CN101481422B

CN101481422B - Antineoplastic target fusion protein with release switch and use thereof

Info

Publication number: CN101481422B
Application number: CN2009100780110A
Authority: CN
Inventors: 孙妙囡
Original assignee: Individual
Current assignee: Sun Miaonan
Priority date: 2009-02-09
Filing date: 2009-02-09
Publication date: 2011-05-18
Anticipated expiration: 2029-02-09
Also published as: CN101481422A

Abstract

The invention discloses fusion protein and an encoding gene and application thereof. The fusion protein consists of three parts which are a targeting carrier, a drug release switch and a warhead; one end of the drug release switch is connected with the targeting carrier, the other end is connected with the warhead; the targeting carrier is polypeptide containing the first-sixty fifth amino acid residues of sequence 2 in a sequence table; the warhead is polypeptide containing the sixty eighth-ninety third amino acid residues of the sequence 2 in the sequence table; and drug the release switch is polypeptide which can be specifically cut by serine protease (for example, urokinase, fibrinolysin and thrombin) or metalloprotein. The recombinant targeted drug protein is characterized by high identification specificity, smaller molecular weight, no immunogenicity, no anaphylactic reaction, nontoxicity and good permeability, and can carry targeted warhead to surfaces of tumor cells to kill the tumor cells, and has no affinity and toxicity to other normal cells of humans.

Description

A kind of antineoplastic target fusion protein and application thereof with release switch

Technical field

The present invention relates to a kind of antineoplastic target fusion protein and application thereof with release switch.

Background technology

The tradition chemotherapeutics also has lethal effect to organism normal cell in killing tumor cell, this is the main reason that its toxic side effect produces.How to reduce the murder by poisoning of organism normal cell and specifically killing tumor cell be the direction that people make great efforts.The Germany Ehrlich of pharmacy man just proposes the conception of targeted therapy as far back as 20 beginnings of the century, with the close tumour carrier of high special the bullet with cytotoxicity is concentrated the guiding tumor locus, and the performance lethal effect reduces the non-special murder by poisoning to host cell.

The antineoplastic target medicine generally is made of two portions, and the one, tumour cell there is the bullet of kill capability, secondly be specific oriented carrier, bullet guiding target site.

1, oriented carrier

Oriented carrier can be divided into following a few class:

1.1 antibody carrier

The most tumors cell has and is different from Normocellular gene expression profile, therefore has significant albumen unconventionality expression or specifically expressing, these albumen are called as tumor associated antigen, tumour specific antigen or are referred to as tumor marker, the antibody of discerning these tumor markers becomes oriented carrier the earliest, after particularly hybridoma technology occurs, provide the monoclonal antibody of a large amount of specially recognizing tumor cells available.Do carrier with mouse source antibody and have following shortcoming: molecular mass is big, penetration power is weak, the specificity intake of tumour antagonist is few, have immunogenicity, repeated application can cause the anti-mouse immune response of people.

1.2 the ligand carrier of special receptor

Deep day by day along with to tumour cell molecular biology and RESEARCH ON CELL-BIOLOGY it is found that tumor cell surface has the acceptor molecule of a lot of overexpressions, and the native ligand of these acceptors has just become oriented carrier of new generation.The main part that uses is a cytokine in basis and the clinical study at present, as EGF, GM-CSF, IL-2, IL-4, IL-6, TGF-a, melanotropin a and Insulin-Like factor I etc.The n cell factor is the human body endogenous protein, can not cause the immune response of body as oriented carrier.Appearance and development along with the phage peptide library triage techniques, people screen tumour cell, obtain the micromolecule polypeptide of special target tumour cell, and with its carrier as targeted drug, these micromolecule polypeptide carriers have kept the target of high special when having reduced gene recombination targeted toxin molecular mass greatly, be the excellent carrier of targeted drug; They are the albumen that body self exists, can avoid the immunogenicity problem of heterologous protein, and these carriers mostly are the polypeptide of small molecular weight, can make the targeted toxin molecule facilitate penetration of physiologic barrier permeates to inside tumor, increase the absorption of tumour cell, improve fragmentation effect solid tumor to it.

2, targeted drug

The bullet of targeted drug mainly contains three classes, i.e. radionuclide, medicine and toxin.At present, the bullet of using in the gene engineered targeted medicine is mainly the toxic protein that derives from different organisms.Mainly be divided into following a few class:

2.1 bacteriotoxin

Diphtheria toxin (DT), Pseudomonas aeruginosa extracellular toxin bacterial exotoxins such as (PE) have the activity of ADP-ribosyltransferase, and they act on elongation factor-2 (EF-2) can make its inactivation, cause the cell protein dyssynthesis and death; There is the glycosylated effect of 28S rRNA N-in shiga toxin A subunit (STXA), can destroy 28S rRNA in the born of the same parents, and blocking protein synthesizes makes cell-lethal.This is the bacteriotoxin that a class is early used, and mainly is to use the molecule of transforming through protein engineering at present, and the molecule through transforming has lot of advantages, comprise and reducing and Normocellular receptors bind, reduce side effect, reduced molecular mass, immunogenicity, and improved killing activity.Except that extracellular toxin, by destroying cytolemma, make its surface form aperture from the phospholipase c (PLC) of bacterium, staphylococcus a hemolysin (a-HL) etc., cause entocyte and leak and finally cause necrocytosis, also be used as bullet.Because this toxoid directly acts on cytolemma, need not enter cell through " endocytosis " and play a role, the target spot of discerning for its oriented carrier has more selection.

2.2 vegetable-protein toxin

The vegetable-protein toxin comprises two classes, and a class is to be connected to form by disulfide linkage by two chains of A, B, comprises Semen Ricini toxin, abrin etc., and wherein the A chain has enzymic activity, can make eukaryotic cell rrna 60S rRNA inactivation after entering endochylema; Another kind ofly be and above-mentioned A chain structure homology, strand toxin that mechanism of action is identical, be called " A chain analogue " again, as Saponaria officinalis toxin, momordin etc., this toxoid molecular mass is generally about 30kDa, is to be alkaline strand glycoprotein.

2.3 humanized's proteotoxin

This is the intravital biological activity protein of people, comprise after birth lytic enzyme, human complement component and rnase etc., merge the targeted toxin hEGF-hpRNase that forms as human epidermal growth factor and people's pancreatic ribonuclease, confirm that in the experiment of cell in vitro poison EGF-R ELISA (EGFR) is crossed the class epithelial cancer cells of expressing tangible specific killing effect.The somebody had made up the CH by the identification TfR in recent years ₂Human mouse chimeric antibody merges the targeted toxin CH that forms with the human angiogenin (angiogenin) with ribonuclease activity ₂Ang, and in the mammary gland of transgenic mice, express, product behind the purifying can be in conjunction with TfR TfR, suppress the growth of kinds of tumor cells such as SF539, HS578T, mdrl, MDA-MB-231, MALME effectively, the good clinical application prospect is arranged, the new way that also provides targeted drug to produce simultaneously.

3, carrier and bullet is connected

Making up one of gordian technique of targeted drug, is oriented carrier and bullet are coupled together and to keep the constant method of attachment of each several part function.First-generation targeted drug adopts the method for chemically crosslinked crosslinked oriented carrier and bullet, and used bullet mainly is radionuclide or chemotherapeutics etc., and oriented carrier is an antibody molecule.The targeted drug of chemically crosslinked preparation has poor stability, molecular mass is big, immunogenicity is strong, poor permeability, the first-class shortcoming of structure inequality.Strong, the good penetrability of targeted drug stability that adopts gene engineering method to make up has better tumour cell guiding specificity; And directly recombinate at gene level, make that people are easy to it is transformed, obtain better proterties; And the preparation method is simple, and mass production is the main object of clinical experimental study in a short time.

Early the oncotherapy targeting toxins medicine that gets the Green Light is the Ontak (DAB389-IL-2) of Ligand Pharmaceuticals company, now clinical application in the treatment of stubbornness or renaturation cutaneous T cell lymphoma, simultaneously also in the clinical treatment test of carrying out other tumours.The clinical effectiveness of targeting toxins pharmacological agent tumour shows that in the treatment of hematological system tumor, because target is discerned the molecular specificity height and medicine arrives easily, so curative effect is better.In addition, can comprise lung cancer, liver cancer, colorectal carcinoma and melanoma, mammary cancer, neurospongioma etc., but the curative effect of solid tumor be can not show a candle to hematological system tumor with the tumour in the body cavity that also has of targeted drug treatment.Main toxic side effects was during the targeting toxins clinical drug used: the blood vessel that the non-special damage of blood vessel endothelium is caused oozes out syndromes (vascular-leak syndrome); During a large amount of medication, cytotoxic activity is to the damage of healthy tissues such as liver, renal function, and the body anaphylaxis that causes of immunogenicity etc.What these symptoms produced mainly is because targeted drug is perfect inadequately, and tumour itself character caused.Present situation in conjunction with present basis and clinical study, the targeting toxins medicine still has its advantage and characteristics in oncotherapy, manage to make do the combined utilization of the exhibition of repeating transmission and other therapies, in combined therapy of tumour as radiotherapy, assisting therapy after chemotherapy and the bone marrow transplantation, dwindle focus before the surgical excision, behind the surgical excision to the means of the removing of the MRD that may exist in the patient body and prevention of recurrence etc., on route of administration, use topical, can adopt interventional therapy and conduit to introduce, nasal cavity suction etc., this is strengthening drug effect, reduce on the toxic side effects its superiority is arranged.

At present, the problem of gene recombination targeted drug existence mainly contains several several aspects down:

1, the immunogenicity of gene recombination targeted drug existence is reused clinical being difficult to

Immunogenicity for carrier part, adopting humanized antibody or natural human body self albumen is carrier, can weaken or remove, the main alloimmunization originality of gene recombination targeted drug is from toxin moiety, the toxin major part of using is to derive from bacterium, plant etc. at present, can reduce its immunogenicity after the transformation of process genetic modification, but can't avoid fully.Except further existing toxin being carried out the protein engineering transformation, the functional protein that also can adopt human body self is a toxin, such as above-mentioned rnase family member etc.

2, targeted drug is difficult to pass physiologic barrier arrival target tissue

Targeted drug is especially poor to the wetting property of large-scale solid tumor, and during by intravenous administration, every gram tumor tissue only absorbs 0.01%～0.001% of injection volume in the solid tumor.On the one hand may be the specificity of the target spot of discern and affinity is not high or the existence of the expression in tumor tissues heterogeneity, also might be that the tumour antigen of being discerned comes off from cell surface, in plaing a part in circulation and targeted drug.The molecular mass of targeted drug is big in addition, also is that it is difficult to pass the reason that physiologic barrier arrives tumor locus.

3, specificity and affinity are not high

The not high meeting of the specificity of targeted drug and affinity causes targeted drug injuring normal cell before the arrival target tissue, has consumed drug effect.

At the problems referred to above, from now in the research of targeted drug, at first will select the high tumour antigen of specificity is target spot, also to carry out appropriate design to targeted drug in addition by protein engineering, reduce molecular mass, improve avidity and specificity, and in the I clinical trial phase, also should take suitable route of administration, increase the concentration and the distribution that reduce in healthy tissues of medicine at tumor tissues, utilization is expressed at the time phasic property at particular target position and is further improved specificity and drug effect, reduces Normocellular toxic side effect.

Summary of the invention

The purpose of this invention is to provide a kind of fusion rotein and encoding gene thereof with antitumor action.

Fusion rotein with antitumor action provided by the present invention is made up of oriented carrier, release switch and bullet three parts;

One end of described release switch links to each other with described oriented carrier, and the other end links to each other with described bullet;

Described oriented carrier derives from pallas pit viper in the Usu (Gloydius ussurinsis), is the polypeptide that comprises the 1-65 amino acids residue of sequence 2 in the sequence table;

Described bullet derives from apis mellifera (Apis mellifera ligustica), is the polypeptide that comprises the 68-93 amino acids residue of sequence 2 in the sequence table;

Described release switch is can be by the polypeptide of serine protease or the cutting of metalloprotein enzyme spcificity, and described serine protease is urokinase, plasmin or zymoplasm.

In the above-mentioned fusion rotein, described release switch is the dipeptides with following structural formula: Arg-X;

Wherein, Arg is an arginine, and X is a nonpolar amino acid.

Described release switch specifically can be: Arg-Val;

Wherein, Arg is an arginine, and Val is a Xie Ansuan.

Above-mentioned fusion rotein specifically can be following 1) or 2) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have antitumor action by 1) deutero-protein.

In order to make 1) in protein be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 2 is formed in by sequence table or C-terminal connect.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag?II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 2) but in the protein synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) proteinic encoding gene can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 1 in, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

Above-mentioned encoding gene with fusion rotein of antitumor action also belongs to protection scope of the present invention.

Encoding gene with fusion rotein of antitumor action specifically can be following 1)-3) in arbitrary described gene:

1) its nucleotide sequence is the sequence 1 in the sequence table;

2) under stringent condition, can hybridize and the above-mentioned dna molecular of encoding with the dna sequence dna that sequence in the sequence table 1 limits with fusion rotein of antitumor action;

3) with 1) gene have the homology more than 90% and the above-mentioned dna molecular of encoding with fusion rotein of antitumor action.

Gene in the described step 3) is with 1) gene homology more than 95% is preferably arranged.

Sequence 1 in the sequence table is by 279 based compositions, and its open reading frame (ORF) is from 5 ' terminal 1-279 bit base, and encoding amino acid sequence is a sequence 2 in the sequence table.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

Increase above-mentioned fusion rotein encoding gene total length with antitumor action or arbitrary segmental primer to also belonging to protection scope of the present invention.

Contain above-mentioned recombinant vectors, expression cassette, transgenic cell line and reorganization bacterium and also belong to protection scope of the present invention with fusion rotein encoding gene of antitumor action.

Another object of the present invention provides a kind of medicine with antitumor action.

Medicine with antitumor action provided by the present invention, its activeconstituents are above-mentioned fusion roteins.

Above-mentioned fusion rotein can be used for preparing the medicine with antitumor action.

As oriented carrier, this oriented carrier contains arginine-glycine-aspartic acid sequence (RGD sequence) with the polypeptide that derives from viper venom in the Usu in the present invention, integrin alpha that can specific combination tumor cell surface unconventionality expression _vβ ₃Thereby, bullet is anchored on tumor cell surface; This oriented carrier itself is again effective target therapeutic agent simultaneously, its molecular weight is less, the targeted drug of doing oriented carrier preparation with it facilitates penetration of physiologic barrier and permeates to inside tumor, increase the density of medicine at tumor cell surface, when having reduced gene recombination targeted drug molecular mass greatly, also kept the target of high special; Raising is to the fragmentation effect of solid tumor; Because this oriented carrier can specially stick the integrin alpha of tumor cell surface unconventionality expression _vβ ₃Thereby, can the antagonism integrin alpha _vβ ₃Function, show the effect that intensive suppresses tumor-blood-vessel growth, metastases and entity tumor growth; This oriented carrier also target suppresses its hydrolytic activity to stromatin in the metalloprotease-II of tumor surface (mmp-2), and metalloprotein endonuclease capable matrix degradation is assisted cancer metastasis, diffusion.These character make above-mentioned N-GEECDCGSPENPCCD be suitable as very much " carrier " and are used for preparing targeted drug.

Metastases and invasion need the effect of sticking mutually between cell and the extracellular matrix.In the metastases process, tumour cell can cause that vascular endothelial cell shrinks, and exposes basilar membrane, part basilar membrane protein binding on the special receptor of cell surface, thereby tumour cell is anchored on the extracellular matrix protein effectively.The proteic cell surface receptor of these binding matrix is mainly formed integrin alpha by integrating element _vβ ₃Be a special marking on malignant cell surface, this adhesion receptor plays crucial effects in the malignant growth process, integrin alpha _vβ ₃Expression of gene and consequent adhesion phenomenon are participated in the malignant tumour diffusion directly.

Vasculogenesis is the bioprocess from the new blood vessel of original vascularization, all there is new vessel to generate in entity tumor growth, wound healing and the inflammation recovery process, in this process, the cell adhesion molecule that is produced by smooth muscle cell and vascular endothelial cell plays keying action.The vasculogenesis regulation and control are unbalance to be the main driver that tumor neogenetic blood vessels generates, and has found the vasculogenesis approach of two dependent cells factors, is respectively α _vβ ₃And α _vβ ₅Approach, the vasculogenesis vascular cell performance keying action of these two kinds of approach for expressing.In the tumor-blood-vessel growth process, Prostatropin, tumour necrosis factor, vascular endothelial growth factor and tumor fragment can be induced α _vβ ₃And α _vβ ₅Express α _vβ ₃And α _vβ ₅Stimulate angiogenic growth and differentiation.

The degraded of tumour cell pair cell epimatrix depends on the proteolytic degradation enzyme, and known and extracellular matrix degradation proteins associated enzyme is mainly matrix metalloproteinase (mmp-2) at present, by suppressing the transfer that MMP activities can suppress tumour.

The bullet of fusion rotein of the present invention derives from the mellitin of apis mellifera, the polypeptide of being made up of 26 amino-acid residues.Mellitin mainly is hydrophobic from C-terminal 1-20 amino acids residue, and mainly be hydrophilic from C-terminal 21-26 amino acids residue, the amino acid structure of mellitin makes it become the strong basicity peptide, in neutral aqueous solution, as monomer is to exist with at random coiled structure, the mellitin oneself is crosslinked, form the tetramer structure of a spiral, the amphipathic of mellitin is the feature of film binding peptide and membranin transbilayer helix, this characteristic decision mellitin both can be water-soluble, again can with film natural combination, and then dissolved cell.The molecule of mellitin is little, compares the low and difficult generation anaphylaxis of immunogenicity with the toxin that Ricin or larynx toxalbumin equimolecular quantity are bigger; Mellitin is lower to Normocellular toxicity, so can heavy dose ofly use.Because of mellitin is by destroying the death that cytolemma causes cell, need not enter cell through " endocytosis " and play a role, so it need not enter in the cell, can demonstrating its tumoricidal toxicity in the extracellular.These character make mellitin be suitable as very much " bullet " and are used for preparing targeted drug.

Easy and the Normocellular cytolemma of mellitin tetramer structure, particularly erythrocytic cytolemma combination, and then cause cell rupture, cause serious toxic side effect, the present invention organically blends targeting vector and mellitin by release switch, the mellitin monomer is existed with the form of fusion rotein, can not form the tetramer in transit in vivo, reduce mellitin normal tissue cell and erythrocytic destruction.When mellitin is towed to the target spot of tumour cell, promptly be anchored at tumor cell surface, this moment, the serine protease (as urokinase, plasmin, zymoplasm) or the metalloprotease of tumor cell surface cut down mellitin at the release switch place, mellitin is assembled into the tetramer of hollow rapidly, insert cytolemma, make entocyte by flowing out in the born of the same parents, play the effect of killing tumor cell or mimicry blood vessel, after the lysis, the G protein binding in mellitin and the entocyte loses the rupture of membranes effect.And do not have integrin alpha on the normal tissue cell surface _vβ ₃Biological missile is difficult to be anchored on cell surface, even be anchored on cell surface once in a while, because the normal cell surface does not have the proteolytic enzyme (as urokinase, plasmin, zymoplasm or metalloprotease) of abnormal secretion, mellitin as bullet can not be disintegrated down, can not be assembled into polymer, thereby healthy tissues is had no side effect, have no drug resistance with destruction cytolemma ability.

Fusion rotein of the present invention has that identification specificity height, molecular weight are less, the characteristics of non-immunogenicity, no anaphylaxis, nontoxicity and good penetrability, can carry the bullet target and be attached to tumor cell surface it is killed and wounded, simultaneously other normal cell of human body not had affinity or toxic action.

Description of drawings

Fig. 1 is the SDS-PAGE electrophoresis detection result of fusion rotein

Fig. 2 is the HPLC color atlas of fusion rotein

Fig. 3 is that fusion rotein is to HeLa cell inhibiting exercising result

Fig. 4 is that fusion rotein is to SMMC-7721 cell inhibiting exercising result

Fig. 5 is that fusion rotein is to SGC-7901 cell inhibiting exercising result

Embodiment

Below in conjunction with specific embodiment fusion rotein of the present invention is described further, but is not limited to fusion rotein among the embodiment.Experimental technique described in the following embodiment if no special instructions, is ordinary method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.

Embodiment 1, has the acquisition of the fusion rotein and the encoding gene thereof of antitumor action

One, the acquisition of oriented carrier

Get pallas pit viper in the Usu (Gloydius ussurinsis) (available from chair mountain, Huinan, Jilin Province She Chang), peel off poison gland, extract its total RNA, reverse transcription obtains cDNA, with this cDNA is template, with 5 '-taggatccgactctcctgcaaatccgtg-3 ' and 5 '-atgaattcggcatggaagggatttctgg-3 ' is a primer, carries out pcr amplification; Obtain the encoding gene of viper venom peptide in the Usu, with this encoding gene as oriented carrier.

Two, the acquisition of mellitin

Get apis mellifera (Apis mellifera ligustica) (available from the happy apiary of containing of Jilin Province's huge rock), peel off poison gland, extract its total RNA, reverse transcription obtains cDNA, with this cDNA is template, with 5 '-taggatccggcattggcgcggtgctgaa-3 ' and 5 '-tacttaagctgctgacgtttacgtttaa-3 ' is a primer, obtains the encoding gene of apis mellifera mellitin, with the encoding gene of this mellitin as bullet.

Three, the acquisition that has the fusion rotein and the encoding gene thereof of antitumor action

1, the acquisition that has antitumor action fusion rotein and encoding gene thereof

The encoding gene of the oriented carrier that obtains according to above-mentioned steps one and the encoding gene of the bullet that above-mentioned steps two obtains, hobby codon according to intestinal bacteria DL-21 and yeast GS115, the encoding gene of synthetic fusion rotein, its DNA sequence is shown in sequence in the sequence table 1, and its amino acid sequence coded is shown in sequence in the sequence table 2; Sequence 2 is made up of 93 amino-acid residues in the sequence table, and the 1-65 amino acids residue of sequence 2 is an oriented carrier; 66th, 67 amino acids residues are release switch, and 68-93 amino acids residue is a bullet.

2, the expression of fusion rotein in e. coli bl21

The fusion rotein encoding gene that above-mentioned steps 1 is obtained carries out double digestion with BamH| and EcoR| (available from Dalian TAKARA bio tech ltd), enzyme is cut product and is inserted in the multiple clone site of plasmid pUC18, changes the recombinant expression vector that obtains over to escherichia coli DH5a and increases; Extract plasmid, carrying out electrophoresis behind BamH| and the EcoR| double digestion identifies, cut the target DNA fragment about glue recovery 300bp, insert again in the multiple clone site of plasmid pGEX2-T (available from the couple stars bio tech ltd), the recombinant expression vector that obtains is changed in the e. coli bl21, and IPTG induces Expression of Fusion Protein.

3, the expression of fusion rotein in Pichia yeast

The fusion rotein encoding gene that above-mentioned steps 1 is obtained carries out double digestion with Eco RI and Not I (available from Dalian TAKARA bio tech ltd), enzyme is cut product and is inserted in the multiple clone site of plasmid pPIC9K (available from the couple stars bio tech ltd), changing the recombinant expression vector that obtains over to Pichia yeast GS115 (available from the couple stars bio tech ltd) increases, under the condition that Histidine lacks, utilize the G418 of different concns to screen, obtain G418 is had the bacterial strain of resistance, the methanol induction Expression of Fusion Protein.

4, the purifying of fusion rotein and evaluation

The fusion rotein that above-mentioned steps 3 is obtained is respectively 30 with molecular weight cut-off, the ultrafiltration membrance filter removal of impurity of 000Dal and 3000Dal, concentrated; Fusion rotein after concentrating adopts the Ni post to carry out purifying; Collect the peak at target protein place, utilize Sephadex G-25 chromatography column to carry out desalting treatment, use enteropeptidase (available from the couple stars bio tech ltd) that fusion rotein is cut then, the Ni post is removed 5 amino acid of 6 His and enteropeptidase recognition site, with integrin alpha _vβ ₃(available from U.S. Sigma chemical reagents corporation) is coupled to Sepharose 4B FF (available from U.S. GE company) and carries out affinity chromatography, and wash-out obtains fusion rotein.

The recombinant target pharmaceutical protein that above-mentioned steps 3 obtains detects through adopting SDS-PAGE behind the above chromatographic technique purifying, and the result as shown in Figure 1.Among Fig. 1, swimming lane 1 is a protein molecular weight standard, and swimming lane 2 and swimming lane 3 are the fusion rotein that obtains behind the above-mentioned purifying.R according to protein molecular weight standard and fusion rotein _fValue, the molecular weight that calculates the fusion rotein that obtains behind the above-mentioned purifying by linear regression equation is about 12,000Dal.

Utilize high performance liquid chromatography to identify the purity of the fusion rotein that obtains behind the above-mentioned purifying, the result as shown in Figure 2.The result shows that the purity of the fusion rotein that obtains behind the above-mentioned purifying has reached 96.8%.

The acute toxicity test of embodiment 2, fusion rotein

1, chmice acute toxicity test trial test

The fusion rotein that the foregoing description 1 is obtained dissolves with physiological saline, makes its concentration reach 300mg/ml.To 10 Kunming mouses (body weight 18～22g, male and female half and half) above-mentioned concentration of tail vein injection is the fusion rotein of 300mg/ml, and injected dose is the 0.5ml/20g body weight, observes the survival condition of mouse.The result shows that within 14 days, mouse is dead successively, so carry out the LD of fusion rotein ₅₀Determination test.

2, mouse peritoneal drug administration by injection acute toxicity test

With 20 Kunming mouse (body weight 18～22g, male and female half and half) fasting be can't help water after 16 hours, by the 0.5ml/20g body weight/time dosage respectively abdominal injection concentration be the fusion rotein that the foregoing description 1 of 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml, 800ug/ml or 1000ug/ml obtains, every injection in 2 hours 1 time, inject altogether 8 times.According to the weight, observe mouse at once and day by day 14 days after the administration, dead mouse is performed an autopsy on sb, measure LD ₅₀Value.Three repetitions are established in experiment, and the result shows, LD ₅₀Average out to 6mg/kg body weight.

The hemolytic test of embodiment 3, fusion rotein

The healthy rabbits back of the body position that body weight is about 2.3kg is fixed on the rabbit platform, put to death back separation arteria carotis communis intubate and get blood in centrifuge tube, stir gently with glass stick and to defibrinate, adding physiological saline shakes up, supernatant liquor is removed in centrifugal hypsokinesis, obtain red blood corpuscle three times so repeatedly, it is standby red blood corpuscle to be diluted to 2% red cell suspension.

Get 7 test tubes, numbering is also pressed the various solution of adding shown in the table 2, shake up gently, place 37 ℃ of water-baths, observe and write down the result who added behind the solution 0.5,1,2,3,4 hour respectively, observation has or not haemolysis and hemagglutination, places under the room temperature and continues after 24 hours to observe to have or not haemolysis and hemagglutination.

The hemolytic test of table 2 fusion rotein

Test tube number	1	2	3	4	5	6	7
								Fusion rotein (ml, 5mg/ml)	0.2	0.4	0.8	1.2	2.0	-	-
0.9% sodium chloride injection (ml)	2.3	2.1	1.7	2.1	0.5	2.5	-
								Distilled water (ml)	-	-	-	-	-	-	2.5
2% red cell suspension (ml)	2.5	2.5	2.5	2.5	2.5	2.5	2.5
								The result
0.5h	-	-	-	-	-	-	+
								1h	-	-	-	+	+	-	+
2h	-	-	-	+	+	-	+
								3h	-	-	+	+	+	-	+
4h	-	-	+	+	+	-	+

In the table 2, "-" no haemolysis of expression and red cell agglutination, "+" expression has haemolysis.

Three repetitions are established in experiment, and the result shows that the fusion rotein that the foregoing description 1 obtains is observed after 4 hours in 37 ℃ of water-baths, and positive control the 7th pipe is obvious pink haemolysis, and no red corpuscle sinks; 1, haemolysis and hemagglutination did not all appear in No. 2 test tubes at 0.5,1,2,3,4 hour, and the visible red cell sinks, shake test tube after, the red corpuscle come-up of sinking, show cell agglutination not, room temperature is observed did not also have haemolysis and hemagglutination in 24 hours; No. 3 haemolysis and hemagglutination did not all appear in test tube at 0.5,1,2 hour, and the visible red cell sinks, shake test tube after, the red corpuscle come-up of sinking, not show cell agglutination; Haemolysis and hemagglutination all occurred at 3,4 hours, no red corpuscle sinks, shake test tube after, show cell come-up; 4, haemolysis and hemagglutination all appearred in No. 5 test tubes at 1,2,3,4 hour, and the show cell sinks, shake test tube after, no red corpuscle floats.

Extract healthy people's venous blood, stir gently with glass stick and defibrinate, add physiological saline and shake up, supernatant liquor is removed in centrifugal hypsokinesis, obtains human red cell.After human red cell usefulness PBS damping fluid washing 4 times, be suspended in the PBS damping fluid, obtain human red cell suspension (10 ⁷Individual cell/ml suspension).The fusion rotein that the foregoing description 1 is obtained is made into storing solution in the PBS damping fluid, the concentration of fusion rotein is 20mg/ml in the storing solution.In above-mentioned storing solution, add PBS damping fluid to the concentration of fusion rotein and be respectively 2mg/ml, 4mg/ml, 6mg/ml, 8mg/ml, 10mg/ml, shake up, 37 ℃ of insulation 60min, the centrifugal 10min of 4000rpm then, get supernatant liquor colorimetric under 414nm, while, as blank, being suspended among the TritonX-100 with human red cell was 100% haemolysis with above-mentioned human red cell suspension.Three repetitions are established in experiment, and the result shows, the fusion rotein 50% hemolytic concentration (HC that the foregoing description 1 obtains ₅₀) be 6mg/ml, 100% hemolytic concentration is 20mg/ml.

The sensitivity test of embodiment 4, fusion rotein

Get 18 (body weight 260～289g of healthy guinea pig, male and female half and half), be divided into 3 groups at random, be negative control group (0.9% sodium chloride injection), positive controls (1% Ovum Gallus domesticus album) and fusion rotein group, every group 6, the concentration that the Ovum Gallus domesticus album of the sodium chloride injection of abdominal injection 0.9% quality percentage composition, 1% volumn concentration and the foregoing description 1 obtain the next day of respectively is each 0.5ml of fusion rotein of 1mg/ml, injects altogether 3 times.Again every group of 6 cavys are divided into 2 groups, every group 3, one group after injecting 14 days for the first time, one group is the fusion rotein 1ml of 1mg/ml by the sodium chloride injection of vena femoralis injection 0.9% quality percentage composition, the Ovum Gallus domesticus album of 1% volumn concentration and the concentration that the foregoing description 1 obtains respectively after injecting 21 days for the first time, observes to inject in back 30 minutes to have or not allergic phenomena.

Three repetition are established in experiment, and the result shows, inject after the 14th day and the 21st day once more vena femoralis injection the first time after, allergic symptom does not all appear in negative control group and fusion rotein group, animal activity is as usual, none death; And the cavy of positive controls occur unpeaceful, continuous dry cough, with pawl grab nose, perpendicular hair, four limbs feel like jelly and symptom such as expiratory dyspnea.Positive controls after being attacked by the femoral vein administration after the first administration in 14 days, and spasm death appears in 3 cavys; Spasm death appearred in 2 cavys by after the femoral vein administration attack in 21 days after the positive controls first administration.

Above result shows, the fusion rotein anaphylaxis feminine gender that the foregoing description 1 obtains.

The muscle of embodiment 5, fusion rotein, blood vessel irritation test

1, the muscle irritation of fusion rotein test

2 of healthy rabbits, body weight 2.3～2.5kg after breeding observing 1 day is no abnormal, is divided into 2 groups: fusion rotein group and blank group (physiological saline).The concentration that two leg quadricepss muscle of thigh are injected the foregoing description 1 acquisition respectively with aseptic method about rabbit is fusion rotein 1ml or the physiological saline of 1mg/ml, inject after 24 hours sacrifice of animal, dissect and take out quadriceps muscle of thigh, vertically cut along the injection site, observe the injection site and have or not the local excitation reaction, and according to the form below is converted into corresponding reaction order.

Reaction order	Irritant reaction
		0	No considerable change
1	Mild hyperaemia, scope is below 0.5 * 1.0cm

2	Moderate hyperemia, scope is below 0.5 * 1.0cm
		3	Severe hyperemia is with myodegeneration
4	Necrosis occurs, the brown sex change is arranged
		5	Occur extensively downright bad

Three repetitions are established in experiment, and the result is as shown in table 3.

The local excitation reaction of table 3 visual inspection injection back

With the above-mentioned conventional fixing back section statining of musculus quadriceps muscle of respectively organizing rabbit, carry out pathological observation under the mirror.

The result shows, compare with control group, the fusion rotein that injection the foregoing description 1 obtains, the musculus quadriceps muscle visual inspection of rabbit does not have obvious local excitation reaction, pathologic finding and control group no significant difference under the mirror show that the fusion rotein that the foregoing description 1 obtains does not have obvious local irritation.

2, the blood vessel irritation of fusion rotein test

Get 4 of healthy rabbits (body weight 2.3～2.5kg, male and female half and half).Be divided into 0.9% sodium chloride injection control group and fusion rotein group by body weight and sex, 2 every group.The fusion rotein that the foregoing description 1 is obtained carries out intravenous drip in rabbit left side ear ear edge by the concentration of 1mg/ml according to the dosage of 10ml/kg body weight, and drip velocity is 1ml/min, instils continuous drip 7 days every day 1 time.Control group is the sodium chloride injection of intravenous drip 0.9% quality percentage composition by the same way.Observe the administration topical manifestations during except that each administration and after the administration, cut the administration exterior feature of picking up the ears after the last intravenous drip, pin proximal part 1cm place is gone in the distance intravenous drip in conventional fixing back, cuts the wide sample of 0.5cm every 2cm, pathological observation under the mirror is carried out in section statining.

Three repetitions are established in experiment, and pathological examination results shows under naked eyes and the mirror, and the fusion rotein intravenous drip that the foregoing description 1 obtains does not have local irritation.

The extracorporeal anti-tumor cell growth function of embodiment 6, fusion rotein

The fusion rotein that adopts 1 acquisition of mtt assay detection the foregoing description is drawn and is suppressed curve the effect of HeLa cell inhibiting, observes the lethal effect of certain density fusion rotein to the HeLa cell simultaneously.

Three repetitions are established in experiment, and the result shows that the fusion rotein that the foregoing description 1 obtains acted on the HeLa cell after 24 hours, and fusion rotein can suppress HeLa cell proliferation in the dose-dependently mode, and fusion rotein suppresses half effect quantity (IC of HeLa cell proliferation ₅₀) be 0.33 μ g/ml, and the HeLa cellular form changes, and concrete experimental result is shown in Fig. 3 and table 4.Among Fig. 3, A is for adding the precellular form of fusion rotein, and B is the form of cell behind the adding fusion rotein.

Table 4 fusion rotein acts on the growth-inhibiting effect behind the HeLa cell 24h

Employing detects the restraining effect of the fusion rotein of the foregoing description 1 acquisition to SMMC-7721 cell (available from Shanghai Christian Dior bio tech ltd) with quadrat method, and the result is shown in Fig. 4 and table 5.Among Fig. 4, A is for adding the precellular form of fusion rotein, and B is the form of cell behind the adding fusion rotein.

Table 5 fusion rotein acts on the growth-inhibiting effect behind the SMMC-7721 cell 24h

Three repetitions are established in experiment, and the result shows that the fusion rotein that the foregoing description 1 obtains can suppress SMMC-7721 cell proliferation in the dose-dependently mode, its IC ₅₀Value is 0.30 μ g/ml, and can cause the change of SMMC-7721 cellular form.

Employing detects the restraining effect of the fusion rotein of the foregoing description 1 acquisition to SGC-7901 cell (available from Shanghai Christian Dior bio tech ltd) with quadrat method, and the result is shown in Fig. 5 and table 6.Among Fig. 5, A is for adding the precellular form of fusion rotein, and B is the form of cell behind the adding fusion rotein.

Table 6 fusion rotein acts on the growth-inhibiting effect behind the SGC-7901 cell 24h

Three repetitions are established in experiment, and the result shows that the fusion rotein that the foregoing description 1 obtains can also suppress the propagation of SGC-7901 cell, its IC in the dose-dependently mode ₅₀Value is 0.30 μ g/ml, and can cause the change of SGC-7901 cellular form.

The solid tumor resisting growth function of embodiment 7, fusion rotein

Murine sarcoma S180 cell (available from Shanghai Christian Dior bio tech ltd) is inoculated in the intraperitoneal of Kunming mouse, selects 7～10 days well-grown mouse of venter posterior water of inoculation murine sarcoma S180 cell, take off cervical vertebra and put to death as knurl source animal.

Under aseptic condition, extract the ascites of above-mentioned knurl source mouse, calculate tumor cell number, tumour cell is diluted to 1 * 10 with aseptic physiological saline ⁷Individual cell/ml puts in the frozen water and deposits.Get 80 Kunming mouse (body weight 18～22g again, male and female half and half), mouse is divided into 4 groups at random, is respectively: 1. blank group, 2. fusion rotein high dose group (dosage is 10 μ g/kg body weight), 3. dosage group (dosage is 5 μ g/kg body weight) and 4. fusion rotein low dose group (dosage is 2.5 μ g/kg body weight) in the fusion rotein.Murine sarcoma S180 cell 0.2ml after every above-mentioned dilution of mouse right fore oxter subcutaneous injection, injection murine sarcoma S180 cell is after 24 hours, distinguish the fusion rotein of the foregoing description 1 preparation of abdominal injection various dose again, inject once every day, injects altogether 10 times.After the last administration 24 hours, the body weight of weighing mouse is taken off mouse cervical vertebra then and is put to death, and strips knurl body tissue and weighs.Calculate the heavy inhibiting rate of knurl by following formula.Three repetitions are established in experiment, and the result is as shown in table 7.

Table 7 recombinant target medicine is to transplantability mouse S ₁₈₀The influence of knurl bulk-growth

Group	Before number of animals (only) administration/administration after	The knurl weight (X ± S, g)	Tumour inhibiting rate (%)
				The blank group	20/20	1.58±0.42	-
The fusion rotein high dose group	20/20	0.71±0.17	55.06
				Dosage group in the fusion rotein	20/20	0.91±0.26	42.41
The fusion rotein low dose group	20/20	1.23±0.19	22.15

The result shows that the fusion rotein of the foregoing description 1 preparation has the effect of obvious suppression tumor growth to murine sarcoma S180 cell, and has certain dose-effect dependence.

The anti metastasis function of embodiment 8, fusion rotein

With B ₁₆Oncocyte (available from Shanghai Christian Dior bio tech ltd) is inoculated in C ₅₇The intraperitoneal of mouse (available from Jilin University's Experimental Animal Center) is selected inoculation B ₁₆The well-grown C of oncocyte ascites 7～10 day after tomorrow ₅₇Mouse takes off cervical vertebra and puts to death as knurl source animal.

Get 80 Kunming mouse (body weight 18～22g, male and female half and half), mouse is divided into 4 groups at random, is respectively: 1. blank group, 2. fusion rotein high dose group (dosage is 10 μ g/kg body weight), 3. dosage group (dosage is 5 μ g/kg body weight) and 4. fusion rotein low dose group (dosage is 2.5 μ g/kg body weight) in the fusion rotein.Extract the ascites of above-mentioned knurl source mouse, calculate tumor cell number, tumour cell is diluted to 1 * 10 with aseptic physiological saline ⁷Individual cell/ml puts in the frozen water and deposits.With the fusion rotein of the foregoing description 1 preparation of difference amount and the B after the above-mentioned dilution ₁₆Oncocyte mixes, and the tail vein is input in the mouse body, and be administered once every day, injects altogether 10 times.After the last administration 24 hours, the body weight of weighing mouse is taken off mouse cervical vertebra then and is put to death, and strips lungs and makes tissue slice, microscopic examination.

Three repetitions are established in experiment, the result shows, the phenomenon that tumour cell shifts to lung has all appearred in the blank group, and low dose group has 2 mouse the phenomenon that tumour cell shifts to lung to occur, and the phenomenon that tumour cell shifts to lung does not all appear in middle dosage group and high dose group.

Embodiment 9, fusion rotein are to the growth inhibition function of metastatic tumour

Lewis lung cancer oncocyte (available from Shanghai Christian Dior bio tech ltd) is inoculated in C ₅₇The intraperitoneal of mouse (available from Jilin University's Experimental Animal Center) is selected the well-grown C of inoculation Lewis lung cancer oncocyte ascites 7～10 day after tomorrow ₅₇Mouse takes off cervical vertebra and puts to death as knurl source animal.

Under aseptic condition, strip knurl source C ₅₇The tumor tissue of mouse shreds after weighing, and the amount that adds 3 ml physiological salines according to every gram oncocyte is mixed, and with aseptic homogenizer cell is ground to form homogenate, put deposit in the frozen water standby.

Get healthy C ₅₇Mouse, every mouse tail vein injection 1 * 10 ⁶Individual above-mentioned Lewis lung cancer cell suspension.Behind the mice-transplanted tumor cell 96 hours, the Lewis lung cancer cell is at lung's implantation of mouse.Mouse is divided into 4 groups at random, is respectively: 1. blank group; 2. fusion rotein high dose group (dosage is 10 μ g/kg body weight); 3. dosage group (dosage is 5 μ g/kg body weight) in the fusion rotein; 4. fusion rotein low dose group (dosage is 2.5 μ g/kg body weight).The fusion rotein tail vein of the foregoing description 1 preparation of difference amount is input in the mouse body, and be administered once every day, injects altogether 10 times.After the last administration 24 hours, mouse is taken off cervical vertebra put to death, do lung's section, microscopic examination.

Three repetitions are established in experiment, the result shows, control group is found tumour implantation or growth, and dosage group and fusion rotein low dose group are not all found tumour implantation or growth in the fusion rotein high dose group, fusion rotein, and the fusion rotein that shows the foregoing description 1 preparation has significant inhibition metastases and shifts the function of back growth.

Sequence table

＜110〉Sun Miaonan

＜120〉a kind of antineoplastic target fusion protein and application thereof with release switch

<130>CGGNARZ92070

<160>2

<210>1

<211>279

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

gactctcctg?caaatccgtg?ctgcgatgct?gcaacctgta?aactgagacc?aggggcacag 60

tgtgcagaag?gactgtgttg?tgagcagtgc?agatttatga?aagaaggaac?agtatgccgg 120

atagcaaggg?gtgatgacat?ggatgattac?tgcaatggca?tatctgctgg?ctgtcccaga 180

aatcccttcc?atgcccgtgt?gggcattggc?gcggtgctga?aagtgctgac?caccggcctg 240

ccggcgctga?ttagctggat?taaacgtaaa?cgtcagcag 279

<210>2

<211>93

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>2

Asp?Ser?Pro?Ala?Asn?Pro?Cys?Cys?Asp?Ala?Ala?Thr?Cys?Lys?Leu?Arg

1 5 10 15

Pro?Gly?Ala?Gln?Cys?Ala?Glu?Gly?Leu?Cys?Cys?Glu?Gln?Cys?Arg?Phe

20 25 30

Met?Lys?Glu?Gly?Thr?Val?Cys?Arg?Ile?Ala?Arg?Gly?Asp?Asp?Met?Asp

35 40 45

Asp?Tyr?Cys?Asn?Gly?Ile?Ser?Ala?Gly?Cys?Pro?Arg?Asn?Pro?Phe?His

50 55 60

Ala?Arg?Val?Gly?Ile?Gly?Ala?Val?Leu?Lys?Val?Leu?Thr?Thr?Gly?Leu

65 70 75 80

Pro?Ala?Leu?Ile?Ser?Trp?Ile?Lys?Arg?Lys?Arg?Gln?Gln

85 90

Claims

1. a fusion rotein is made up of the aminoacid sequence shown in the sequence in the sequence table 2.

2. the encoding gene of the described fusion rotein of claim 1.

3. encoding gene according to claim 2 is characterized in that: the nucleotide sequence of described encoding gene is the sequence 1 in the sequence table.

4. the recombinant expression vector that contains claim 2 or 3 described genes.

5. contain claim 2 or 3 described expression of gene boxes.

6. the reorganization bacterium that contains claim 2 or 3 described genes.

7. the transgenic cell line that contains claim 2 or 3 described genes.

8. medicine with antitumor action, its activeconstituents is the described fusion rotein of claim 1.

9. the described fusion rotein of claim 1 has application in the medicine of antitumor action in preparation.