneously, Lane1) with the independent proUK (Fig. 7 that adds, Lane3) compare, the area of the transparent strip that the former produces is significantly less than the latter, prompting is when rhATF exists, reduce with U937 cell surface bonded proUK amount, illustrate that rhATF can block the keying action of proUK and uPAR.

4, Fig. 8 shows that rhATF can suppress the activation of UK to plasminogen.Fig. 9 shows that rhATF can suppress the activation of plasmin to proUK.And above-mentioned two kinds of restraining effect all are dose-dependence with rhATF, and this result suppresses proUK inductive plaminogen activation consistent [28] with ATF.

The present invention is an expression vector with pET-29a (+), with BL21 (DE3) is that the rhATF expression strain that the host bacterium makes up has higher expression efficiency, through renaturation and CM-cellulose, Superdex G-75 purification step, can obtain the rhATF of 15mg from every liter of fermented liquid, sample purity surpasses 95%.Determination of activity is the result show, the rhATF of this method preparation not only can block combining of proUK and U937 cell surface uPAR, and can suppress plasmin to the activation of proUK and the UK activation to plasminogen.Prepare ATF with the UK hydrolysis method and compare [8], adopt gene engineering method to produce ATF and have advantages such as can not polluting UK in simple, the with low cost and sample of step, be more suitable for being applied to the preparation of ATF and further research.Be used for suppressing the sticking of cell, migration and chemical chemotactic process as: ATF; ATF is used for suppressing tumor vascular endothelial cell growth and migration; ATF is used for suppressing growth of tumour cell and migration; ATF is used for joining to come the targeted inactivation tumour cell with some biotoxins or medicine coupling; ATF is used for suppressing the release with virus particle of duplicating of HIV virus; The application of ATF aspect the treatment acquired immune deficiency syndrome (AIDS); ATF is used for suppressing the enzymatic reaction of proUK or UK participation.

Although what the present invention adopted is that Bacillus coli cells is expressed ATF, but the applicant has pIZ/V5-His carrier and pcDNA3.1 carrier on hand, later work is the ATF gene to be inserted in above-mentioned two kinds of carriers go, and transforms respectively and transfection yeast cell and Chinese hamster ovary celI.Respectively behind the expression and purification and carry out the comparison of functional experiment.

Definition:

Common molecular biology

According to the present invention, in the technology of this area, should comprise traditional molecular biology, microbiology, recombinant DNA technology, protein expression separates and purification technique, enzyme kinetics method, zymograph technology etc.These technology have detailed explanation in the literature.See, for example, work such as " fine works molecular biology experiment guide " Ao Sibai, Jin Dongyan school Science Press: 1998; " molecular cloning experiment guide " second edition gold winter wild goose school; " modern medicine molecular biology " paddy will is far outstanding, People's Medical Officer Press: 1998; " biological chemistry " third edition first volume Wang Jing rock Zhu Xu Changfa in holy heptan chief editor, Higher Education Publishing House: 2002; " microbiology study course " second edition Zhou Deqing chief editor, Higher Education Publishing House: 2003; The strong Wu Junzheng chief editor of " cell cultures " Si Tuzhen, world book publishing company: 2004; Work such as " round pcr experiment guide " Di Fenhe, Huang Peitang etc. translate Science Press: 2003; " Biochemistry and Molecular Biology experiments frequently-used data handbook Wu Guan chief editor such as rue, Science Press: 1998; " molecular cloning experiment guide " third edition J. Sa nurse Brooker D.W. Russell work, Science Press: 2002; " protein electrophorese experimental technique " Guo Yaojun compiles, Science Press: 2003.

" carrier " is meant and is used to hold any instrument that external source is treated expressing gene and is transferred to competent escherichia coli cell.Used among the present invention is the pET carrier of Novagen company.Carrier comprises virus vector and non-virus carrier.Virus vector comprises retrovirus, adeno-associated virus, poxvirus, vaccinia virus, baculovirus, hsv, Epstein-Barr virus and adenovirus carrier.Non-virus carrier comprises plasmid, liposome, electrically charged lipid, DNA-protein complex and biopolymer.

" external source " is meant not to be that this treats that expressing gene just links to each other with carrier under the native state.

" conversion " is meant the process that foreign gene is imported bacterium.In addition " transfection " be meant foreign gene imported eukaryotic process.

" competence " is the state that the cytolemma of phalangeal cell is easy to accept foreign DNA.

" ultrasonic " be meant with Ultrasonic Cell Disruptor in certain damping fluid with bacterium cracked process.

" inclusion body " is meant owing to the great expression of protein in endochylema, have little time folding or can not correctly fold the structure of a kind of densification of back formation owing to lack molecular chaperones.It is insoluble to the general aqueous solution.There is not vigor.Must behind the first sex change repeatability function be arranged.

" molecular chaperones " is meant another molecule that helps certain molecular folding to become correct structure.

" sex change " be meant with containing denaturing agent (example hydrochloric acid guanidine or urea) thus damping fluid handle protein solution and destroy hydrogen bond and make it the deactivated process of peptide chain-unfolding.

" renaturation " is meant that protein solution removes denaturing agent, and it is correct folding that the peptide chain of stretching, extension is formed again, thus the process that rejuvenates.

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Claims (7)

1, a kind of rhATF's preparation method is characterized by the structure of expression plasmid, the screening of positive colony, rhATF's abduction delivering and purifying.

2, a kind of method of measuring rhATF's determination of activity, after it is characterized by rhATF and treated U937 cell and proUK insulation, infer the active method of rhATF thereby measure the dissociated proUK content of cell surface with the zymograph method.

3, a kind of method of measuring the rhATF is characterized by with the enzyme kinetics method and measures the rhATF activates proUK to UK plasminogen activation (plasminogen) and plasmin (plasmin) influence.

4, the ATF that the present invention produced suppress HIV virus duplicate and virus particle release aspect effect and in the application aspect the treatment acquired immune deficiency syndrome (AIDS).

5, effect and the effect inhibition growth of tumour cell and migration aspect of the ATF that the present invention produced aspect inhibition of endothelial cell proliferation and migration.

6, the ATF that the present invention produced is used for and some biotoxins or medicine coupling join to come the effect of targeted inactivation tumour cell aspect.

7, the ATF that the present invention produced is used for suppressing the application of the enzymatic reaction aspect that proUK or UK participate in.

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