CN1303947A - Method for producing human angiogenesis inhibine - Google Patents

Abstract

The present invention provides a method for expressing truncated human angiogenesis inhibine (AGN K1-3) protein in colibacillus, specially provides the expression vector including AGN K1-3 code sequence and colibacillus thermostable enterotoxin signal sequence (STII) fused with the above-mentioned AGN K1-3 code sequence and colibaccilus alkaline phosphatase (pho A) promoter, colibacillus cell transformed by the described epxression vector, and the method for high-level production of correctly folded and processed bio-active AGN polypeptide.

Description

Produce the method for human angiogenesis inhibin

The invention provides the proteic method of human angiogenesis inhibin (AGN K1-3) in the expression in escherichia coli brachymemma, particularly provide to comprise AGN K1-3 encoding sequence and the intestinal bacteria thermostability enterotoxin signal sequence (ST II) that merges with it and the expression vector of escherichia coli alkaline phosphatase (phoA) promotor, the Bacillus coli cells that is transformed by said expression vector reaches the method that produces the biologic activity AGN polypeptide that correctly unfolds and process with high level.

Early seventies, Folkman have proposed to treat notion (Folkman J., the Am.Surg.175:409-416 of noumenal tumour by suppressing vasculogenesis based on its result of study; 1972).Over nearly 20 years,, found multiple angiogenesis factor (AGF) and Angiostatin (AIF), and proved tumor growth and shifted dependence vasculogenesis along with the foundation of vasculogenesis in vitro study method and perfect.For example the someone finds, in the blood vessel early stage that tumour forms, tumour can only rely on freely to spread obtains nutrition, when tumour has formed self vascular system, i.e. intravasation after date, just ramp and begin remote transfer (Gimbrone of tumour, MA.et al., J.Exp.Med., 73:461-473,1972).Find in addition, intraperitoneal injection bFGF (Hori, A.et al., Cancer Res., 51:6180-6184,1991) or vascular endothelial growth factor (VEGF) can promote tumor-blood-vessel growth, and increasing tumor vascular density, anti-bFGF or VEGF antibody then can suppress growth and transfer (Kim KJ, the et al of tumour cell, Nature (London) 362:841-844,1993)).

It is necessary that vasculogenesis is not only tumor growth, and the contacting or cause the remote major reason (Blood and Zetter, PNAS, 1032:89-118,1990) that shifts of tumour of tumour cell and new vessel system.During transfer, tumour cell comes off and passes through the basilar membrane intravasation from primary tumor, escapes immunosurveillance and also survives in the recycle system, is positioned the capillary blood vessel of target organ then and enters target organ, and then bring out vasculogenesis in the metastasis.Therefore, vasculogenesis is that the termination of transfer process initial sum is necessary.

Vasculogenesis is an extremely complex physical process, and is subjected to the adjusting of multiple angiogenesis factor and Angiostatin.Known angiogenesis factor comprises bFGF, VEGF, PD-ECGF, HGF and IL-8 etc.Known Angiostatin comprises IFN-γ, TBS-1, PE-4 etc.These factors are collaborative mutually or restriction in the different links of angiogenesis, keeps normal blood vessels and generates and regulation mechanism.In tumour takes place, vasculogenesis may be the vasculogenesis stimulating factor with supressor measure and functioning efficiency on loss of equilibrium the result (Folkman J, Semin.Cancer Biol., 3:65-67.1992).

Therefore, might carry out auxiliary diagnosis and prognostic analysis to tumour based on the detection of tumor tissues vascularization degree and angiogenesis factor, Angiostatin and endothelial cell adhesion molecule isoreactivity molecular level.According to the relation between tumour cell and the endotheliocyte in the tumor tissues vasculogenesis, might suppress tumor-blood-vessel growth by following four kinds of approach, reach the purpose that suppresses tumor growth: 1) suppress the secretion of tumor angiogenesis factor; 2) activity of blocking-up tumor angiogenesis factor; 3) mitotic division of prevention tumor angiogenesis factor inductive vascular endothelial cell; 4) stop the blood vessel tumor tissues of growing into.

People such as O ' Reily are when the molecule mechanism of rapid more this phenomenon of the cut back of research noumenal tumour primary tumor metastatic tumor growth, from the serum of tumor model animal and urine, separates and purifying obtains that a kind of to have an inhibition of specificity vascular endothelial cell proliferation active, name for " angiostatin " (AGN) protein (O ' Reilly MS, et al., Cell 79:315-328,1994).The amino acid sequence analysis result shows that the proteic interior segments of mouse AGN and mouse Profibrinolysin (PLG) is that amino acid sequence number 98 to 440 has 98% homology, and is equivalent to Kringke 1 to 4 district (K1-4) in the PLG structure.Studies have shown that further AGN has special inhibition activity to the growth of vascular endothelial cell, and find to have equally the activity of the capillary endothelial cell propagation that suppresses vitro culture with the resulting K1-4 fragment in pancreatic elastase hydrolysis people PLG (hPLG) back.Some in vivo test results of study have also proved the tumor growth and the metastases restraining effect (Fidler IJ.and Ellie LM, Cell 79:185-188,1994) of this biologically active proteins.

People such as Cao (Cao Y, et al., J.Biol.Chem, 271:29461-29467, discovering 1996), the endothelial cell proliferation that each Kringle zone and their various combination can show in various degree in the AGE molecule suppresses active, and the active order of single area and combination zone is: K1＞K3＞K2; K1+K2＞K1; K2+K3＞K3＞K2; K1-3＞K1-4, wherein K1-3 activity (ED ₅₀=70mM) be K1-4 (ED ₅₀=135mM) 2 times.The interregional amino acid sequence homology of each Kringle is approximately 45% in the AGN molecule, and wherein except that K4, K1,2 and 3 zones all have stronger endothelial cell proliferation and suppress active.People such as Cao find that also the K-5 district of PLG also has tangible endothelial cell growth and suppresses active.In addition, the not end glycosylation of also finding PLG or AGN has protected protein matter and avoids suffering proteasome degradation, prolongs the effect of its circulating half-life.

The method for preparing AGN with the DNA recombinant technology is known (for example referring to International Patent Application WO 97/15667).Ordinary method generally is based in the intestinal bacteria endochylema and expresses this protein with insoluble form (being so-called inclusion body form).Can not form necessary disulfide linkage but the shortcoming of expressing is institute's synthetic protein in endochylema, so that can not normally unfold.Therefore, express the AGN further oxidation and folding again during preparation that is generated in the endochylema.If this refolding process can not take place effectively, can cause some not segregative by products, as going back the generation of ortho states product, oligomer and unusual folding product.Another problem of expressing in the endochylema is to have only the formed N of small part translate duration not hold methionine(Met) to be cut from the AGN molecule.In order to overcome these shortcomings, a kind of feasible way is to utilize to make expression product (AGN) be secreted into expression vector in the cell pericentral siphon by cytolemma.It is known that a kind of once to be successfully used at expression in escherichia coli human growth factor receptor expression/secretion box be construct (the Fuh et al. that comprises alkaline phosphatase promoter (pboA) and intestinal bacteria thermostability enterotoxin II signal sequence (ST II), J.Biol.Chem.265:3111-3115,1990).

In addition, from the purifying aspect of expression product,, preferably use the method that does not need to carry out sex change, renaturation and precipitation process in order to obtain the protein of natural folding.Traditional method need be carried out a series of purification steps such as immune affinity layer folding, reversed phase chromatography (RPC), cation-exchange chromatography, ultrafiltration and gel permeation chromatography.Wherein the most important thing is to have the immunoaffinity chromatography step of high selectivity.Yet the immunoaffinity chromatography step need be used the expensive monoclonal antibody of price, so that has increased to obtaining the required production cost of high purity hAGN.Therefore, need to set up a kind of sex change/settling step that saves in the present technique field, and needn't use immunoaffinity chromatography can obtain the method for high purity purpose product.

The invention provides a kind of in intestinal bacteria with the proteic recombinant expression vector of soluble form secreting, expressing AGN K1-3, be characterised in that said carrier contains the signal sequence that is connected to the intestinal bacteria thermostability enterotoxin II on AGN K1-3 (AGN K1-3) encoding sequence.

According to recombinant expression vector of the present invention, wherein employed expression controlling elements is the escherichia coli alkaline phosphatase promotor that is connected said signal sequence upstream.

According to recombinant expression vector of the present invention, wherein also include the ribosome bind site of ST II gene.

Fig. 1 shows the gene mapping of recombinant expression plasmid pCE-AGN K1-3.Wherein the EcoR of plasmid pAT153 I-Hind III fragment is replaced by the expression cassette of pAGN K1-3.

Can use any plasmid that can in Escherichia coli, stably keep and copy heavy as making up the present invention The parent material of group carrier. The nucleotide sequence that is used for coding ST II gene signal peptide be known (as referring to Picken et al., Infection and Immunity 42:269-275,1983). Those skilled in the art can according to The degeneracy principle of genetic codon is utilized partially under the condition of the amino acid sequence that does not change said signal peptide The property codon preparation sequence variant that is more suitable in particular host cell, expressing. Perhaps, can utilize known Sudden change or induced mutations (codon replaces, disappearance, insert and add) method design and preparation ST II base Mutant because of signal peptide sequence.

The cDNA sequence of human plasminogen (hPLG) be known (as referring to Forsgren M, et al., FEBS Lett., 213:254-260,1987). The Angiogenesis that contains different Kringle zones of being derived by hAGE presses down The amino acid sequence that system is plain and its nucleotide coding sequence, and the combination in different Kringle zones is lived with it Property also is known (referring to the people's such as above-mentioned Cao document).

Preferred AGN sequence is to consist essentially of the strong blood vessel of having of Kringle 1-3 zone among the present invention Generate the AGN that suppresses active. SEQ ID NO:5 and 6 has provided respectively and has consisted essentially of AGN K1-3 Amino acid sequence and nucleotide coding sequence thereof. It will be appreciated by those skilled in the art that, can use and SEQ The sequence of ID NO:6 has the dna sequence dna of high homology to express AGN K1-3 or its in prokaryotic Mutant, these AGN K1-3 analogs or mutant also will comprise within the scope of the invention. This area The technical staff can determine homology degree (Beltz et al., the Meth. between dna sequence dna according to known technique Enzymol.100:266-285,1983). Known nucleic acid hybridization, for example filter hybridization is to determine homology Common technology. The people such as Beltz (Meth.Enzymol.100:266-285,1983) have instructed with the filter hybridization method true Decide wash conditions, probe length, guanine/cytimidine (V/C) content of homology, the ion of cleaning solution Intensity and wash temperature etc. In addition, ability technical staff can use known method (Owen et al., Chem. Meth.Bact.Systemat.pp.67-93,1985) determine the level of DNA-DNA hybridization. Therefore, this In the bright method and construct, preferably the nucleotide sequence with SEQ ID NO:6 has about 80% at least The nucleotide sequence coded AGN K1-3 function equivalent of homology.

Among the present invention, preferred expression control sequenc is escherichia coli alkaline phosphatase (phoA) promoter sequence, and the ribosome bind site that is incorporated into the ST II gene in the genetic expression construct of the present invention (plasmid).People such as Chang (Gene 44:121-125,1986) disclose the sequence of phoA promotor; People such as Pichen (Ifection and Immunity 42:269-275,1983) disclose the sequence of ST II ribosome bind site.Promotor and ribosome bind site sequence can be connected in the expression construct and so as to expressing the AGN gene.Can use methods known in the art (Sambrook et al., MolecularCloming, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) conversion, the transformant of finishing structure, the e. coli host cell of carrier cultivated and the extraction of product.For example, can use intestinal bacteria W3110 bacterial strain (wild-type e. coli K12, f-, λ-, IN (rrnD-rrnE) 1) as expressing the proteic host cell of AGN K1-3 among the present invention.Can in the LB substratum, cultivate primary culture, and the amount of infeeding of monitoring oxygen and nutrient substance is with preparation master culture (OD ≈ 260-280).

Surprisingly, we find ST II signal sequence is connected on the AGN gene, might set up a stable expression/excretory system.Construct of the present invention is preferably in the control of phoA promotor and expresses down, and preferably integrates a ribosome bind site that is beneficial to the ST II gene of expression.Under the situation of using the phoA promotor, needn't add other chemical substances for activation/abduction delivering, in case and expression regulation sequence be in state of activation and can obtain high level expression.Expressed protein can be secreted in the cell pericentral siphon in a large number in endochylema.And the protein of being secreted out can be able to correct folding, promptly contains the N-terminal sequence of true E and correct disulfide bridge bond.In fact, we analyze demonstration to the SDS-PAGE of intestinal bacteria (W3110) expression product, and nearly 42% AGN is correctly processed, and the final certification product is excretory AGN protein.

In order to extract the AGN K1-3 protein of expressing justacrine with high yield, preferably be suspended in the dilute acetic acid by the biomass of homogenizer with acid activation, stir the poly-ethamine that adds about 0.25% (W/V) down and mixture is transferred to acid pH, the centrifugal then somatic cells of removing.

The invention provides the rapid chromatography purification method of multistep, wherein preferred chromatographic step is successively: silica gel adsorption chromatography, hydrophobic interaction chromatography, cation-exchange chromatography, anion-exchange chromatography.Can use the gel coat of 950W preparing gel silica gel column chromatography, with Tris-HCI (pH7.2) balanced gel post (flow velocity is about the 20ml/ branch), and with about 600mM tetraethylammonium chloride (TMAC) as elutriant.Hydrophobic interaction chromatography preferably uses in advance with the Tris damping fluid equilibrated phenyl Sepharose that contains ammonium sulfate (30%) ^TMPost, and be about 25% ethylene glycol gradient elution with final concentration.For example can use Toyopearl ^TMTSK SP5PW (Tosohaas) sulfopropyl ion exchange resin is filled out post and is used the linear salt gradient wash-out AGN that adds 15% ethylene glycol of having an appointment.Preferably use DEAE-Sepharose ^TM, as Sepharose Fast Flow ^TM(Pharmacia) gel column of preparation anion-exchange chromatography, and sample and wash-out under about pH5.5-6.0 condition.The most handy 15mM Tris buffer solution elution that contains 0.5M NaCl and 0.1%Tween-20.

Behind the purifying, use the reversed-phase HPLC analytical method, for example use the composition and the purity of BakerBond-WP-C18 post analytic sample.The solvent that uses in the chromatography comprises the acetonitrile solution of trifluoroacetic acid aqueous solution and trifluoroacetic acid.Can under reductive condition, further carry out electrophoretic analysis to the AGN of purifying with SDS-PAGE gel electrophoresis (15% gel).Wherein employed protein reductive agent is two thin basic threitols.

Below by specific embodiment the present invention is described for example further, but these embodiment do not constitute the await the reply restriction of claim scope to the present invention.The digestion with restriction enzyme of the DNA that relates in the following example, end are filled and led up being connected of the phenol extraction of reaction, DNA and ethanol sedimentation, agarose gel electrophoresis and product wash-out, dna molecular, the conversion of host cell and the steps such as separation of plasmid DNA and are all finished (referring to Sambrook et al. by the standard method that second is known, Molecular Cloning, A Laboratory Mannual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Embodiment 1: the structure of expression vector and the conversion of bacterial cell

From plasmid pAT153 (Twigg ﹠ Slierrant, Nature 283:216-218,1980) preparation pCE-2.It contains escherichia coli alkaline phosphatase promotor (phoA) (Cheing et al., Gene 44:121-125,1986) and ST II leading peptide coding region (Picken et al., Infection and Immunity42:269-275,1983) and people IFNwl gene (Hauptmann et al., Nucl.AcidsRes.13:4739-4749,1985).

Prepare expression cassette through two step PCR reaction by phoA promotor, AGN (K1-3) sequence and ST II leader sequence.For this reason, the plasmid pPLGKG (being provided by doctor Heden of Sweden Lund university) that will contain hPLG cDNA with the Hind III is cut into linearity, with Eco R I with Hind III digested plasmid pCE2-DNA and separate big fragment.Carry out sequence amplification according to people such as Ho (Gene 77:51-59,1989) described " overlap and extend cut-grafting " PCR method (SOE-PCR).

The amplification of AGN K1-3: with the linearizing pPLGKG DNA of 100ng, 30pmol primer 1:5 '-CGACGAATTCAATGTATCTCTCAGAGTGCAAC-3 ' (SEQ ID NO:1) and 30 pmol primer 2: 3 '-GGTGTCTGGATGCAGGTGGTGCTGTGGGAGCCAATT-5 ' (SEQ ID NO:2) are added in E and contain 50mM KCl, 10mM Tris-HCI (pH8.0), 1.5mM MgCl2, in the 50 μ l damping fluids of 0.01% gelatin and four kinds of dNTP of each 0.2mM and 1.25 Taq of unit polysaccharases, carry out the PCR reaction with the hot thread ring of TC-1 type instrument: 94 ℃ of insulations are after 3 minutes, carry out 15 and took turns thermal cycling (94 ℃ 40 seconds, 55 ℃ 30 seconds, 72 ℃ 90 seconds).

The amplification of PhoA promotor+ST II leader sequence: the big fragment of the linearizing pCE2 of 100ng, 30pmol primer 3:5 '-CGTCTTCAAGAATTCGAGATTATCG-3 ' (SEQ ID N0:3) and 30pmol primer 4:3 '-GGCAGATCACATGCATAGGCATTTGTAGCAATAG-5 ' (SEQ ID NO:4) are carried out pcr amplification by above-mentioned same reaction conditions and in same damping fluid.

The dna fragmentation (being respectively about 830bp and 374bp) that the above-mentioned PCR reaction of gel (being added in 1.5% low melting-point agarose gel in the tbe buffer liquid) electrophoretic separation obtains.Each 5 μ l agarose/dna solution is incorporated in the solution that 100 μ l contain primer 1 and primer 4 (each 50pmol) carries out thermal cycle reaction: 94 ℃ of heating were carried out 25 circulations (94 ℃ 40 seconds after 5 minutes in above-mentioned same damping fluid, 55 ℃ 30 seconds, 72 ℃ 5 minutes).After the amplification, extract and the ethanol sedimentation purify DNA through phenol/chloroform.Dissolving PCR product also cuts with Hind III and EcoR I in corresponding damping fluid.

Cut Bluscribe M13+ (Strtagene) also with the big fragment of 1.2% sepharose purifying with Hind III and EcoR I double digestion.Containing 50mM Tris-HCI, 10mM MgCl ₂, 20mM DTT, 1mM ATP, 50mg/ml BSA and 2 T of unit ₄In the 10 μ l solution of dna ligase, make 10ng BluscribeM13 ⁺With 0 ℃ of reaction of the 50ng PCR product of cutting with EcoR I/Hind III enzyme 1 hour, under room temperature, continue reaction 3 hours then.With 10 μ l reaction mixture transformed competence colibacillus intestinal bacteria JM101 cells.Selection contains the positive colony of expression cassette, and DNA isolation is also carried out sequential analysis.Institute's DCRP named be pDH-AGNK1-3.

With the Ssp I with Pst I double digestion pAT153 and separate big fragment.Cut pDH-AGN K1-3 and use the Klenow fragment of archaeal dna polymerase that end is filled and led up with the EcoR I.After extracting linear pDH-AGN K1-3 DNA, cut this DNA and from 1.2% sepharose, isolate the fragment that contains phoA promotor, ST II leader sequence and AGN K1-3 gene with the Pst I.The fragment that makes big fragment of the pAT153 that so obtains and 40ng contain expression cassette in the presence of the T4DNA ligase enzyme is carried out ligation then, and with ligation mixture (5 μ l) transformed competence colibacillus intestinal bacteria HB101 cell.The bacterium colony that selection is transformed on the LB agar plate that contains 10 μ g/ml tsiklomitsins.Separate the plasmid DNA of each bacterium colony that so obtains and carry out restricted enzyme cutting analysis.Selecting wherein, each fragment connects correct plasmid and it is named into pCE-AGN (referring to accompanying drawing 1).According to conventional CaCl ₂Method plasmid pCE-AGN K1-3 transformed into escherichia coli W3110.

Embodiment 2: the fermentation culture of transformant and the extraction of expression product

The LB substratum (800ml) that A. will contain the 5mg/ml tsiklomitsin is added in 2 liters of culturing bottles that contain standby culture (OD560 ≈ 0.02), cultivates 12 hours for 37 ℃ under stirring and aeration condition.

B. the substratum that uses in the Primary Fermentation is basically by (NH4) ₂HPO ₄, (NH4) ₂SO ₄, K ₂HPO ₄, MgSO ₄, NaCl, NH ₄Inorganic salt such as Cl, Trisodium Citrate, Fe ³⁺, Zn ²⁺, Co ²⁺, Cu ²⁺, Mn ²⁺Deng trace element, and carbon source such as glucose, VitB1, tryptophane, methionine(Met) and nitrogenous source are formed.Add suitable defoamer in case of necessity.Fermentation condition is: with 1000rpm stirring, air flow 1vvm, and uplift pressure 0.3 bar, 37 ± 0.1 ℃ of temperature, pH is about 6.7 ± 0.1.In 24 hours filler speed by the 2.5g/ liter in when beginning/hour bring up to gradually 5.0g/ liter/hour, remain to fermenting process then and finish.

In order to activate biomass, mixture is cooled to about 10 ℃ and with dilute hydrochloric acid pH is transferred to 3.0.Centrifugation biomass and freezing preservations under-70 ℃.

C. will stir 1 hour in about 500ml 1% acetate of biomass suspension of acid activation and in 0 ℃.Adding poly-ethamine to final concentration is 0.25% (W/V).With 3N NaOH suspension pH being transferred to 6.0 back 0 ℃ of continuation stirred 2 hours.And then pH is transferred to 7.5 and the centrifugal thalline of removing.

The purifying of embodiment 3:AGN

A. behind the isolated cell throw out, the supernatant liquor that will contain AGN is added on to be used on the silicagel column (Grace) that 20mM Tris-HCI (pH7.5) and 100mM TMCA balance cross.Initial damping fluid with 20 times of column volumes is washed post.With the cumulative TMAC wash-out AGN of concentration to 600mM.

B. in steps A, add in the material of wash-out (NH4) ₂SO ₄To final concentration be 15% (W/V), go up the Phenyl Sepharose post of having crossed with 20mM Tris-HCI+30% ammonium sulfate balance then.With 20MmTris-HCl+30% ethylene glycol (pH7.0) linear gradient elution (10ml/ branch).The purity of AGN is about 70% in the resulting eluate.

C. the eluate of step B thoroughly after the dialysis, is transferred to 4.0 with HCI with its pH with the 20mM sodium succinate, and on the sulfopropyl ion exchange resin column crossed with 20mM sodium succinate balance.The flow velocity that divides with 10ml/ is with 20mM succsinic acid+400mM NaCl+10% ethylene glycol (pH6.0) linear gradient wash-out AGN from the post.The purity of gained AGN is about 94% behind this step purifying.

D. after the eluate of cation-exchange chromatography being dialysed to 10mM bisTris (pH5.8), on DEAE-Sepharose post (the DEAE Sepharose Fast Flow that crossed with same damping fluid balance, Pharmacia), the flow velocity wash-out AGN that divides with 5ml/ with 10mM bisTris+400mM NaCl+0.1%Tween 20 linear gradients.Behind this step chromatography purification, the purity of AGN reaches 98% in the eluate.

The evaluation of embodiment 4:AGN product

(125 * 4.5mm) carry out the reversed-phase HPLC analysis in 37 ℃ to AGN albumen with Supersphere 120-4 C-18 post (Merck).Wherein solvent for use A is 0.1% trifluoroacetic acid aqueous solution, and solvent B is the acetonitrile solution of 0.1% trifluoroacetic acid.As seen the result begins the about 30 minutes eluates in back in washing and is rendered as simple spike.

Under reductive condition, on the 15%SDS-polyacrylamide gel, analyze the AGN sample.With DTT raw sample also, electrophoresis is after the blue dyeing of coomassie manifests protein band before the electrophoresis.As seen behind three step purifying, the AGN of purifying is shown as the single band of an about 36.8KD of molecular weight to the result.

Embodiment 5: the biologic activity analysis of the AGN K1-3 of purifying

A. chick chorioallantoic membrane (CAM) analytical method:

Basically detect the extracorporeal blood vessel generation inhibition activity of AGN K1-3 of the present invention according to the described CAM analytical method of people such as Ausprunk (Am.J.Pathology, 79:597-628,1975).Briefly, make with 6 days that chick chorioallantoic membrane is a target tissue, observed behind the AGN K1-3 that drips different concns 48 hours, to the influence of target tissue new capillary vessel density in the filter disc, the tissue that stimulates with 30ng bFGF is as positive control.As seen the result suppresses the generation of new capillary vessel among the CAM significantly in dosage dependence mode by the people AGN K1-3 of the inventive method preparation.On the contrary, compare with the blank group, as seen the CAM that bFGF handles then has the radial new capillary vessel bud of comparatively dense to form.

B. murine melanoma growth inhibition test

The Babl/c mouse hypodermic inoculation B16 melanoma cell (10 of the about 25g of body weight ⁶Cell/only), when tumor growth arrives the about 150mm of major diameter, press the dosage intraperitoneal injection AGN K1-3 of the present invention of per kilogram of body weight 50-200mg after 10 days.With the animal of injection equal-volume PBS as negative control.Be administered once every day, the back observation of 2 weeks and record subcutaneous tumor volumes and weight.The result as seen, the growth of the B16 cell that suppresses to transplant in the body in dosage dependence mode by the AGN K1-3 of the inventive method preparation.

Sequence table (1) general information (ⅰ) applicant: Biopharmaceutial Research ﹠ Development Center of Jinan University (ⅱ) denomination of invention: method (ⅲ) sequence number of producing human angiogenesis inhibin: 5 (ⅳ) address:

(A) contact person: Li Jiaokun

(B) street: Tianhe District Stone steles

(C) city: Guangzhou

(D) country: the People's Republic of China (PRC)

(E) postcode: 510632 (ⅴ) computer-reader form:

(A) amboceptor type: 3.5 inches floppy disks

(B) computer: IBM Premium III

(C) operating system: WIN.97

(D) software: WORD97 (ⅳ) telecommunication information:

(A) phone: 020-85223275

(B) fax: the information of 86-20-85223275 (2) SEQ ID NO:1: (ⅰ) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linear (ⅱ) molecule type: CDNA (ⅲ) sequence description: SEQ ID NO:1

The information of CGACGAATTCAATGTATCTCTCAGAGTGCAAC (2) SEQ ID NO:2: (ⅰ) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linear (ⅱ) molecule type: eDNA (ⅲ) sequence description: the information of SEQ ID NO:2 GGTGTCTGGATGCAGGTGGTGCTGTGGGAGCCAATT (2) SEQ ID NO:3: (ⅰ) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linear (ⅱ) molecule type: eDNA (ⅲ) sequence description: the information of SEQ ID NO:3 CGTCTTCAAGAATTCGAGATTATCG (2) SEQ ID NO:4: (ⅰ) sequence signature:

(A) length: 34 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: CDNA

(ⅲ) sequence description: SEQ ID NO:4

The information of GGCAGATCACATGCATAGGCATTTGTAGCAATAG (2) SEQ ID NO:5: (ⅰ) sequence signature:

(A) length: 748 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ⅱ ) ：CDNA ( ⅲ ) ：SEQ ID NO:5AC GAATTCA ATG TAT CTC TCA GAC TGC AAG ACT GGG AAT GGA AAG AACTAC ATA GGG ACG ATG ACC AAA ACA AAA AAT GGC ATC ACC TGT CAA AAATGG AGT TCC ACT TCT CCC CAC AGA CCT AGA TTC TCA CCT GCT ACA CACCCC TCA GAG GGA CTG GAG GAG AAC TAC TGC AGG AAT CCA GAC AAC GATCCG CAG GGG CCC TGG TGC TAT ACT ACT GAT CCA GAA AAG AGA TAT GACTAC TGC GAC ATT CTT GAG TGT GAA GAG GGA TGT ATG CAT TGC AGT GGAGAA AAC TAT GAC GGC AAA ATT TCC AAG ACC ATG TCT GGA CTG TGC CAGGCC TGG GAC TCT CAG AGC CCA CAC GCT CAT GGA TAG ATT CCT TCC AAATTT CCA AAC AAG AAC CTG AAG AAG AAT TAC TGT CGT AAC CCC GAT AGGGAG CTG CGG CCT TGG TGT TTC ACC ACC GAC CCC AAC AAG CGC TGG GAACTT TGC GAC ATC CCC CGC TGC ACA ACA CCT CCA CCA TCT TCT GGT CCCACC TAC CAG TGT CTG AAG GGA ACA GGT GAA AAC TAT CGC GGG AAT GTGGCT GTT ACC GTT TCC GGG CAC ACC TGT CAG CAC TGG AGT GCA CAG ACCCCT CAC ACA CAT AAC AGG ACA CCA GAA AAC TTC CCC TGC AAA AAT TTGGAT GAA AAC TAC TGA CGC AAT CCT GAC GGA AAA AGG GCC CCA TGGTGC CAT ACA ACC AAC AGC CAA GTG CGG T

Claims (3)

1, a kind of in intestinal bacteria with the proteic recombinant expression vector of soluble form secreting, expressing AGN K1-3, be characterised in that said carrier contains the signal sequence of the intestinal bacteria thermostability enterotoxin II that is connected on AGN K1-3 (AGNK1-3) encoding sequence.

2, according to the recombinant expression vector of claim 1, wherein employed expression controlling elements is the escherichia coli alkaline phosphatase promotor that is connected said signal sequence upstream.

3,, wherein also include the ribosome bind site of ST II gene according to the recombinant expression vector of claim 1.

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