CN1903875A - Antitumour integrator alpha V beta 3 antigen and its coding gene and application - Google Patents

Abstract

The present invention discloses a anti-tumor integrant alpha V beta3 antigen, its coding gene and application. Said invention is aimed at providing antigen of integrant alpha V beta3, its coding gene and its application in preparation of vaccine for preventing and curing tumor. Said antigen has amino acid residue sequence of SEQ ID No.1 in sequence table, and it coding gene has the DNA sequence of SEQ ID No.2 in sequence table. The invented integrant alpha V beta3 antigen and its coding gene have obvious tumor-inhibiting effect.

Description

Antitumor integrin alpha V β 3Antigen and encoding gene thereof and application

Technical field

The present invention relates to integrate plain antigen and encoding gene thereof and application, particularly relate to a kind of integrin alpha V β ₃Antigen and encoding gene and its application in preventing and/or treating property of preparation tumor vaccine and medicine.

Background technology

Malignant tumour is one of most important killer who threatens human health.The vascularization of the generation of tumour, development and transfer and tumour is closely related, and concerning the postoperative patient of radical operation, 5 years recurrence rates of patient of no capillary blood vessel invasion and attack are 11%, and the 5 years recurrence rates of patient that have capillary blood vessel invasion and attack are up to 50%.Because it is identical that the tumor area blood vessel has, therefore anti-angiogenic formation treatment is applicable to multiple noumenal tumour.With the blood vessel is target spot, inhibition vascularization, thereby suppresses tumor proliferation and shift to have become a new oncotherapy approach.

Studies show that, vascular endothelial growth factor (vascular endothelialgrowth factor is not only depended in the formation of blood vessel, VEGF) and acceptor (vascular endothelial growth factor receptors, VEGF-R), also be subjected to simultaneously extracellular matrix (extracellular matrix, ECM) influence [the Kerbel RS.A cancer therapy resistant to resistance[J] .Nature of protein receptor-integration plain (integrin), 1997,390 (6658): 335-336.Ellis LM..Angiogenesis and its role incolorectal tumor and metastasis formation[J] .Semin Oncol, 2004,31 (6): 3-9.].Wherein, VEGF-E, VEGF-R ₂With α V β ₃The closest with the relation that tumor vessel forms, can be used as and suppress the direct target that pathologic vessels forms.

In recent years, more and more perfect to the research of vascularization mechanism.Discover that endothelial cell growth factor (ECGF) and acceptor thereof are not only depended in tumor vascular formation, also be subjected to extracellular matrix (extracellular matrix, ECM) protein receptor-integrin alpha V β ₃Influence [Stromblad S, Becker JC, Yebra M, et al.Suppression of p53activity and p21WAF1/CIP1expression by vascular cellintegrin alphaVbeta3 during angiogenesis[J] .J Clin Invest, 1996,98 (2): 426-433.].The neonatal blood vessels endotheliocyte is expressed high-caliber α V β in the tumor tissues ₃, do not express or some organizes utmost point low expression level and have in adult's normal blood vessels endotheliocyte.It can cause the activity of MMP-2 matrix degradation at endothelial cell surface, and suppresses the p53 and the proteic activity of the inductive p21 of institute thereof of endotheliocyte, thereby shows anti-apoptosis effect.A large amount of experiments show, use integrin alpha V β in the animal body ₃Antagonist can cause participating in generating the apoptosis of the endotheliocyte of blood vessel; Anti-α V β ₃Antibody or α V β ₃The antagonist new vessel that can destroy quail embryo, mouse retina, rabbit corneal generate; Use α V β in the tumor model ₃Antagonist not only stoped the relevant vasculogenesis of tumour, and can cause shrinking back of tumour.α V β ₃Be by α V chain and β ₃The non-covalent transmembrane glycoprotein in conjunction with a heterodimer that constitutes of chain is an energy and the acceptor of ECM broad incorporation, and the ECM albumen of the every RGD of having sequence (Arg-Gly-Asp) all can combine with it.

Summary of the invention

The purpose of this invention is to provide a kind of integrin alpha V β with antineoplastic vascular formation effect ₃Antigen.

Integrin alpha V β provided by the present invention ₃Antigen derives from Africa xenopus, has SEQ ID № in the sequence table: 1 amino acid residue sequence.

SEQ ID № in the sequence table: 1 is made up of 142 amino-acid residues.

Above-mentioned integrin alpha V β encodes ₃Antigenic gene also belongs to protection scope of the present invention, can have SEQ ID № in the sequence table: 2 dna sequence dna.

SEQ ID № in the sequence table: 2 by 426 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 1-426 bit base.

Contain integrin alpha V β of the present invention ₃The expression vector of antigen gene, transgenic cell line and host bacterium all belong to protection scope of the present invention.

Increase arbitrary segmental primer in the described antigen gene to also within protection scope of the present invention.

With above-mentioned integrin alpha V β ₃Antigen-specific bonded antibody also belongs to protection scope of the present invention.

Described antibody comprises monoclonal antibody and polyclonal antibody, all can prepare according to ordinary method.

Another object of the present invention provides the above-mentioned integrin alpha V of a kind of expression β ₃Antigenic method.

The above-mentioned integrin alpha V of expression provided by the present invention β ₃Antigenic method is to contain above-mentioned integrin alpha V β ₃The recombinant expression vector of antigen gene imports host cell, expresses to obtain integrin alpha V β ₃Antigen.

The carrier that sets out that is used to make up described recombinant expression vector can be at expression in escherichia coli expression of exogenous gene carrier, as pGEX-4T-2, pET-3a, pET-30a, pET-28a, pET-28b or pET-28c, is preferably pGEX-4T-2.

Be the carrier that sets out with pGEX-4T-2, structure contain described integrin alpha V β ₃The recombinant expression vector of antigen gene is pGEX-α V β ₃

Above-mentioned recombinant expression vector all can make up according to ordinary method.

Described host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.

Described intestinal bacteria can be E.coli BL21 (DE3), E.coli JM109, E.coli HB101 or E.coliTop10 etc.

Can adopt the conventional culture condition that makes up the used starting strain of engineering bacteria that engineering bacteria is cultivated, when described engineering bacteria is recombination bacillus coli, need to add the IPTG inductor, add IPTG concentration be 0.8-1.2mmol/L, be preferably 1mmol/L, inducing temperature is 35-39 ℃, is preferably 37 ℃, induction time is 2-4 hour, is preferably 3 hours.

Integrin alpha V β of the present invention ₃Antigen can be used for preventing and/or treating property of preparation tumor vaccine, coding integrin alpha V β ₃Antigenic gene can be used for preventing and/or treating property of preparation tumour dna vaccination.

Integrin alpha V β in described the preventing and/or treating property tumour dna vaccination ₃Antigen gene can be present in the carrier for expression of eukaryon.

Be used for making up and carry integrin alpha V β ₃The carrier that sets out of the carrier for expression of eukaryon of antigen gene can be any one can be in Mammals the expression vector of expression alien gene, be preferably pCI-GPI, its physical map is seen Figure 18.

Be the carrier that sets out with pCI-GPI, structure carry integrin alpha V β ₃The recombinant expression vector of antigen gene is pCI-α V β ₃

When needing, can also incorporate the gene of one or more cytokines such as granular leucocyte-macrophage colony stimulating factor (GM-CSF), interferon-(IFN-γ), interleukin-22 (IL-2), TGF-β 4 or protein in the vaccine with the vascular endothelial growth factor VEGF-E antigen preparation as the molecular immune adjuvant.

The invention provides integrin alpha V β ₃Antigen and encoding gene thereof.To contain integrin alpha V β of the present invention respectively ₃The eukaryon expression plasmid of respective section is as the immunogen immune mouse in antigen gene and the mouse, the result compares with PBS, empty carrier and isoantigen control group, all produced the antibody of higher level in the antigen group mice serum, the T lymphocyte quantity of secretion of gamma-IFN and IL-4 obviously increases CD4 in the antigen group mouse boosting cell ⁺/ CD8 ⁺Ratio also obviously raise and integrin alpha V β of the present invention ₃Antigen more can excite the humoral immune reaction of mouse than the corresponding antigens of mouse; After immune mouse is accepted the subcutaneous transplantation knurl and attacked, compare with PBS, empty carrier and isoantigen control group, the one-tenth knurl time lengthening of antigen group mouse, tumor growth slowly and knurl volume-diminished, tumour inhibiting rate obviously improve and integrin alpha V β of the present invention ₃Antigen is more remarkable than the tumor killing effect of the corresponding antigens of mouse, and tumour inhibiting rate can reach more than 80%.Above-mentioned experimental result shows integrin alpha V β of the present invention ₃Antigen and encoding gene thereof have significant tumor killing effect, and have the expression amount height, easy purifying, and with short production cycle, industrial scale is big, and the advantage that cost is low can be prepared into preventing and/or treating property tumour medicine and vaccine.The present invention has bigger practical significance and wide application prospect 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 for dividing five step pcr amplification integrin alpha V β with the center die plates method ₃The mode chart of antigen gene

Fig. 2 is pcr amplification integrin alpha V β ₃The agarose gel electrophoresis detected result of antigen gene

Fig. 3 is the m α V β of pcr amplification ₃The agarose gel electrophoresis detected result of gene fragment

Fig. 4 is the RT-PCR qualification result of stable transfected cells mRNA

Fig. 5 is the immunofluorescence detected result of stable transfected cells

Fig. 6 is the Western Blotting qualification result of stable transfected cells

Fig. 7 is integrin alpha V β ₃Antigen gene construction of prokaryotic expression vector schema

Fig. 8 is pCI-α V β ₃Agarose gel electrophoresis detected result with pGEX-4T-2 plasmid SalI and NotI double digestion product

Fig. 9 is the integrin alpha V β of prokaryotic expression and purifying ₃Antigenic SDS-PAGE detected result

Figure 10 is the ELISPOT detected result of the IFN-γ and the IL-4 of immune mouse

Figure 11 is CD in the immune mouse ₄ ⁺And CD ₈ ⁺The cells were tested by flow cytometry result of T cell count ratio

Figure 12 is CD in the immune mouse ₄ ⁺And CD ₈ ⁺The histogram of T cell count ratio

Figure 13 is the average one-tenth knurl time of each group immune mouse subcutaneous transplantation knurl

Figure 14 attacks the back knurl cubing result who respectively organized immune mouse in 50 days for the subcutaneous transplantation knurl

Figure 15 attacks the back knurl remeasurement result who respectively organized immune mouse in 50 days for the subcutaneous transplantation knurl

Figure 16 for the B group be control group respectively organize immune mouse press down tumour rate statistics

Figure 17 attacks for the subcutaneous transplantation knurl and afterwards respectively organized the survival condition of immune mouse in 50 days, at body knurl volume and external knurl volume

Figure 18 is the physical map of carrier pCI-GPI

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.All primers synthesize and examining order is finished by the living worker's biotechnology in Shanghai company limited.

Embodiment 1, integrin alpha V β ₃The clone of antigenic design and encoding gene thereof

One, integrin alpha V β ₃Antigenic design

By literature search on a large scale and sequence have relatively obtained a series of integrin alpha V β both at home and abroad ₃The aminoacid sequence of functional region and the homology difference in different plant species thereof.Choose Africa xenopus α V β ₃Functional fragment in the total length comprises: from aminoterminal (N end) 118-131,126-129,217-231,211-221,109-118,99-118, the sequence that the 209-220 position is made up of 142 amino-acid residues, i.e. SEQ ID № in the sequence table: 1.

Two, integrin alpha V β ₃The clone of antigen gene and mouse source corresponding gene thereof

Integrin alpha V β according to the step 1 design ₃Antigenic aminoacid sequence is derived the nucleotide sequence of this sequence of coding, be the SEQ ID № in the sequence table: 2, by 426 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 1-426 bit base.Full gene is divided into two sections, utilizes the center die plates method to divide synthetic this gene of 5 steps, clone mouse α V β simultaneously ₃Corresponding fragment is called for short m α V β in contrast ₃, concrete grammar is as follows:

1, integrin alpha V β ₃The clone of antigen gene

According to the integrin alpha V β that derives ₃The nucleotide sequence of antigen gene designs the pcr amplification primer, and adds restriction enzyme Xho I and EcoR V recognition site respectively at primers F 5, R5 two ends, and primer sequence is as follows:

Leading portion F1 5 '-ATGATTTAATTAAGATCCAAACCCTGGGCACTAGTTTGTCAGAGA-3 '

Leading portion R1 5 '-ATCCGCAGGTTACTAGTTAGCCGGCGCATCCTCTCTGACAAACTA-3 '

Leading portion F2 5 '-ACCTTATGGACCTTTCCTATTCCATGAAGGATGATTTAATTAAGA-3 '

Leading portion R2 5 '-ATCGGCTTGTCAACGAATGCACCAAATCCAATCCGCAGGTTACTA-3 '

Leading portion F3 5 '-AAGTAGAAGACTATCCTGTGGACATATACTACCTTATGGACCTTT-3 '

Leading portion R3 5 '-TCAGGAGGTGACATGAACATGTACGGTGACATCGGCTTGTCAACG-3 '

Leading portion F4 5 '-GTCTTCAACCTACAGGTGAGGCAAGTAGAAGACTATC-3 '

Leading portion R4 5 '-ATAGCATGGGTTCTTGATGACTTCAGGAGGTGACATG-3 '

Back segment F1 5 '-GCACGTCTTGACTTTGACAGAGGAAGTCTTGCTGTTTAACGAGGAGGT-3 '

Back segment R1 5 '-GAGAATCCCGGTTTCGGGAGACCTTCTGTTTCTGGACCTCCTCGTTAA-3 '

Back segment F2 5 '-TCAATACCGAGTGCATGCCGACATTCGGATATAAGCACGTCTTGACTT-3 '

Back segment R2 5 '-GCTGCCTGTAGCACCGCATCAAATCCACCCTCTGGAGAATCCCGGTTT-3 '

Back segment F3 5 '-CCTGAAGTCATCAAGAATCCATGCTATGAATTCAATACCGAGTGC-3 '

Back segment R3 5 '-ATTTCTCCAGCCAATCTTCTCGTCGCATACTGCTGCCTGTAGCAC-3 '

F5 5 '-GC CTCGAGGTCTTCAACCTACAGGTGAGGCAAG-3 ' (band underscore base is a restriction enzyme XhoI recognition site)

R5 5 '-GC GATATCATTTCTCCAGCCAATCTTCTCGTCG-3 ' (band underscore base is a restriction enzyme EcoRV recognition site)

Adopt the center die plates method to divide five step pcr amplification goal gene, synthesis model figure sees Fig. 1 (F: upstream primer, the R downstream primer), full gene is divided into forward and backward two sections, two fragments all obtain goal gene with four step PCR synthesis methods, utilize PCR method that the rear and front end is stitched together then, concrete grammar is as follows: the first step amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl ₂(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of primer R1, F1 (20 μ M), TaqDNA polysaccharase (5U/ μ l) 1 μ l adds deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 5 circulations; Last 72 ℃ are continued to extend 10min.

Second step, the 3rd step and the amplification of the 4th step are carried out as template after all diluting 50 times with previous step PCR product.Amplification system is the same, and just primer second goes on foot pcr amplification and selects primer and F2 for use, and the 3rd step was selected primer R3 and F3 for use, and the 4th step was selected primer R4 and F4 for use.The PCR reaction conditions is: 94C 1min, 55C1min, 72C 1min, totally 30 circulations.Two sections goal gene of gained behind four pcr amplifications, called after leading portion and back segment respectively.

Final step pcr amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl ₂(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of synthetic primer (20 μ M) F5 and R5, Taq archaeal dna polymerase (5U/ μ l) 1 μ l, the leading portion 1 μ l after diluting 50 times, the back segment 1 μ l after diluting 50 times adds deionized water to 50 μ l.The PCR reaction conditions is with step 2 to four.Behind five pcr amplifications, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, detected result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 2; Swimming lane 1-4. leading portion substep PCR product; Swimming lane 5. α V β ₃PCR end product (426bp); Swimming lane 6-8. back segment substep PCR product), the result has obtained the gene fragment (arrow indication fragment among Fig. 2) that length is about 426bp through pcr amplification, conforms to expected results." the quick glue of PCR segment reclaims test kit " and reference reagent box specification sheets with vast Tyke, Beijing biological gene technology limited liability company reclaim and this purpose fragment of purifying, it is cloned in the pMD18-T carrier (TaKaRa company), check order to containing the pulsating recombinant vectors of recovery, result's segment that increases has SEQ ID № in the sequence table: 2 nucleotide sequence, extension increasing sequence is correct.

2, the pulsating amplification of corresponding gene in the mouse

1) design primer

Integrin alpha V β according to mouse among the GeneBank ₃(sequence number: L-13591) design amplification respective regions is (with this fragment called after m α V β for gene order ₃) primer, and introduce the recognition site of restriction enzyme enzyme Xho I and EcoR V respectively at the two ends of upstream and downstream primer, primer sequence is as follows:

F6 (upstream primer) 5 '-GC GCTAGCATCTTCTCACTTCAAGTGCGGCAGGT-3 ' (band underscore base is a restriction enzyme Xho I recognition site)

R6 (downstream primer): 5 '-GC GATATCATTCCTCCAGCCAATTTTTTCATCAC-3 ' (band underscore base is a restriction enzyme EcoR V recognition site).

2) preparation mouse embryo tissue homogenate

Disconnected neck is put to death pregnant mouse, is soaked in 5min in 75% ethanol; Aseptic taking-up uterus and wherein embryo, placenta are rejected fat and reticular tissue as far as possible and are shredded with scissors; The tissue that shreds is put in adds the liquid nitrogen porphyrize in the mortar to Powdered, be collected in the glass homogenizer of ice precooling, add 1mL Trizol, homogenate is to transparent; Tissue homogenate changes in the aseptic RNase-free EP pipe standby rapidly.

3) the Trizol method is extracted total RNA of tissue homogenate

Add chloroform 200 μ l in the EP pipe of results tissue homogenate, put upside down mixing 15s immediately, room temperature leaves standstill 5min; 4 ℃/12000rpm, centrifugal 15min; Carefully upper phase is moved in the 1.5mL EP pipe of another new aseptic RNase-free, add the Virahol of equivalent, put upside down mixing, room temperature leaves standstill 10min; 4 ℃, the centrifugal 10min of 12000rpm; Abandon supernatant, add 75% ethanol 1mL, put upside down mixing, 4 ℃, the centrifugal 5min of 7500rpm abandon supernatant; Repeat thoroughly to abandon supernatant after the step, will precipitate seasoning, add DEPC water 10 μ l dissolution precipitations, be directly used in reverse transcription reaction.

4) reverse transcription reaction

Total RNA of the mouse embryo tissue that step 3) is extracted is a template, be primer with olig (dT) 20, and the SuperScript III RNase H-Reverse Transcriptase of usefulness American I nvitrogen also synthesizes cDNA with reference to the product description reverse transcription.

5) m α V β ₃Pcr amplification

CDNA is a template with step 4) reverse transcription synthetic, under the guiding of primers F 6 and R6, carry out pcr amplification, 100 μ l PCR reaction systems are: 2 * GC damping fluid I (the La Taq of TaKaRa company is subsidiary), 50.0 μ l, dNTPs mixture (2.5mM) 16.0 μ l, TaKaRa La Taq (5U/ μ l) 1.0 μ l, reverse transcription product 2.0 μ l, F6 (20 μ M) 2.0 μ l, R6 (20 μ M) 2.0 μ l replenish cumulative volume to 100 μ l with the sterilization deionized water.PCR reaction conditions: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ were extended 10 minutes.After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, (swimming lane 1 is DL-2000Marker to detected result as shown in Figure 3; Swimming lane 2 is amplification gene), the result has obtained the gene fragment that length is about 426bp through pcr amplification, conforms to expected results." the quick glue of PCR segment reclaims test kit " and reference reagent box specification sheets with vast Tyke, Beijing biological gene technology limited liability company reclaim and this purpose fragment of purifying, it is cloned in the pMD18-T carrier (TaKaRa company), check order to containing the pulsating recombinant vectors of recovery, extension increasing sequence is correct as a result.

Embodiment 2, integrin alpha V β ₃Antigen gene Construction of eukaryotic and evaluation thereof

One, integrin alpha V β ₃The antigen gene Construction of eukaryotic

Integrin alpha V β with embodiment 1 amplification ₃After antigen gene carries out double digestion with restriction enzyme Xho I and EcoR V, form with on the eukaryon expression plasmid pCI-neo+ basis of carrier pCI-GPI[in Promega company of same enzyme double digestion, making up.Mainly be between former multiple clone site Nhe I of pCI-neo+ and Xba I restriction enzyme site, to insert the universal sequence structure to form.The gene order of universal sequence is 5 '-Nhe I-signal peptide-Xho I-EcoR V-EcoRI-IgG Fc-GPI-Xba I-3 ', and the foreign immunologic molecular gene can select proper restriction site to insert between Nhe I-EcoR I.PCI-GPI is except that containing human IgG Fc section (GenBank number: Z17370) and glycosylation phosphatidylinositols GPI (GenBank number: XM676434) the encoding sequence, the main skeleton structure that also contains the pCI carrier, as the CMV early promoter, chimeric intron, the Neo selection markers, SV40 enhanser and ammonia benzyl resistant gene Ampr+ etc., physical map is seen Figure 18] connect 12-24 hour down at 16 ℃, linked system is: 10 * T4DNA connects damping fluid 1 μ l, T4DNA ligase enzyme (12u/ μ l) 1 μ l, PCR purified product 4 μ l, pCI-GPI carrier 1 μ l adds sterilization distilled water to 10 μ l.To connect product transformed into escherichia coli DH5 α competent cell, transformant be coated the enterprising row filter of LB resistant panel that contains the 100mg/mL penbritin.Single bacterium colony of growing on resistant panel of picking carries out bacterium colony PCR and identifies then: single colony inoculation is contained 100mg/mL penbritin (Amp in 3mL ⁺) the LB liquid nutrient medium in enlarged culturing 3h.After cultivating end, get 1 μ l bacterium liquid and dilute, boil 10min, the centrifugal 3min of 12000rpm with 100 μ l water, get supernatant and do template, carry out pcr amplification under the guiding of primers F 5 and R5,25 μ l PCR reaction systems are: supernatant 5 μ l, each 1 μ l of primers F 5 and R5,10 * PCR damping fluid, 2.5 μ l, MgCl ₂2.5 μ l, dNTPs 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l adds sterilization distilled water to 25 μ l.PCR reaction conditions: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ were extended 10 minutes.After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, can obtain the positive recon that length is about the dna fragmentation of 426bp through pcr amplification.The plasmid of the positive recombinant that extraction identifies through PCR is done further to identify with sequence measurement, and sequencing result shows that the sequence of goal gene and the position in carrier are all correct, has obtained vascular endothelial growth factor receptor VEGF-R ₂The carrier for expression of eukaryon of antigen gene, called after pCI-α V β ₃Simultaneously with the mouse m α V β of same procedure with embodiment 1 amplification ₃Gene fragment also connects among the carrier pCI-GPI, obtains m α V β ₃Carrier for expression of eukaryon, called after pCI-m α V β ₃

Two, the evaluation of eukaryon expression plasmid

1, transient transfection RenCa cell

Carry carrier for expression of eukaryon pCI-α V β respectively with what step 1 obtained ₃With pCI-m α V β ₃The positive colony bacterium be inoculated in the LB liquid nutrient medium, cultivated 12-24 hour down at 37 ℃, extract test kit in a small amount and, use the transfection reagent SuperFect of QIAGEN company then with the plasmid of QIAGEN company with reference to product description extraction plasmid ^Transfection Reagent and with reference to product description with pCI-α V β ₃With pCI-m α V β ₃Plasmid transfection renal carcinoma cell line RenCa is contrast with pCI-neo (Promega company).

2, the screening of stable transfected cells

Transfectional cell is cultivated about 48 hours when cell density reaches 50-70% and merges, abandon nutrient solution, use RPMI 1640 substratum (Gibco company) that contain G-418 (400 μ g/mL) instead and carry out the resistant cell screening and culturing, the RenCa cell with untransfected compares simultaneously.When control cells was almost all dead, G-418 concentration can be reduced to 200 μ g/mL to keep the screening effect.After two weeks, as seen have resistance clone to form, treat that it increases gradually after, with the pancreatin of 0.25% (mass percentage concentration) cell dissociation is got off, with limiting dilution assay picking mono-clonal and carry out enlarged culturing.

3, the evaluation of stable transfected cells

1) RT-PCR of stable transfected cells mRNA identifies

Pancreatin with 0.25% is made single cell suspension with the monoclonal cell digestion of enlarged culturing, is collected in the glass centrifuge tube, and the centrifugal 5min of 1000rpm abandons supernatant; Inhale fully after PBS gives a baby a bath on the third day after its birth time and abandon supernatant; Re-suspended cell and counting, the whole density that makes cell is 1 * 10 ⁶/ mL; Get the 1mL cell suspension and add 1mL Trizol mixing, extract cell total rna, with primers F 5 and R5 transfection is had pCI-α V β then ₃The cell of plasmid carries out RT-PCR and identifies with primers F 6 and R6 transfection is had pCI-m α V β ₃The cell of plasmid carries out RT-PCR and identifies that qualification result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 4; Swimming lane 1. α V β ₃Swimming lane 2.m α V β ₃), all obtained the fragment that length is about 426bp through amplification, show integrin alpha V β ₃Gene has pCI-α V β in transfection ₃On the mRNA level, express m α V β in the RenCa cell of plasmid ₃Gene has pCI-m α V β in transfection ₃Also on the mRNA level, express in the RenCa cell of plasmid.

2) immunofluorescence of stable transfected cells detects

The stable transfected cells that step 1) is identified is (with pCI-neo ⁺Transfectional cell and RenCa cell are contrast) trysinization with 0.25% is prepared into single cell suspension; After PBS washes 3 times, encapsulant (3%BSA) room temperature sealing 20-30 minute; Remove encapsulant, add fluorescein-labeled mouse-anti human IgG (available from China fir Golden Bridge Technology, Inc. in Beijing), 4 ℃ of lucifuges were hatched 12-24 hour; PBS washes 3 times, adjusts cell concn to 5 * 10 ⁵/ mL; Cell suspension drips on the slide glass, mountant (available from China fir Golden Bridge Technology, Inc. in Beijing) mounting.Detected result is (1.pCI-α V β as shown in Figure 5 ₃Transfectional cell; 2.pCI-m α V β ₃Transfectional cell; 3.pCI-neo ⁺Transfectional cell; 4.RenCa cell), show integrin alpha V β ₃Antigen gene, m α V β ₃Gene obtains respectively to express in transfectional cell separately.

3) the Western Blotting of stable transfected cells identifies

With step 2) stable transfected cells identified is (with pCI-neo ⁺Transfectional cell and RenCa cell are contrast) use 0.25% trysinization, collecting cell, PBS washed twice; With the TBS that contains 1%Triton X-100 (50mMTris-Cl, pH7.6,150mM NaCl) lysing cell 30min; 4 ℃, the centrifugal 10min collection of 12000rpm supernatant; After testing sample carried out the 15%SDS-PAGE electrophoretic separation, albumen is transferred to nitrocellulose filter (voltage 15V shifted 1 hour) from polyacrylamide gel with half-dried transfer instrument; With the TBST that contains 5% skim-milk (TBS+0.05%Tween 20) room temperature sealing 1 hour; Mouse-anti human IgG monoclonal antibody (available from China fir Golden Bridge Technology, Inc. in Beijing, by 1: 500 dilution proportion) with the HRP mark was hatched 12-24 hour for 4 ℃; After the TBST washing, add two anti-goat anti-mouse IgG (available from China fir Golden Bridge Technology, Inc. in Beijing, by 1: the 2000 dilution proportion) incubated at room 2 hours of horseradish peroxidase-labeled; After the TBST washing, with the ECL colour developing that exposes.Detected result is (swimming lane 1.pCI-α V β as shown in Figure 6 ₃Transfectional cell; Swimming lane 2.pCI-m α V β ₃Transfectional cell; Swimming lane 3.pCI-neo ⁺Transfectional cell; Swimming lane 4.RenCa cell), further prove integrin alpha V β ₃Antigen gene, m α V β ₃Gene obtains respectively to express in transfectional cell separately.

Embodiment 3, integrin alpha V β ₃The prokaryotic expression of antigen gene

One, integrin alpha V β ₃The antigen gene construction of prokaryotic expression vector

Make up integrin alpha V β referring to Fig. 7 ₃The prokaryotic expression carrier of antigen gene, concrete grammar is: with the pCI-α V β of purifying ₃Plasmid and pGEX-4T-2 (Pharmacia company) carry out double digestion with restriction enzyme Sal I and Not I respectively, enzyme is cut product carry out the detection of 1.2% agarose gel electrophoresis, detected result is (swimming lane M1.DNA relative molecular mass standard DL-15000 as shown in Figure 8; Swimming lane M2.DNA relative molecular mass standard DL-2000; Before swimming lane 1.pGEX-4T-2 enzyme is cut; After swimming lane 2.pGEX-4T-2 enzyme is cut; Swimming lane 3.pCI-α V β ₃Endonuclease bamhi), with " the quick glue of PCR segment reclaims test kit " of vast Tyke, Beijing biological gene technology limited liability company and reference reagent box specification sheets reclaims and the pGEX-4T-2 of linearization of purifying and the pCI-α V β of 426bp ₃Endonuclease bamhi (arrow indication fragment among Fig. 8), the purpose fragment with purifying is connected 12-24 hour for 16 ℃ with linearizing pGEX-4T-2 carrier then, linked system is: 10 * ligase enzyme damping fluid, 1 μ l, T ₄Dna ligase 1 μ l, goal gene 4 μ l, pGEX-4T-2 carrier 1 μ l is with distilled water postreaction system to 10 μ l.To connect product transformed into escherichia coli DH5 α competent cell, transformant is coated the enterprising row filter of LB resistant panel that contains the 100mg/mL penbritin, picking can obtain the positive recon that length is about the dna fragmentation of 426bp through pcr amplification at the single bacterium colony that grows on the resistant panel and carry out bacterium colony PCR and identify under the guiding of primers F 5 and R5 then.The plasmid of the positive recombinant that extraction identifies through PCR is done further to identify with sequence measurement, and sequencing result shows that the sequence of goal gene and the position in carrier are all correct, has obtained integrin alpha V β ₃The prokaryotic expression carrier of antigen gene, called after pGEX-α V β ₃

Two, a large amount of preparations of the acquisition of engineering bacteria and inclusion body

With pGEX-α V β ₃Transformed into escherichia coli BL21 (DE3) competent cell, filter out positive recombinant bacterial strain with the method identical with step 1, in 1: 100 ratio the positive bacterium of recombinating is seeded in the 1000mL LB liquid nutrient medium that contains the 100mg/mL penbritin and cultivates in a large number under 37 ℃ then, adding chemical inducer IPTG when being cultured to logarithmic phase is 1mmol/L to final concentration, and continuation was cultivated 12-24 hour under 37 ℃.After cultivating end, 4 ℃, the centrifugal 15min collection of 6000rpm thalline, (20mmol/L, pH8.0) resuspended bacterial sediment add N,O-Diacetylmuramidase (1mg/mL) in room temperature magnetic agitation 10min with 100mL TE damping fluid; The broken thalline of ultrasonic wave in the ice bath (30s/ opens, and 20s/ stops, 15 circulations); 4 ℃, the centrifugal 20min of 12000rpm are collected inclusion body precipitation and supernatant respectively.NaCl (TE preparation) with 1mol/L washs 1 time with the inclusion body precipitation, 4 ℃, the centrifugal 20min of 12000rpm are to remove the foreign protein in the inclusion body, wash 2 times with the TE damping fluid after abandoning supernatant, 4 ℃, the centrifugal 20min collecting precipitation of 12000rpm carry out purifying with following step again.

Three, the purifying of target protein

With the urea of 8mol/L dissolving inclusion body, add mercaptoethanol to 1% (concentration expressed in percentage by volume) behind 20 ℃, the centrifugal 10min of 12000rpm; Go up sample after the TE damping fluid balance with Q-Sepharose Fast Flow anion-exchange column (available from Pharmacia company) usefulness 6mol/L urea; After going out to pass the peak with the TE buffer solution elution of 6moI/L urea,, collect relevant elution peak with NaCl (the TE damping fluid preparation of the 6mol/L urea) wash-out of different concns (concentration is 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.5M and 1M); Each peak sampling carrying out SDS-PAGE identifies, according to electrophoresis result, the target protein component of 43KD is carried out S-Sepharose Fast Flow cationic exchange coloum again and SephardexG-50 post (available from Pharmacia company) carries out purifying, at last purified target protein being carried out SDS-PAGE detects, detected result as shown in Figure 9, show and obtained the purified target protein of 43KD, i.e. integrin alpha V β ₃Antigen.

Embodiment 4, integrin alpha V β ₃Antigenic antitumor activity detects

One, grouping of animal and immunity

6 the week age Balb/c mouse be divided into 5 groups, 9 every group.The A group is the blank group; The B group is the PBS control group; The C group is pCI-neo+ empty carrier group; The E-1 group is pCI-m α V β ₃Group, the E-2 group is pCI-α V β ₃Group.

With three kinds of plasmid DNA (pCI-neo, pCI-m α V β ₃(embodiment 2 makes up), pCI-α V β ₃(embodiment 2 makes up)) (m: mixed v), vibration back room temperature was placed 15 minutes gently, obtained LipofectAMINE-plasmid dna complex compound, as immunity DNA to press 1: 1 with transfection reagent LipofectAMINE respectively.Quadriceps muscle of thigh muscle multi-point injection is adopted in immunity, 100 μ g/ only: at first inject 100 μ l, 0.25% bupivacaine pre-treatment inoculation position, behind the 72h at the same area initial immunity, respectively at the 3rd week and the 6th all each booster immunizations 1 time.

Two, the ELISA of serum antibody titer detects

Every group of mouse got blood through the tail vein in each immunity back in 2 weeks, the centrifugal 10min of 3000rpm behind the room temperature placement 3h, get upper serum and detect antibody titers with the ELISA method, to verify antigenic humoral immune reaction, set up the negative control and the PBS blank of the preceding mouse serum of immunity simultaneously, the ELISA detection method is as follows:

It is 5 μ g/mL that the albumen of embodiment 3 preparation is diluted to final concentration with coating buffer, and bag is by elisa plate, and 4 ℃ left standstill 12-24 hour; PBST washing: get rid of the coating buffer of abandoning in the elisa plate hole, PBST washing 1 time; With 2% casein solution room temperature sealing 4h, discard confining liquid, pat dry elisa plate; To add each hole behind the immune mouse serum usefulness PBS doubling dilution, the 100ul/ hole, 37 ℃ leave standstill 30min; With the sheep anti-mouse igg (available from China fir Golden Bridge Technology, Inc. in Beijing) of PBST washing back adding HRP mark, 37 ℃ of reaction 20min; After the PBST washing, with the TMB 10min that develops the color, 2M sulfuric acid termination reaction; Detect the OD value of 450nm at last with SPECTRAIII enzyme connection instrument.The result judges: the OD value of testing sample is positive more than or equal to 2.1 (P/N 〉=2.1) with the ratio of the OD value of negative control, shows that the maximum antibody dilution of positive findings is the antibody titer of immune serum.Antibody titer detected result in the immune serum is as shown in table 1,

Antibody titer in table 1 immune serum

	Initial immunity	2 immunity	3 immunity
				A group (blank) B group (PBS) C group (pCI-neo +Empty carrier) E-1 group (pCI-m α V β 3) E-2 organizes (pCI-α V β 3)	- ＜1∶100 ＜1∶100 ＜1∶100 1∶100 ※	- ＜1∶100 ＜1∶100 ＜1∶100 1∶1600 ※※	- ＜1∶100 ＜1∶100 ＜1∶100 1∶3200 ※※

Annotate: ^※With PBS, pCI-neo ⁺With comparing with species antigen control group of mouse source, this group mouse all produces specific antibody (P＜0.05)

^{※ ※}The twice booster immunization obviously antibody titer of enhancing immunity mice serum (P＜0.05) compared with the empty carrier control group with PBS, all produced the antibody of higher level in antigen group (E-1 group, the E-2 group) mice serum, twice booster immunization be the antibody titer of enhancing immunity mice serum obviously, in addition, integrin alpha V β of the present invention ₃Antigen more can excite the humoral immune reaction of mouse than the corresponding antigens of mouse.

Three, the ELISPOT of the IFN-γ of immune mouse and IL-4 detects

1, the separation of immune mouse splenic lymphocyte

Every group of immune mouse got three, aseptic separation splenic lymphocyte, and method is: disconnected neck is put to death mouse, and the 5min that sterilizes in 75% ethanol puts into super clean bench immediately and becomes right arm reclining; Aseptic taking-up spleen is put in the plate (the 8mL serum-free RPMI1640 nutrient solution of interior dress precooling), rejects fat and reticular tissue as far as possible, pushes the spleen tissue gently with grinding rod on 200 order stainless (steel) wires; Splenocyte suspension 8mL slowly is equipped with in the transparent centrifuge tube of 4mL Ficoll lymphocyte separation medium (available from Pharmacia company) the centrifugal 20min of 2000rpm level along tube wall slow the adding; The careful tunica albuginea layer of drawing is washed (1000rpm, 10min) 2 times with the substratum of 10mL precooling, cell is resuspended in the RPMI1640 substratum that contains 20% foetal calf serum, adjusts cell concn to 1 * 10 ⁷/ mL.

2, the ELISPOT of the IFN-γ of immune mouse and IL-4 detects

The splenic lymphocyte of the isolating immune mouse of step 1 is detected with the ELISPOT that Murine IFN γ and IL-4ELISPOT test kit and reference reagent box specification sheets carry out IFN-γ and IL-4, and detected result sees Table 2 and shown in Figure 10,

The ELISPOT of table 2 immune mouse IFN-γ and IL-4 detects

A group (blank) B group (PBS) C group (pCI-neo empty carrier) E-1 group (pCI-m α V β 3) E-2 organizes (pCI-α V β 3)	Spot number/1 * 10 6Individual splenocyte
			IFN-γ 0 5.73±1.31 9.48±0.18 59.53±8.09 ※ 89.27±11.88 ※※	IL-4 0 4.08±0.76 8.65±1.09 47.01±5.78 ※ 97.40±15.03 ※※

Annotate: ^※Compare with B, C control group, ELISPOT detects this group mouse spot number and increases P＜0.05, ^{※ ※}P＜0.01 is compared with the empty carrier control group with PBS, and the T lymphocyte quantity of secretion of gamma-IFN and IL-4 all obviously increases with the control group ratio in antigen group (E-1 group, the E-2 group) mouse boosting cell, and integrin alpha V β of the present invention ₃Antigen more can excite the humoral immune reaction of mouse than the corresponding antigens of mouse.

Four, the T Lymphocyte Subsets Determination of immune mouse splenic lymphocyte

Get mouse boosting cell 1 * 10 ⁶/ mL-2 * 10 ⁶/ mL, PBS wash twice; The anti-CD that adds the PE mark respectively ₄ ⁺The anti-CD of mAb and FITC mark ₈ ⁺Each 20ul of mAb (available from the brilliant U.S. biological company limited in Beijing), 4 ℃ of lucifuge reaction 30min behind the mixing; After PBS washes twice, add 0.5mL fluorescence and preserve liquid (0.15mol/L PBS, 20g/L glucose, 10g/L formaldehyde and 1g/L NaN ₃) re-suspended cell, with flow cytometry analysis T cell subsets, immune mouse CD ₄ ⁺And CD ₈ ⁺The cells were tested by flow cytometry result of T cell count ratio such as table 3 and Figure 11, shown in Figure 12.

The immune mouse CD of table 3 cells were tested by flow cytometry ₄ ⁺And CD ₈ ⁺The ratio of T cell count

	CD 4 +And CD 8 +The ratio of T cell
		A organizes (blank)	1.8

B group (PBS) C group (pCI-neo empty carrier) E-1 group (pCI-m α V β 3) E-2 organizes (pCI-α V β 3)

1.96 1.67 5.66 ※ 13.2 ※※

Annotate: ^※Compare this group mouse CD with B, C control group ₄ ⁺/ CD ₈ ⁺Ratio significantly increase P＜0.05, ^{※ ※}P＜0.01 is compared with the empty carrier control group with PBS, CD4 in antigen group (E-1 group, the E-2 group) mouse boosting cell ⁺/ CD8 ⁺Ratio obviously raise and integrin alpha V β of the present invention ₃Antigen more can excite the humoral immune reaction of mouse than the corresponding antigens of mouse.

Five, detect the provide protection that immune mouse is attacked the subcutaneous transplantation knurl

A week remaining 6 immune mouses of each group being carried out the subcutaneous transplantation knurl after the last immunity attacks.The RenCa cell of results logarithmic phase, the preparation single cell suspension washes twice with physiological saline, adjusts cell concn to 1 * 10 ⁷/ mL is 1 * 10 in mouse back right side subcutaneous vaccination 100 μ l cell suspensions ⁶/ only.After the subcutaneous transplantation knurl is attacked, observe the growing state of mice-transplanted tumor.Treat that tumor nodule forms back (can lay one's hand on and, the about 2-3mm of major diameter), write down the one-tenth knurl time of every group of mouse, observe the survival condition of mouse simultaneously.Attack back 50 days execution mouse in tumour cell, take pictures; With the vertical major diameter (a) and vertical minor axis (b) of vernier caliper measurement tumour, calculate the transplanted tumor volume according to formula; It is heavy to take by weighing knurl, calculates the inhibiting rate of tumour according to formula.

Each average one-tenth knurl time of organizing immune mouse subcutaneous transplantation knurl sees Table 4 and Figure 13, the subcutaneous transplantation knurl is attacked the back survival condition of respectively organizing immune mouse in 50 days, see that at body knurl volume and external knurl volume Figure 17 (from left to right is the mouse survival condition successively, show gross tumor volume at body, exsomatize and show gross tumor volume), wherein the survival rate statistics sees Table 5, the knurl cubing the results are shown in Table 6 and Figure 14, the knurl remeasurement the results are shown in Figure 15, each tumour rate statistics that presses down of organizing immune mouse sees Table 7, be that the histogram that presses down tumour rate statistics of respectively organizing immune mouse of control group is seen Figure 16 wherein with the B group

Table 4 is respectively organized the average one-tenth knurl time of immune mouse subcutaneous transplantation knurl

	The average one-tenth knurl time (my god)
		A group (blank) B group (PBS) C group (pCI-neo empty carrier) E-1 group (pCI-m α V β 3) E-2 organizes (pCI-α V β 3)	/ 8.67±1.03 9.33±1.03 11±1.09 14±1.26 ※

Annotate: ^※With B, C control group relatively this group mouse become knurl prolongation of latency (P＜0.05)

Table 5 subcutaneous transplantation knurl is attacked the back survival condition of respectively organizing immune mouse in 50 days

	Death toll (only/6)	Survival rate (%)
			A group (blank) B group (PBS)	0 4	100 33.3

C group (pCI-neo empty carrier) E-1 group (pCI-m α V β 3) E-2 organizes (pCI-α V β 3)

4 1 1

33.3 83.3 ※ 83.3 ※

Annotate: ^※With B, C control group relatively this group mouse survival rate obviously increase (P＜0.05)

Table 6 subcutaneous transplantation knurl is attacked the back knurl volume of respectively organizing immune mouse in 50 days

	Gross tumor volume (cm 3)
		A group (blank) B group (PBS) C group (pCI-neo empty carrier) E-1 group (pCI-m α V β 3) E-2 organizes (pCI-α V β 3)	/ 6.47±0.88 7.19±0.69 2.57±0.96 ※ 0.27±0.28 ※

Annotate: ^※With B, C control group relatively this group mouse tumor volume obviously dwindle (P＜0.001)

Table 7 is respectively organized the restraining effect of immune mouse to tumour

	Knurl heavy (g)	B is a control group	Tumour inhibiting rate (%)
						D-1 is a control group	E-1 is a control group	F-1 is a control group
			A group (blank) B group (PBS) C group (pCI-neo empty carrier) E-1 group (pCI-m α V β 3) E-2 group (pCI-α V β 3)	/ 5.09±0.33 5.93±0.96 3.34±0.65 ※ 0.54±0.38 ※※	/ - 0 34.32 ※ 89.34 ※※				/ / / / /	/ / / - 83.78 ※※	/ / / / /

Annotate: ^※Compare with control group, this group mouse is to the restraining effect highly significant (P＜0.01) of tumour; ^{※ ※}After P＜0.001 immune mouse is accepted the attack of subcutaneous transplantation knurl, compare with the empty carrier control group with PBS, the one-tenth knurl time lengthening of antigen group (E-1 group, E-2 group) mouse, tumor growth slowly and knurl volume-diminished, tumour inhibiting rate obviously improve and integrin alpha V β of the present invention ₃Antigen is more remarkable than the tumor killing effect of the corresponding antigens of mouse.

Sequence table

<160>2

<210>1

<211>142

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>1

Val Phe Asn Leu Gln Val Arg Gln Val Glu Asp Tyr Pro Val Asp Ile

1 5 10 15

Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Ile Lys

20 25 30

Ile Gln Thr Leu Gly Thr Ser Leu Ser Glu Arg Met Arg Arg Leu Thr

35 40 45

Ser Asn Leu Arg Ile Gly Phe Gly Ala Phe Val Asp Lys Pro Met Ser

50 55 60

Pro Tyr Met Phe Met Ser Pro Pro Glu Val Ile Lys Asn Pro Cys Tyr

65 70 75 80

Glu Phe Asn Thr Glu Cys Met Pro Thr Phe Gly Tyr Lys His Val Leu

85 90 95

Thr Leu Thr Glu Glu Val Leu Arg Phe Asn Glu Glu Val Gln Lys Gln

100 105 110

Lys Val Ser Arg Asn Arg Asp Ser Pro Glu Gly Gly Phe Asp Ala Val

115 120 125

Leu Gln Ala Ala Val Cys Asp Glu Lys Ile Gly Trp Arg Asn

130 135 140

<210>2

<211>426

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

gtcttcaacc tacaggtgag gcaagtagaa gactatcctg tggatatcta ctaccttatg 60

gacctttcct attccatgaa ggatgattta atcaagatcc aaaccctggg cactagttta 120

tcagaaagga tgcgccggct aactagtaac ctgcggattg gatttggtgc atttgttgac 180

aagcctatgt caccgtacat gtttatgtca cctcctgaag tcatcaaaaa cccctgctat 240

gaatttaaca ccgagtgcat gcccactttt ggatataaac acgtcttgac tttgacagag 300

gaagtcttga gatttaacga ggaggtccag aaacagaagg tctcccgaaa ccgggattct 360

ccagagggtg gatttgatgc ggtgctacag gcagcagtat gcgacgagaa gattggctgg 420

agaaat 426

Claims (10)

1, integrin alpha V β ₃Antigen has SEQ ID № in the sequence table: 1 amino acid residue sequence.

2, the described integrin alpha V of coding claim 1 β ₃Antigenic gene.

3, gene according to claim 2 is characterized in that: described gene has SEQ ID № in the sequence table: 2 dna sequence dna.

4, contain claim 2 or 3 described expression carrier, transgenic cell line and host bacterium.

5, a kind of expression integrin alpha V β ₃Antigenic method is to contain claim 2 or 3 described integrin alpha V β ₃The recombinant expression vector of antigen gene imports host cell, expresses to obtain integrin alpha V β ₃Antigen.

6, method according to claim 5 is characterized in that: the carrier that sets out that is used to make up described recombinant expression vector is pGEX-4T-2, pET-3a, pET-30a, pET-28a, pET-28b or pET-28c; Be the carrier that sets out with pGEX-4T-2, structure contain described integrin alpha V β ₃The recombinant expression vector of antigen gene is pGEX-α V β ₃Described host is E.coli BL21 (DE3), E.coli JM109, E.coli HB101 or E.coli Top10.

7, with the described integrin alpha V of claim 1 β ₃Antigen-specific bonded antibody.

8, the described integrin alpha V of claim 1 β ₃The application of antigen in preventing and/or treating property of preparation tumor vaccine and medicine.

9, claim 2 or 3 described integrin alpha V β ₃The application of antigen gene in preventing and/or treating property of preparation tumour dna vaccination and medicine.

10, application according to claim 9 is characterized in that: the integrin alpha V β in the described dna vaccination ₃Antigen gene is present in the carrier for expression of eukaryon; The described integrin alpha V β that carries ₃The carrier for expression of eukaryon of antigen gene is pCI-α V β ₃

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