CN101070349B - Fusion protein with function of selective killing endothelial cells in tumor neogenetic blood vessels and use thereof - Google Patents

Abstract

This invention relates to a fusion protein that possess action of selectively kill tumour rebirth blood vessel endotheliocyte, and its application. This fusion protein through connecting peptide connect VEGF121 with amido end of sponge gourd seed ribosome inactivating protein(RIP) Luffin Alpha. The vascular endothelial cell growth factor VEGF121 act as means of delivery, make this fusion toxin idiosyncratic incorporate with vascular endothelial cell growth factor receptor F1k1/ KDR, thereby optionally ingress tumour rebirth vascular endothelial cell; the polypeptide possess amphipathic molecule character, by destroy lysosome membrane to promote the release of lysosome inside dissociate toxin molecule; the toxin carboxyl terminal endoplasmic reticulum loacting signal led toxin molecule( sponge gourd seed RIP Luffin Alpha) to target site to exert toxic effect, so to destroy rebirth blood vessel of tumour organization, cut off tumour blood supply, achieve the end of restraining tumor.

Description

Fusion rotein and application with function of selective killing endothelial cells in tumor neogenetic blood vessels

Technical field

The present invention relates to fusion rotein and encoding gene thereof and application, particularly relate to fusion rotein and encoding gene thereof with function of selective killing endothelial cells in tumor neogenetic blood vessels, and this Expression of Fusion Protein method and the application in the preparation antitumor drug.

Background technology

Cancer is one of major disease of serious threat human health.Death reaches more than 700 ten thousand people to the newly-increased tumor cases in the whole world all more than 1,000 ten thousand every year, and at present, global tumour patient has exceeded 4,000 ten thousand people.According to Ministry of Health statistics, China's tumor incidence is overall ascendant trend, in recent years, annual newly-increased tumour patient 2,000,000, dead more than 130 ten thousand, at present, national tumour patient Estimate of Total Number reaches 4,500,000 people approximately.

The tumor-targeting of (excision and put, chemotherapy) is poor because the traditional tumour methods of treatment, and toxic side effect is big, thereby tumor-targeting drug has become the important trend of following cancer therapy drug development.It is the hot fields of neoplasm targeted therapy that the specificity antineoplastic new vessel generates.Studies show that tumor growth must rely on the formation of new vessel around it, as long as can destroy tumor neogenetic blood vessels, tumour will stop growing because obtaining enough nutrition, and atrophy rapidly, apoptosis, necrosis, is finally eliminated by body.Therefore, the anti-angiogenic treatment of tumour provides the brand-new treatment thinking at a plurality of links in tumour generation, the evolution, has a good application prospect.

Vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF) be the carbohydrate protein that at first purifying comes out in Niu Chuiti folliculus stellate cell vitro culture liquid such as Ferrara in 1989, vascularization is had specificity, and have functions such as the vascular endothelial cell proliferation of promotion, vasculogenesis, increase vascular permeability, quickening blood flow.VEGF and receptor family thereof, especially II receptor (F1k1/KDR) is close with growth of tumor, infiltration and transfer relationship, is the important target of cancer therapy drug design in recent years.Human VEGF is because the different montages of mRNA produce 7 kinds of different VEGF molecules, that is: VEGF ₁₂₁, VEGF ₁₄₅, VEGF ₁₄₈, VEGF ₁₆₅, VEGF ₁₈₃, VEGF ₁₈₉And VEGF ₂₀₆Wherein, VEGF ₁₂₁Be the most common VEGF, it comprises 121 amino-acid residues, and different with other member is, it can with II type vegf receptor (F1k1/KDR) with the high-affinity specific combination, and with the basic debond of I type vegf receptor (Flt1).Confirm that the F1k1/KDR acceptor has tyrosine kinase activity,, and almost can't detect at the vascular endothelial cell of adjacent healthy tissues in the vascular endothelial cell surface overexpression of multiple malignant tumor tissue.

(ribosome-inactivating protein is extensively to be present in a botanic class toxalbumin RIP) to ribosome inactivating protein, is eukaryotic protein synthetic inhibitor.It acts on 28S rRNA on the Mammals large ribosomal subunit by RNA N-Glycosylase, and produces and take off the VITAMIN B4 effect, thereby destroys ribosomal structure, the translation of arrestin matter; RIP also participates in apoptotic adjusting simultaneously, has huge pharmaceutical potential.

According to the difference of RIP primary structure, can be divided into two classes: I type and II type.I type RIP is made up of a peptide chain; II type RIP is made up of two polypeptide chains, is called A chain and B chain, and two chains link to each other by disulfide linkage.A chain and I type RIP have RNA N-glycosidase activity, can make the rrna inactivation.The sponge gourd seed ribosome deactivated protein is typical case's representative of I type RIP, and II type RIP mainly comprises ricin, Semen Abri Precatorii albumen and fragrant camphor tree toxalbumin etc.I type RIP is particularly suitable for doing the warhead section of cytotoxicity medicine, RIP can be connected on the part of monoclonal antibody or tumour antigen, RIP is taken in the target cell, kill tumor cell optionally, thereby have higher curative effect and lower toxicity.

The present inventor has obtained a kind of ribosome inactivating protein from the sponge gourd seed, name is called Luffin α, and its amino acid residue sequence is shown in the SEQ ID NO:1 in the sequence table, and its encoding sequence can be shown in the SEQ IDNO:2 in the sequence table.

Summary of the invention

The purpose of this invention is to provide a kind of II of having type vegf receptor specificity, the fusion rotein of alternative killing tumor cells new vessel endotheliocyte effect.

Fusion rotein provided by the present invention, called after VEGF ₁₂₁/ LuffinKDEL is to connect VEGF by connection peptides at the aminoterminal (N end) of the ribosome inactivating protein Luffin of sponge gourd seed α ₁₂₁Fusion rotein.

The selection of described connection peptides is diversified, the polypeptide that its amino acid residue sequence can the grade shown in the SEQ ID NO:3 in the sequence table has amphiphilic character, can promote of the release of free lps molecule by the lysosome membrane that destroys target cell, thereby strengthen the lethal effect of toxin target cell.

Described connection peptides is also replaceable to be its analogue, and the difference of these analogues and described connection peptides can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or other known Protocols in Molecular Biology.Analogue also comprises having the analogue that is different from natural L-amino acid residue (as D-amino acid), and has that non-natural exists or the amino acid whose analogue of synthetic.

Specifically, the amino-acid residue of described connection peptides can carry out following change (amino-acid residue after the residue number replacement): 4Gly; 5Ser; 6Lys, Ala; 7Lys, Ala, D-Ile, Arg.Leu, Val, His, Net; 8Lys, Ala, Trp, Arg, His; 9D-Lys, Ala, Arg, His; 10D-Phe, Ala, Lys, Trp, Leu, Ile, Val, Met; 11 D-Leu, Lys, Ala, Ile, Val, Met; 12Lys, D-His, Ala, Arg, Ile, Val, Net; 13Ala, Lys, D-Scr, Trp, Met, Ile, Arg, His, Thr, Leu, Val; 14Lys, D-Ala, Trp, Arg, His, Leu, Ile, Val; 15D-Lys, Lys Ala, Trp, Arg, His, Leu, Ile, Val; 16D-Lys, Ars, His; 17D-Phe, Lys, Trp, Arg, His; 18Ala, Lys, Trp, Met, Arg, His, Phe, Leu, Ile, Val; 19Ala, D-Lys, Arg, His; 20Lys, Trp, D-Ala, Arg, His, Phe; 21Ala, Lys, D-Phe, Ile, Val, Met, Leu; 22Ala, Lys, D-Val, Trp, Arg, His, Met, Leu; 23Ala, Lys, TrP, Arg, His, Leu, Met; 24Ala, Lys, D-Glu, Gly, Arg, His, Leu, Ile, Phe, Asn; 25D-Ile, Ala, Lys, Trp, Leu, Phe, Val, Het; 26Lys, Pro, Ala, His, Leu, Arg, Ile, Phe; 27Lys, Ala, D-Asn, Gln, Arg, His; 28D-Ser, Lys, Ala, Thr, Gly, Leu, Ile, Gln, Asn; 32Gly; 33Ser.

Specifically, described fusion rotein with function of selective killing endothelial cells in tumor neogenetic blood vessels is one of following amino acid residue sequences:

1) the SEQ ID NO:4 in the sequence table;

2) with the amino acid residue sequence of SEQ ID NO:4 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of function of selective killing endothelial cells in tumor neogenetic blood vessels.

SEQ ID NO:4 in the sequence table is made up of 405 amino-acid residues, is VEGF from aminoterminal 1-122 amino acids residue ₁₂₁, be the connection peptides sequence from aminoterminal 123-157 amino acids residue, be the ribosome inactivating protein of sponge gourd seed from aminoterminal 158-405 amino acids residue.

Above-mentioned gene (VEGF with fusion rotein of function of selective killing endothelial cells in tumor neogenetic blood vessels encodes ₁₂₁/ LuffinKDEL), be one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:5 in the sequence table;

2) dna sequence dna of SEQ ID NO:4 in the code sequence tabulation;

3) with sequence table in the nucleotide sequence that limits of SEQ ID NO:5 have 90% above homology and have the nucleotide sequence of function of selective killing endothelial cells in tumor neogenetic blood vessels;

4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID NO:5 in the sequence table.

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

SEQ ID NO:5 in the sequence table is by 1215 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID NO:4 in the sequence table from 5 ' end 1-1215 bit base, is VEGF from 5 ' end 1-366 bit base ₁₂₁Encoding sequence, be the encoding sequence of connection peptides from 5 ' end 367-471 bit base, be the encoding sequence of sponge gourd seed ribosome deactivated protein from 5 ' end 472-1215 bit base.

For obtaining higher specificity, increase fusion toxin albumen VEGF of the present invention ₁₂₁/ LuffinKDEL its target point--the concentration of endoplasmic reticulum, the C of sponge gourd seed ribosome deactivated protein end also is connected with the endoplasmic reticulum signal for locating in the described fusion toxin albumen, and the amino acid residue sequence of described endoplasmic reticulum signal for locating is shown in the SEQ ID NO:6 in the sequence table.

Specifically, the described fusion rotein with function of selective killing endothelial cells in tumor neogenetic blood vessels that is connected with the endoplasmic reticulum signal for locating is one of following amino acid residue sequences:

1) the SEQ ID NO:7 in the sequence table;

2) with the amino acid residue sequence of SEQ ID NO:7 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of function of selective killing endothelial cells in tumor neogenetic blood vessels.

SEQ ID NO:7 in the sequence table is made up of 409 amino-acid residues, is VEGF from aminoterminal 1-122 amino acids residue ₁₂₁, be the connection peptides sequence from aminoterminal 123-157 amino acids residue, be the ribosome inactivating protein of sponge gourd seed from aminoterminal 158-405 amino acids residue, be the endoplasmic reticulum signal for locating from aminoterminal 406-409 amino acids residue.

The above-mentioned gene of encoding with fusion rotein of function of selective killing endothelial cells in tumor neogenetic blood vessels

(VEGF ₁₂₁/ LuffinKDEL), be one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:8 in the sequence table;

2) dna sequence dna of SEQ ID NO:7 in the code sequence tabulation;

3) with sequence table in the nucleotide sequence that limits of SEQ ID NO:8 have 90% above homology and have the nucleotide sequence of function of selective killing endothelial cells in tumor neogenetic blood vessels;

4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID NO:8 in the sequence table.

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

SEQ ID NO:8 in the sequence table is by 1227 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID NO:7 in the sequence table from 5 ' end 1-1227 bit base, is VEGF from 5 ' end 1-366 bit base ₁₂₁Encoding sequence, from 5 ' end 367-471 bit base is the encoding sequence of connection peptides, from 5 ' end 372-1215 bit base is the encoding sequence of sponge gourd seed ribosome deactivated protein, is the encoding sequence of endoplasmic reticulum signal for locating from 5 ' end 1215-1227 bit base.

Described VEGF ₁₂₁The fragment of/LuffinKDEL, derivative and analogue also are that the present invention will protect, and are meant to keep VEGF of the present invention ₁₂₁Identical biological function of/LuffinKDEL or active polypeptide.Polypeptide fragment of the present invention, derivative or analogue may be defined as: 1) by one or more conservative or polypeptide that non-conservative amino acid residues (being preferably the conservative amino acid residue) is replaced, and the amino-acid residue of such replacement can be, also can not encoded by genetic code; 2) in one or more amino-acid residues, has the polypeptide of substituted radical; 3) mature polypeptide and another compound merge formed polypeptide; 4) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as is used for the sequence of this polypeptide of purifying, or the fusion rotein of antibody fragment or other antigen ligand sequence), or the nucleotide sequence of another polypeptide of will encoding (or its part) merges the encoding sequence that just can obtain fusion polypeptide with nucleotide sequence of the present invention (or its part), make the encoding sequence of this fusion polypeptide obtain to express again, just can produce fusion polypeptide.The technology of described generation fusion polypeptide is well known in the art, comprises the encoding sequence that connects coded polypeptide, thereby makes them in same reading frame, and makes the expression of fusion polypeptide be controlled by identical promotor and terminator.

Described VEGF ₁₂₁The analogue of/LuffinKDEL and VEGF ₁₂₁The difference of/LuffinKDEL can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or other known Protocols in Molecular Biology.Analogue also comprises having the analogue that is different from natural L-amino acid residue (as D-amino acid), and has that non-natural exists or the amino acid whose analogue of synthetic.The amino acid residue sequence that should be understood that fusion rotein of the present invention is not limited to the above-mentioned representative sequence that exemplifies.

Described VEGF ₁₂₁/ LuffinKDEL fusion rotein also can be the process modification, or the modified VEGF that has improved its anti-proteolysis performance or optimized solubility property ₁₂₁/ LuffinKDEL polypeptide.(the not changing primary structure usually) form of modifying comprises: 1) in the body or the chemically derived form of external polypeptide, as acetylize or carboxylated; 2) glycosylation, carry out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic of polypeptide and processing or further, this modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to; 3) has the sequence of phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).

Code book invention VEGF ₁₂₁The polynucleotide of/LuffinKDEL can be dna form or rna form.Dna form comprises cDNA or artificial-synthetic DNA, can be strand or double-stranded, also can be coding strand or noncoding strand.

The present invention also provides the varient of described fusion rotein polynucleotide, its coding and VEGF ₁₂₁/ LuffinKDEL has polypeptide or polypeptide fragment, analogue and the derivative of identical aminoacid sequence.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place, and also can comprise replacing varient, deletion mutation body and inserting varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

Contain gene VEGF of the present invention ₁₂₁The expression vector of/LuffinKDEL, transgenic cell line and host bacterium all belong to protection scope of the present invention.

Amplification VEGF ₁₂₁Arbitrary segmental primer is to also within protection scope of the present invention among the/LuffinKDEL.

Another object of the present invention provides a kind of above-mentioned fusion rotein VEGF with function of selective killing endothelial cells in tumor neogenetic blood vessels that expresses ₁₂₁The method of/LuffinKDEL.

Fusion rotein VEGF provided by the present invention ₁₂₁The expression method of/LuffinKDEL is with described fusion rotein VEGF ₁₂₁/ LuffinKDEL gene, this gene variant or contain fusion rotein VEGF ₁₂₁The recombinant expression vector of/LuffinKDEL gene transforms or the transduction host cell, cultivates host cell, and separation and purification albumen from substratum or cell obtains fusion rotein VEGF ₁₂₁/ LuffinKDEL.

Described fusion rotein VEGF with function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁/ LuffinKDEL gene can be inserted in the recombinant expression vector.Make up the carrier that sets out of described recombinant expression vector, can be any one and refer to that the bacterial plasmid that carries out exogenous gene expression well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.Described carrier includes but not limited to: the expression vector based on the T7 promotor of expressing in bacterium (AH Rosenberg, et al.Vectors for selectiveexpression of cloned DNAs by T7RNA polymerase.Gene.1987,56 (1): 125-135); The pMSXND expression vector of in mammalian cell, expressing (SJ Lee and D Nathans.Proliferinsecreted by cultured cells binds to mannose 6-phosphate receptors.J.Biol.Chem.1988; 263:3521-3527) with at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain copy-point, promotor, marker gene and translation controlling elements usually.

Be the carrier that sets out with pET-30a (+), structure contain described antigen-4 fusion protein gene VEGF with function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁The recombinant expression vector of/LuffinKDEL is pET-30a (+)-VEGF ₁₂₁/ LuffinKDEL.

Can adopt method well known to those skilled in the art to make up and contain described antigen-4 fusion protein gene VEGf with function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁The recombinant expression vector of/LuffinKDEL, as the extracorporeal recombinant DNA technology, (Sambrook, et al Molecular cloing such as the interior recombinant technology of DNA synthetic technology and body, aLaboratory Manual.Cold spring harbor laboratory.New York, 1989).Described antigen-4 fusion protein gene VEGF with function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁The dna sequence dna of/LuffinKDEL gene can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA's.Described promotor can be: colibacillary lac or trp promotor, phage promoter, retrovirus and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector also can comprise one or more selected markers, to be provided for selecting the phenotype shape of transformed host cells, to cultivate dihydrofolate reductase gene, neomycin resistance gene and green fluorescent protein (GFP) gene of usefulness or be used for colibacillary tsiklomitsin or ampicillin resistance gene etc. as eukaryotic cell.

Described host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; The low eukaryotic cell that waits is as yeast cell; Higher eucaryotic cells is as mammalian cell.Representative example has: intestinal bacteria, streptomycete; The bacterial cell of Salmonella typhimurium; Eukaryotic cell such as yeast, vegetable cell; Insect cells such as fruit bat S2 or Sf9; Zooblasts such as CHO, COS, 293 cells or Bowes melanoma cell.

When polynucleotide of the present invention are expressed in higher eucaryotic cells,, will make to transcribe to be enhanced if in carrier, insert enhancer sequence.Enhanser is the cis acting factor of DNA, and length is generally 10-300 base pair, acts on promotor transcribing with enhancing gene.As the length in replication origin side in late period one be about the SV40 enhanser of 100-270 base pair, at the polyoma enhanser of replication origin side in late period one or adenovirus enhanser etc.

Available routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell, is cultivated transformant, the abduction delivering target protein, and recombinant protein advanced separation and purification.

Cultivation contains the fusion rotein VEGF that the present invention has function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁The substratum of the host cell of/LuffinKDEL encoding gene and culture condition all can be substratum and the culture condition of cultivating the host that sets out.

Described fusion rotein VEGF ₁₂₁/ LuffinKDEL and encoding gene thereof can be used for preparing antitumor drug, thereby the present invention also provides a kind of antitumor drug.

Antitumor drug provided by the present invention, its activeconstituents are above-mentioned fusion rotein VEGF with function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁/ LuffinKDEL or its encoding gene.

Described fusion rotein VEGF with function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁/ LuffinKDEL gene can be present in the carrier for expression of eukaryon.

When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent and the absorption carrier etc. of pharmaceutical field routine.

Medicine of the present invention can be made various ways such as injection liquid or lyophilisate.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.

The consumption of above-mentioned protein drug and genomic medicine can adopt the routine dose of protein drug and genomic medicine in the pharmaceutical field, and can be according to the practical situation adjustment.The tumor bearing nude mice test-results shows, fusion rotein VEGF of the present invention ₁₂₁When/LuffinKDEL is the 15mg/kg body weight in injected dose, the effect of selective killing endothelial cells in tumor neogenetic blood vessels can be brought into play, and growth of tumor can be obviously delayed.

The invention provides a kind of fusion rotein toxin VEGF ₁₂₁/ LuffinKDEL and encoding gene thereof.This albumen is to utilize VEGF ₁₂₁With the vegf receptor F1k1/KDR high special bonded characteristics of malignant tumour high expression level, the fusion rotein toxin that is formed by connecting by connection peptides with the ribosome inactivating protein Luffin α that derives from the sponge gourd seed.Blood vessel endothelial cell growth factor VEGF ₁₂₁As launch vehicle, can make this fusion toxin and vascular endothelial growth factor receptor F1k1/KDR specific combination and internalization intravasation endotheliocyte, sponge gourd seed ribosome deactivated protein Luffin α brings into play its effects of toxins then, optionally enter and killing tumor cells new vessel endotheliocyte, and then the new vessel of destruction tumor tissues, cut off tumor blood supply, reach the purpose that suppresses tumour.In addition, this proteic expression condition is simple, is easy to purifying, can carry out large-scale industrial production, thereby, can this fusion rotein be that activeconstituents is prepared into antitumor drug.The present invention will play a significant role in medical science and field of biological pharmacy.

Below in conjunction with specific embodiment the present invention is described in further details.

Description of drawings

Fig. 1 is the VEGF at expression in escherichia coli ₁₂₁The 10% denaturing polyacrylamide gel electrophoresis detected result of-LuffinKDEL

Fig. 2 is expression in escherichia coli and purified VEGF ₁₂₁The 10% denaturing polyacrylamide gel electrophoresis detected result of-LuffinKDEL

Fig. 3 is the tumor growth curve of different time after control group and the test group administration

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, Dayid W., Molecular Cloning:A Laboratory Manual, 3 ^RdEdition, 2001, NY, Cold SpringHarbor).The primer synthesizes and examining order is finished by Beijing AudioCodes Bioisystech Co., Ltd.

Embodiment 1, has the fusion rotein VEGF of function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁The acquisition of/LuffinKDEL encoding gene

One, human vascular endothelial growth factor VEGF ₁₂₁The acquisition of encoding gene

1, from people's liver cancer tissue, extracts total RNA

Utilize total RNA of the Trizol reagent extraction people liver cancer tissue of Invitrogen company, method is: get 100mg people's liver cancer tissue (taking from PLA General Hospital) and put into aseptic glass grinding device, add 1mL Trizol reagent, grind rapidly under the condition of ice bath, draw supernatant to the centrifuge tube that does not have the RNA enzyme, 4 ℃, the centrifugal 10min of 12000g, draw supernatant, add 0.2mL chloroform/1mL Trizol, fully leave standstill 2-3min behind the mixing, then water is moved in the new centrifuge tube, add isopyknic Virahol, after putting upside down mixing, precipitation at room temperature 10min, 4 ℃ then, the centrifugal 5min of 7500g repeats rinsing once, drying at room temperature 5-10min behind the removal Virahol adds DEPC water dissolution RNA precipitation.

2, RT-PCR amplification human vascular endothelial growth factor VEGF ₁₂₁Gene

With Invitogen Corporation's Super script II ThermoScript II and according to the specification sheets of test kit, the total RNA of liver cancer that obtains with step 1 is synthetic its cDNA of template reverse transcription, and then to get 5ul reverse transcription synthetic cDNA be template, at primer VEGF ₁₂₁F (upstream primer): 5 '-ATA CATATGgCTCCgATggCAgAAggTggCggg and VEGF ₁₂₁R (downstream primer): 5 '- AAATTTACCAATTCCCAgAAAgCCACCgCCPcr amplification VEGF under the guiding of CCgCCTCggCTTgTCACATTTTTC (is connection peptides (SEQ ID NO:3 in the sequence table) part encoding sequence with single underscore base) ₁₂₁Gene, 50ul PCR reaction system is: 10 * PCR damping fluid (contains 15mM MgSO ₄) 5ul, each 50pmol of upstream and downstream Auele Specific Primer, 10mM dNTPs 1ul, pyrobest polysaccharase (available from TaKaRa company) 1U, moisturizing is to 50ul.The PCR reaction conditions is: 94 ℃ of 30sec of elder generation, 55 ℃ of 40sec, 72 ℃ of 1min, totally 25 circulations; 72 ℃ were extended 7 minutes then.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, it is the dna fragmentation of 400bp that the result has obtained length, consistent with the expection size.Reclaim and this purpose fragment of purifying, connect among the carrier pMD 18-T (TaKaRa company), will connect product transformed into escherichia coli (E.coli) DH5 α competent cell again, screening positive clone, the upgrading grain obtains containing the recombinant plasmid of goal gene, called after pMD 18-VEGF ₁₂₁, it is checked order, sequencing result shows and has obtained the correct human vascular endothelial growth factor VEGF of sequence ₁₂₁Gene.

Two, the acquisition of sponge gourd seed ribosome deactivated protein luffin a gene

1, the extraction of the total RNA of sponge gourd seed

The mortar that the sponge gourd seed places precooling is produced in Beijing, adding liquid nitrogen is ground into powder rapidly, add TRIzol reagent (Invitrogen company), 4 ℃, 120, centrifugal 10 minutes of 000rpm gets supernatant, add 200ul chloroform/1mL TRIzol, violent jolting 15-30 second, room temperature was placed 3 minutes, 4 ℃ again, 120, centrifugal 15 minutes of 000rpm, get the upper strata aqueous phase solution, add the equal-volume Virahol, room temperature was placed 10 minutes behind the mixing, 4 ℃ 120, centrifugal 10 minutes of 000rpm abandons supernatant, adds 75% ethanol (with the preparation of the DEPC water) washing of precooling, be placed to the precipitation transparent after, add the water dissolution precipitation of an amount of nuclease free, obtain the total RNA of sponge gourd seed ,-70 ℃ of preservations are standby.

2, reverse transcription PCR amplification luffin α gene

With Superscript II ThermoScript II (Invitogen company) and with reference to specification sheets, the total RNA of sponge gourd seed that gets the acquisition of 1ug step 1 makes template, and its cDNA is synthesized in reverse transcription.After the end, get the 5ul reverse transcription product and make template, at upstream primer Luffin-linker (F1):

5 ' -ggAgAgATAATgAATTCAggCggTggCTTTCTgggATCCGCTgATgTgAggTTCAgTTTg (being with single underscore base is connection peptides (SEQ ID NO:3 in the sequence table) part encoding sequence) and downstream primer LuffinKDEL/NotI:5 '-gAgT GCggCCgCTCATTA

(be with single underscore base is restriction enzyme Not I recognition site to CgCAACATTTTgTTTgTAATT, base is endoplasmic reticulum signal for locating (SEQ ID NO:6 in a sequence table) encoding sequence in the square frame) guiding under, pcr amplification sponge gourd seed ribosome deactivated protein luffin α gene, the pcr amplification system is: 10 * PCR damping fluid 5ul, 25mM MgSO ₄6ul, each 50pmol of upstream and downstream primer, 10mM dNTPs 1ul, pyrobest polysaccharase 1U, moisturizing to reaction system is 50ul.The PCR reaction conditions is: 94 ℃ of 30sec of elder generation, 55 ℃ of 40sec, 72 ℃ of 1min, totally 25 circulations; 72 ℃ were extended 7 minutes then.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, the amplified fragments size is about 800bp, conforms to expected results.

Three, the fusion rotein VEGF that has function of selective killing endothelial cells in tumor neogenetic blood vessels ₁₂₁The amplification of/LuffinKDEL encoding gene

Pcr amplification product with step 2 is a template, at upstream primer Luffin/MAG (F2):

5 '-ggCggTggCTTTCTgggAATTggTAAATTTTTgCACTCAgCAAAAAAATTTggAAA AgCTTTTgTgggAgAgATAATgAATTCA and downstream primer LuffinKDEL/NotI:

5 '-gAgT GCggCCgCTCATTA

(be with single underscore base is the NotI recognition site to CgCAACATTTTgTTTgTAATT, base is endoplasmic reticulum signal for locating (SEQ ID NO:6 in a sequence table) encoding sequence in the square frame) guiding under carry out pcr amplification, the PCR reaction conditions is: 94 ℃ of 3min, 94 ℃ of 30sec, 55 ℃ of 40sec, 72 ℃ of 60sec, totally 25 circulations.The VEGF that obtains with this pcr amplification product and step 1 again ₁₂₁Gene is a template, makes up its fusion gene with the method for joint-extension PCR, and amplimer is VEGF ₁₂₁F and LuffinKDEL/NotI, the PCR reaction conditions is: 94 ℃ of 3min of elder generation, 94 ℃ of 30sec, 70 ℃ of 10min; 94 ℃ of 40sec then, 55 ℃ of 60sec, 72 ℃ of 90sec, totally 25 circulations.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect.The amplified fragments size is about 1250bp as a result, conforms to expected results.Reclaim and this purpose fragment of purifying, with its with restriction enzyme Nde I with after Not I carries out double digestion be connected through the carrier pET30a of same enzyme double digestion (Novagen company), to connect product transformed into escherichia coli (E.coli) DH5 α competent cell again, screening positive clone, the upgrading grain, obtain containing the recombinant plasmid of goal gene, called after pET30a-VEGF ₁₂₁-LuffinKDEL checks order to it, sequencing result show obtained sequence correct pass through the VEGF that connection peptides connects ₁₂₁With the encoding gene of sponge gourd seed ribosome deactivated protein Luffin alpha fusion protein, be VEGF with this unnamed gene ₁₂₁-LuffinKDEL, nucleotide sequence with SEQ ID NO:8 in the sequence table, by 1227 based compositions, its encoding sequence is from 5 ' end 1-1227 bit base, coding has the protein of the amino acid residue sequence of SEQ ID NO:7 in the sequence table, is VEGF from 5 ' end 1-366 bit base ₁₂₁Encoding sequence, from 5 ' end 367-471 bit base is the encoding sequence of connection peptides, from 5 ' end 472-1215 bit base is the encoding sequence of sponge gourd seed ribosome deactivated protein Luffin α, is the encoding sequence of endoplasmic reticulum signal for locating from 5 ' end 1215-1227 bit base.

Embodiment 2, VEGF ₁₂₁Expression and the purifying of-LuffinKDEL fusion rotein toxin in intestinal bacteria

The engineering bacteria of getting the 10mL incubated overnight is inoculated into 1000mL and contains in the LB liquid nutrient medium of 25ug/mL kantlex, and 37 ℃ are cultured to OD=0.4-0.6, and adding IPTG then is 1mM to final concentration, and the while is contrast with what do not add IPTG, continues inducing culture 5 hours.After cultivating end, get 500ul bacterium liquid, 10000g collected thalline in centrifugal 2 minutes, add the 50ul sample-loading buffer, 100 ℃ of sex change 5 minutes, centrifugal 1 minute of 12000g gets 10ul and carries out 10% denaturing polyacrylamide gel electrophoresis and detect, (swimming lane 1 is a protein molecular weight standard to detected result as shown in Figure 1, swimming lane 2 is the tropina before inducing, and swimming lane 3 is the tropina after inducing), behind the IPTG abduction delivering, having obtained molecular weight is the recombinant protein of 45Kd, consistent with expected results.

Centrifugal collection thalline, thalline is dissolved among the 30mL 10mM TrisHCl (pH6.5), add N,O-Diacetylmuramidase (available from SIGMA company) to final concentration 1mg/mL, proteinase inhibitor PMSF (available from SIGMA company) is to final concentration 100ug/mL, room temperature was placed 30 minutes, ice bath, ultrasonication 10 minutes, centrifugal, collecting precipitation, precipitation is dissolved in the 6M Guanidinium hydrochloride, according to the operation instructions purifying protein of Ni-NTA resin, purified albumen is carried out 10% denaturing polyacrylamide gel electrophoresis detect, after electrophoresis is finished, coomassie brilliant blue staining, decolouring, detected result is (swimming lane 1 is a protein molecular weight standard, and swimming lane 2 is the fusion rotein behind the purifying) as shown in Figure 2, show the purified higher target protein of purity that obtained, with purifying protein in-20 ℃ of preservations.

Embodiment 3, press down the knurl experiment

Get 5 the week age Balb/c nude mice, female, body weight is 18-20g, is divided into 2 groups at random, 5 every group.Vitro culture A375 cell, every nude mice injection 5 * 10 ⁵Humanmachine tumour A375 cancer cells, with inoculated tumour same day be first day, administration in the 3rd day, dosage is 15mg VEGF ₁₂₁-LuffinKDEL/kg body weight is contrast with the physiological saline (saline) of injecting equal volume.Measure control group and test group (VEGF at different time ₁₂₁-LuffinKDEL) the size of mouse tumor.Result data is carried out the variance analysis of the two factors design of a replicate measurement, and the result is than control group (saline), VEGF ₁₂₁P＜0.05 of-LuffinKDEL group shows that the difference between two groups has remarkable statistical significance.Draw control group (saline) and test group (VEGF according to the mean value of every group of mouse tumor size ₁₂₁-LuffinKDEL) the tumor growth curve of different time after the administration, growth curve shows fusion rotein VEGF of the present invention as shown in Figure 3 ₁₂₁-LuffinKDEL has significant inhibitory effect to growth of tumor.

Sequence table

<160>8

<210>1

<211>277

<212>PRT

＜213〉cucurbitaceous plant sponge gourd (Luffa cylindrica)

<400>1

Met?Lys?Arg?Phe?Thr?Val?Leu?Ile?Leu?Ala?Ile?Phe?Leu?Ala?Ala?Ser

1 5 10 15

Thr?Val?Glu?Ala?Asp?Val?Ser?Phe?Ser?Leu?Ser?Gly?Ser?Ser?Ser?Thr

20 25 30

Ser?Tyr?Ser?Lys?Phe?Ile?Gly?Asp?Leu?Arg?Lys?Ala?Leu?Pro?Ser?Asn

35 40 45

Gly?Thr?Val?Tyr?Asn?Ile?Thr?Leu?Leu?Leu?Ser?Ser?Ala?Ser?Gly?Ala

50 55 60

Ser?Arg?Tyr?Thr?Leu?Met?Thr?Leu?Ser?Asn?Tyr?Asp?Gly?Lys?Ala?Ile

65 70 75 80

Thr?Val?Ala?Val?Asp?Val?Thr?Asn?Val?Tyr?Ile?Met?Gly?Tyr?Leu?Val

85 90 95

Asn?Ser?Thr?Ser?Tyr?Phe?Phe?Asn?Glu?Ser?Asp?Ala?Lys?Leu?Ala?Ser

100 105 110

Gln?Tyr?Val?Phe?Lys?Gly?Ser?Thr?Ile?Val?Thr?Leu?Pro?Tyr?Ser?Gly

115 120 125

Asn?Tyr?Glu?Lys?Leu?Gln?Thr?Ala?Ala?Gly?Lys?Ile?Arg?Glu?Lys?Ile

130 135 140

Pro?Leu?Gly?Phe?Pro?Ala?Leu?Asp?Ser?Ala?Ile?Thr?Thr?Leu?Phe?His

145 150 155 160

Tyr?Asp?Ser?Thr?Ala?Ala?Ala?Ala?Ala?Phe?Leu?Val?Ile?Ile?Gln?Thr

165 170 175

Thr?Ala?Glu?Ala?Ser?Arg?Phe?Lys?Tyr?Ile?Glu?Gly?Gln?Ile?Ile?Glu

180 185 190

Arg?Ile?Ser?Lys?Asn?Gln?Val?Pro?Ser?Leu?Ala?Thr?Ile?Ser?Leu?Glu

195 200 205

Asn?Glu?Trp?Ser?Ala?Leu?Ser?Lys?Gln?Ile?Gln?Leu?Ala?Gln?Thr?Asn

210 215 220

Asn?Gly?Thr?Phe?Lys?Thr?Pro?Val?Val?Ile?Thr?Asp?Asp?Lys?Gly?Gln

225 230 235 240

Arg?Val?Glu?Ile?Thr?Asn?Val?Thr?Ser?Lys?Val?Val?Thr?Lys?Asn?Ile

245 250 255

Gln?Leu?Leu?Leu?Asn?Tyr?Lys?Gln?Asn?Val?Ala?Ala?Phe?Asp?Glu?Asp

260 265 270

Ile?Pro?Ala?Lys?His

275

<210>2

<211>831

<212>DNA

＜213〉cucurbitaceous plant sponge gourd (Luffa cylindrica)

<400>2

atgaagagat?ttacagtgct?aattctcgcc?atcttccttg?cagcttcaac?tgttgaagcc 60

gatgtgagct?tcagtttgtc?aggttcttcc?tccacatctt?atagcaagtt?catcggagat 120

ctgaggaaag?cacttccatc?taatgggaca?gtctacaaca?taactctctt?actctcctct 180

gcttcgggcg?caagccgcta?cactctcatg?acactctcca?attacgacgg?caaagccatc 240

acggttgctg?tagatgtaac?caacgtttac?attatgggct?atctcgtcaa?ttcaacatcc 300

tacttcttca?acgagtctga?tgctaaatta?gcttctcaat?acgtattcaa?aggcagtacc 360

atcgtcacgc?ttccatattc?tggcaattac?gaaaagcttc?aaactgctgc?aggcaagata 420

agagagaaga?ttccacttgg?attcccagct?ttggacagtg?cgattaccac?cttgtttcat 480

tacgactcca?cggctgctgc?tgcggcattc?ctcgtaatca?ttcagaccac?tgctgaggct 540

tccagattca?agtatatcga?gggacagatt?attgagagaa?tttcgaaaaa?ccaggtgccg 600

agtctggcga?ctataagttt?agaaaacgaa?tggtctgctc?tctccaaaca?aattcagttg 660

gcacagacga?ataacggaac?gtttaaaact?cccgttgtga?taacggacga?taaaggccaa 720

cgagttgaaa?taaccaacgt?tacgtcgaaa?gttgtaacca?agaacatcca?attgcttcta 780

aattacaaac?aaaatgttgc?ggcttttgac?gaggatattc?ctgcaaaaca?c 831

<210>3

<211>35

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>3

Gly?Gly?Gly?Phe?Leu?Gly?Ile?Gly?Lys?Phe?Leu?His?Ser?Ala?Lys?Lys

1 5 10 15

Phe?Gly?Lys?Ala?Phe?Val?Gly?Glu?Ile?Met?Asn?Ser?Gly?Gly?Gly?Phe

20 25 30

Leu?Gly?Ser

35

<210>4

<211>405

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>4

Met?Ala?Pro?Met?Ala?Glu?Gly?Gly?Gly?Gln?Asn?His?His?Glu?Val?Val

1 5 10 15

Lys?Phe?Met?Asp?Val?Tyr?Gln?Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr

20 25 30

Leu?Val?Asp?Ile?Phe?Gln?Glu?Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe

35 40 45

Lys?Pro?Ser?Cys?Val?Pro?Leu?Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp

50 55 60

Glu?Gly?Leu?Glu?Cys?Val?Pro?Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln

65 70 75 80

Ile?Met?Arg?Ile?Lys?Pro?His?Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser

85 90 95

Phe?Leu?Gln?His?Asn?Lys?Cys?Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala

100 105 110

Arg?Gln?Glu?Lys?Cys?Asp?Lys?Pro?Arg?Arg?Gly?Gly?Gly?Phe?Leu?Gly

115 120 125

Ile?Gly?Lys?Phe?Leu?His?Ser?Ala?Lys?Lys?Phe?Gly?Lys?Ala?Phe?Val

130 135 140

Gly?Glu?Ile?Met?Asn?Ser?Gly?Gly?Gly?Phe?Leu?Gly?Ser?Ala?Asp?Val

145 150 155 160

Arg?Phe?Ser?Leu?Ser?Gly?Ser?Ser?Ser?Thr?Ser?Tyr?Ser?Lys?Phe?Ile

165 170 175

Gly?Asp?Leu?Arg?Lys?Ala?Leu?Pro?Ser?Asn?Gly?Thr?Val?Tyr?Asn?Ile

180 185 190

Thr?Leu?Leu?Leu?Ser?Ser?Ala?Ser?Gly?Ala?Ser?Arg?Tyr?Thr?Leu?Met

195 200 205

Thr?Leu?Ser?Asn?Tyr?Asp?Gly?Lys?Ala?Ile?Thr?Val?Ala?Val?Asp?Val

210 215 220

Thr?Asn?Val?Tyr?Ile?Met?Gly?Tyr?Leu?Val?Asn?Ser?Thr?Ser?Tyr?Phe

225 230 235 240

Phe?Asn?Glu?Ser?Asp?Ala?Lys?Leu?Ala?Ser?Gln?Tyr?Val?Phe?Lys?Gly

245 250 255

Ser?Thr?Ile?Val?Thr?Leu?Pro?Tyr?Ser?Gly?Asn?Tyr?Glu?Lys?Leu?Gln

260 265 270

Thr?Ala?Ala?Gly?Lys?Ile?Arg?Glu?Lys?Ile?Pro?Leu?Gly?Phe?Pro?Ala

275 280 285

Leu?Asp?Ser?Ala?Ile?Thr?Thr?Leu?Phe?His?Tyr?Asp?Ser?Thr?Ala?Ala

290 295 300

Ala?Ala?Ala?Phe?Leu?Val?IleIl?e?Gln?Thr?Thr?Ala?Glu?Ala?Ser?Arg

305 310 315 320

Phe?Lys?Tyr?Ile?Glu?Gly?Gln?Ile?Ile?Glu?Arg?Ile?Ser?Lys?Asn?Gln

325 330 335

Val?Pro?Ser?Leu?Ala?Thr?Ile?Ser?Leu?Glu?Asn?Glu?Trp?Ser?Ala?Leu

340 345 350

Ser?Lys?Gln?Ile?Gln?Leu?Ala?Gln?Thr?Asn?Asn?Gly?Thr?Phe?Lys?Thr

355 360 365

Pro?Val?Val?Ile?Thr?Asp?Asp?Lys?Gly?Gln?Arg?Val?Glu?Ile?Thr?Asn

370 375 380

Val?Thr?Ser?Lys?Val?Val?Thr?Lys?Asn?Ile?Gln?Leu?Leu?Leu?Asn?Tyr

385 390 395 400

Lys?Gln?Asn?Val?Ala

405

<210>5

<211>1215

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

atggctccga?tggcagaagg?aggagggcag?aatcatcacg?aagtggtgaa?gttcatggat 60

gtctatcagc?gcagctactg?ccatccaatc?gagaccctgg?tggacatctt?ccaggagtac 120

cctgatgaga?tcgagtacat?cttcaagcca?tcctgtgtgc?ccctgatgcg?atgcgggggc 180

tgctgcaatg?acgagggcct?ggagtgtgtg?cccactgagg?agtccaacat?caccatgcag 240

attatgcgga?tcaaacctca?ccaaggccag?cacataggag?agatgagctt?cctacagcac 300

aacaaatgtg?aatgcagacc?aaagaaagat?agagcaagac?aagaaaaatg?tgacaagccg 360

aggcggggcg?gtggctttct?gggaattggt?aaatttttgc?actcagcaaa?aaaatttgga 420

aaagcttttg?tgggagagat?aatgaattca?ggcggtggct?ttctgggatc?cgctgatgtg 480

aggttcagtt?tgtcaggttc?ttcctccaca?tcttatagca?agttcatcgg?agatctgagg 540

aaagcacttc?catctaatgg?gacagtctac?aacataactc?tcttactctc?ctctgcttcg 600

ggcgcaagcc?gctacactct?catgacactc?tccaattacg?acggcaaagc?catcacggtt 660

gctgtagatg?taaccaacgt?ttacattatg?ggctatctcg?tcaattcaac?atcctacttc 720

ttcaacgagt?ctgatgctaa?attagcttct?caatacgtat?tcaaaggcag?taccatcgtc 780

acgcttccat?attctggcaa?ttacgaaaag?cttcaaactg?ctgcaggcaa?gataagagag 840

aagattccac?ttggattccc?agctttggac?agtgcgatta?ccaccttgtt?tcattacgac 900

tccacggctg?ctgctgcggc?attcctcgta?atcattcaga?ccactgctga?ggcttccaga 960

ttcaagtata?tcgagggaca?gattattgag?agaatttcga?aaaaccaggt?gccgagtctg 1020

gcgactataa?gtttagaaaa?cgaatggtct?gctctctcca?aacaaattca?gttggcacag 1080

acgaataacg?gaacgtttaa?aactcccgtt?gtgataacgg?acgataaagg?ccaacgagtt 1140

gaaataacca?acgttacgtc?gaaagttgta?accaagaaca?tccaattgct?tctaaattac 1200

aaacaaaatg?ttgcg 1215

<210>6

<211>4

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>6

Lys?Asp?Glu?Leu

1

<210>7

<211>409

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>7

Met?Ala?Pro?Met?Ala?Glu?Gly?Gly?Gly?Gln?Asn?His?His?Glu?Val?Val

1 5 10 15

Lys?Phe?Met?Asp?Val?Tyr?Gln?Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr

20 25 30

Leu?Val?Asp?Ile?Phe?Gln?Glu?Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe

35 40 45

Lys?Pro?Ser?Cys?Val?Pro?Leu?Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp

50 55 60

Glu?Gly?Leu?Glu?Cys?Val?Pro?Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln

65 70 75 80

Ile?Met?Arg?Ile?Lys?Pro?His?Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser

85 90 95

Phe?Leu?Gln?His?Asn?Lys?Cys?Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala

100 105 110

Arg?Gln?Glu?Lys?Cys?Asp?Lys?Pro?Arg?Arg?Gly?Gly?Gly?Phe?Leu?Gly

115 120 125

Ile?Gly?Lys?Phe?Leu?His?Ser?Ala?Lys?Lys?Phe?Gly?Lys?Ala?Phe?Val

130 135 140

Gly?Glu?Ile?Met?Asn?Ser?Gly?Gly?Gly?Phe?Leu?Gly?Ser?Ala?Asp?Val

145 150 155 160

Arg?Phe?Ser?Leu?Ser?Gly?Ser?Ser?Ser?Thr?Ser?Tyr?Ser?Lys?Phe?Ile

165 170 175

Gly?Asp?Leu?Arg?Lys?Ala?Leu?Pro?Ser?Asn?Gly?Thr?Val?Tyr?Asn?Ile

180 185 190

Thr?Leu?Leu?Leu?Ser?Ser?Ala?Ser?Gly?Ala?Ser?Arg?Tyr?Thr?Leu?Met

195 200 205

Thr?Leu?Ser?Asn?Tyr?Asp?Gly?Lys?Ala?Ile?Thr?Val?Ala?Val?Asp?Val

210 215 220

Thr?Asn?Val?Tyr?Ile?Met?Gly?Tyr?Leu?Val?Asn?Ser?Thr?Ser?Tyr?Phe

225 230 235 240

Phe?Asn?Glu?Ser?Asp?Ala?Lys?Leu?Ala?Ser?Gln?Tyr?Val?Phe?Lys?Gly

245 250 255

Ser?Thr?Ile?Val?Thr?Leu?Pro?Tyr?Ser?Gly?Asn?Tyr?Glu?Lys?Leu?Gln

260 265 270

Thr?Ala?Ala?Gly?Lys?Ile?Arg?Glu?Lys?Ile?Pro?Leu?Gly?Phe?Pro?Ala

275 280 285

Leu?Asp?Ser?Ala?Ile?Thr?Thr?Leu?Phe?His?Tyr?Asp?Ser?Thr?Ala?Ala

290 295 300

Ala?Ala?Ala?Phe?Leu?Val?Ile?Ile?Gln?Thr?Thr?Ala?Glu?Ala?Ser?Arg

305 310 315 320

Phe?Lys?Tyr?Ile?Glu?Gly?Gln?Ile?Ile?Glu?Arg?Ile?Ser?Lys?Asn?Gln

325 330 335

Val?Pro?Ser?Leu?Ala?Thr?Ile?Ser?Leu?Glu?Asn?Glu?Trp?Ser?Ala?Leu

340 345 350

Ser?Lys?Gln?Ile?Gln?Leu?Ala?Gln?Thr?Asn?Asn?Gly?Thr?Phe?Lys?Thr

355 360 365

Pro?Val?Val?Ile?Thr?Asp?Asp?Lys?Gly?Gln?Arg?Val?Glu?Ile?Thr?Asn

370 375 380

Val?Thr?Ser?Lys?Val?Val?Thr?Lys?Asn?Ile?Gln?Leu?Leu?Leu?Asn?Tyr

385 390 395 400

Lys?Gln?Asn?Val?Ala?Lys?Asp?Glu?Leu

405

<210>8

<211>1227

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>8

atggcaccga?tggcagaagg?aggagggcag?aatcatcacg?aagtggtgaa?gttcatggat 60

gtctatcagc?gcagctactg?ccatccaatc?gagaccctgg?tggacatctt?ccaggagtac 120

cctgatgaga?tcgagtacat?cttcaagcca?tcctgtgtgc?ccctgatgcg?atgcgggggc 180

tgctgcaatg?acgagggcct?ggagtgtgtg?cccactgagg?agtccaacat?caccatgcag 240

attatgcgga?tcaaacctca?ccaaggccag?cacataggag?agatgagctt?cctacagcac 300

aacaaatgtg?aatgcagacc?aaagaaagat?agagcaagac?aagaaaaatg?tgacaagccg 360

aggcggggcg?gtggctttct?gggaattggt?aaatttttgc?actcagcaaa?aaaatttgga 420

aaagcttttg?tgggagagat?aatgaattca?ggcggtggct?ttctgggatc?cgctgatgtg 480

aggttcagtt?tgtcaggttc?ttcctccaca?tcttatagca?agttcatcgg?agatctgagg 540

aaagcacttc?catctaatgg?gacagtctac?aacataactc?tcttactctc?ctctgcttcg 600

ggcgcaagcc?gctacactct?catgacactc?tccaattacg?acggcaaagc?catcacggtt 660

gctgtagatg?taaccaacgt?ttacattatg?ggctatctcg?tcaattcaac?atcctacttc 720

ttcaacgagt?ctgatgctaa?attagcttct?caatacgtat?tcaaaggcag?taccatcgtc 780

acgcttccat?attctggcaa?ttacgaaaag?cttcaaactg?ctgcaggcaa?gataagagag 840

aagattccac?ttggattccc?agctttggac?agtgcgatta?ccaccttgtt?tcattacgac 900

tccacggctg?ctgctgcggc?attcctcgta?atcattcaga?ccactgctga?ggcttccaga 960

ttcaagtata?tcgagggaca?gattattgag?agaatttcga?aaaaccaggt?gccgagtctg 1020

gcgactataa?gtttagaaaa?cgaatggtct?gctctctcca?aacaaattca?gttggcacag 1080

acgaataacg?gaacgtttaa?aactcccgtt?gtgataacgg?acgataaagg?ccaacgagtt 1140

gaaataacca?acgttacgtc?gaaagttgta?accaagaaca?tccaattgct?tctaaattac 1200

aaacaaaatg?ttgcgaaaga?tgaactg 1227

Claims (11)

1. the fusion rotein that has function of selective killing endothelial cells in tumor neogenetic blood vessels is to connect VEGF by connection peptides at the aminoterminal of the ribosome inactivating protein Luffin of sponge gourd seed α ₁₂₁Fusion rotein, the amino acid residue sequence of described connection peptides is shown in the SEQ ID NO:3 in the sequence table.

2. fusion rotein according to claim 1 is characterized in that: described fusion rotein is the amino acid residue sequence that SEQ ID NO:4 represents.

3. fusion rotein according to claim 1 and 2 is characterized in that: described fusion rotein also includes the endoplasmic reticulum signal for locating, and the amino acid residue sequence of described endoplasmic reticulum signal for locating is shown in the SEQ ID NO:6 in the sequence table.

4. fusion rotein according to claim 3 is characterized in that: described fusion rotein is the amino acid residue sequence that SEQ ID NO:7 represents.

5. the gene of coding claim 1 or 2 or 3 or 4 described fusion roteins.

6. according to the gene of the described fusion rotein of claim 5, it is characterized in that: be the dna sequence dna that SEQ ID NO:8 represents in SEQ ID NO:5 represents in the sequence table dna sequence dna or the sequence table.

7. the expression carrier that contains claim 5 or 6 described fusion roteins.

8. the transgenic cell line that contains the gene of claim 5 or 6 described fusion roteins.

9. the host bacterium that contains the gene of claim 5 or 6 described fusion roteins.

10. each described Expression of Fusion Protein method of claim 1-4, be gene transformation or transduction host cell with claim 5 or 6 described fusion roteins, cultivate host cell, separation and purification albumen from substratum or cell obtains having the fusion rotein of function of selective killing endothelial cells in tumor neogenetic blood vessels.

11. the application of the gene of each described fusion rotein of claim 1-4 or claim 5 or 6 described fusion roteins in the preparation antitumor drug.

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