CN103304668B

CN103304668B - Ultra-VEGF-trap immune fusion protein, its preparation method and application thereof

Info

Publication number: CN103304668B
Application number: CN201210063738.3A
Authority: CN
Inventors: 胡品良
Original assignee: JIANGSU JIANDE BIOLOGICAL PHARMACEUTICAL CO Ltd
Current assignee: Jiangsu Huatai Vaccine Engineering Technology Research Co ltd
Priority date: 2012-03-12
Filing date: 2012-03-12
Publication date: 2015-10-28
Anticipated expiration: 2032-03-12
Also published as: CN103304668A

Abstract

The invention provides a kind of Ultra-VEGF-trap immune fusion protein, described albumen is 1) or 2) in albumen: 1) as SEQ? the protein of the aminoacid sequence composition shown in ID.1; 2) as SEQ? aminoacid sequence shown in ID.1 through replacement and/or disappearance and/or add one or several amino acid and antitumor relevant by 1) derivative protein.Present invention also offers construction process of a kind of Ultra-VEGF-trap immune fusion protein and uses thereof.Immune fusion protein of the present invention has all blocked VEGFs/VEGFRs pathway, has the value of huge treatment Several Kinds of Malignancy.

Description

Ultra-VEGF-trap immune fusion protein, its preparation method and application thereof

Technical field

The present invention relates to a kind of Ultra-VEGF-trap immune fusion protein, its preparation method and application thereof, belong to biological technical field.

Background technology

The unrestricted invasive growth of tumour and transfer thereof depend on vasculogenesis.If do not have new vessel to supply nutrition, gross tumor volume can not more than 2mm ³-3mm ³.Tumor vascular growth can be divided into front blood vessel phase and blood vessel phase.In the front blood vessel phase, tumour cell mainly relies on diffusion fashion to support and excretion metabolism product.Though tumor cell proliferation is rapid, Proliferation and apoptosis is in running balance, and tumor size is generally no more than 1-2mm ³, several months or several years can be kept not to shift.Tumour is once be transformed into the vasculogenesis phase, and tumor angiogenesis factor produces too much, unbalance with supressor, and obtain blood and nutritive substance supply by vasculogenesis, apoptosis obviously reduces, and gross tumor volume increases rapidly and shifts.In addition, discovered in recent years lymphangiogenesis is formed and plays vital effect to the lymphatic metastasis of tumour.A large amount of Clinical pathological study shows that a lot of solid tumor is early stage and just occurs the diffusion of cancer cells to regional lymph nodes, and being shifted by lymphatic vessel is usually the topmost approach of the initial transition phase of tumour.Tumour cell enters people's draining lymph node, then enters human bloodstream, causes distant metastasis widely, threatens patient vitals.Serous canal in tumor tissues can be growth of tumour cell provides nutritive substance especially early stage in metastatic tumor growth, the providing main and provided, when tumor growth is to 1-2mm by the form expanded by diffusion fashion and micro-lymphoglandula, Lymphangiogenesis of its nutritive substance ³time nutritive substance just account for major way by providing of blood vessel.

Vascularization (angiogenesis) is a quite complicated process, by the positive negative regulation of the multiple factor, its VEGF (vascular endothelial growth factor, VEGF) be act on one of the strongest positivity regulatory factor, it is by combining with its specific receptors-vascular endothelial growth factor receptor (vascularendothelial growth factor receptor, VEGFR) formation stimulating new vessel.In healthy tissues, vascular endothelial growth factor and vascular endothelial growth inhibitor: An update exist simultaneously, and keep relative equilibrium, and this balance makes body vessels can normally generation and differenciation.But in Tumor Growth, VEGFs quantity is increased sharply, and the Imbalance between Angiostatin, greatly facilitate division growth and the migration of endotheliocyte, inhibition tumor cell apoptosis, improves vascular permeability, for the growth of tumour and transfer provide good microenvironment.VEGFs is the homodimer glycoprotein of high conservative, has six hypotypes: VEGF-A ,-B ,-C ,-D ,-E, and placenta generates the factor (PLDF), and molecular weight is not from 35 to 44kDa etc.Wherein VEGF-A is due to the different cut mode of mRNA, has 7 kinds of varients, i.e. VEGF-121, VEGF-145, VEGF-148, VEGF-165, VEGF-183, VEGF-189 and VEGF-206 at least.In body, VEGF-165 expresses the highest, but plays a leading role in VEGF-121 angiogenic growth.VEGF-121 and VEGF-165 is solubility secretory protein, is main effects molecule, all with paracrine form mediation endothelial cell mitogen and increase vascular permeability.The microvessel density of the expression of VEGF-A and some solid tumors has dependency, and in tissue, the prognosis of the solid tumor such as concentration and mammary cancer, lung cancer, prostate cancer and colorectal carcinoma of VEGF is relevant.VEGF-B and VEGF-A has the homology of 43%, and ripe VEGF-B albumen has two kinds of hypotype (VEGF-B ₁₆₇, VEGF-B ₁₆₈), respectively containing 167 and 186 amino-acid residues, molecular weight is respectively 21kD and 32kD.VEGF-B ₁₆₇and VEGF-B ₁₈₆heterodimer (J Biol Chem.1995 is formed by disulfide linkage and VEGF-A, 270 (13): 7717-23.), VEGF-B mrna expression (Salven P can be detected in various tumours (the comprising optimum and pernicious) cell of the mankind, et al, Am J Pathol, 1998,153 (1): 103-108).In wound or other damaged tissues, VEGF-B release increases and activates.The growth of VEGF-B and tumour also has close relationship, and VEGF-B and VEGF-A promotes angiogenic growth in local, thus is conducive to the growth of tumour.VEGF-C and D is the main Vascular ar endothelial growth facor-c identified at present, and the two is the secreting glycoprotein of structural similitude, is the ligands specific of tyrosine kinase receptor VEGFR-3/Flt-4.The two has homology with between VEGF-A.VEGF-C optionally induces vasculolymphatic hyperplasia, and participates in drainage, the immunity moderation function of interstitial fluid, closely related with metastases.VEGF-C also has the unique function regulating lymphatic endothelia, in other a few class endothelial permeabilities of adjustment and vasculogenesis, have identical effect with VEGF-A.VEGF-C has very strong angiogenic action in vivo, and comparatively VEGF-A persistent, in the embryo grown, VEGF-C can promote that blood vessel carries out differential growth.Endothelial cell VEGF-C can act on VEGFR-2 and VEGFR-3 acceptor, induction capillary endothelium hyperplasia.VEGF-D is another newcomer of VEGF family, the discovery when computer carries out Homology search, with VEGF-C structural similitude.VEGF-D discovered in recent years its can in inducing entity knurl or lymphangiogenesis generates and or expansion, closely related with the lymphatic metastasis of malignant tumour, Vascular ar endothelial growth facor-c (Kleespies A is called as together with VEGF-C, et al.Onkologie.2005,28 (5): 281-8.).The aminoacid sequence of placenta growth factor (PLGF) 40% is identical with VEGF-A, the new vessel under participation pathological state and the formation of collatoral vessel.VEGF-E Yi Shu VEGF family, is recently existing at parapoxvirus (Orf virus) genome, can be combined specifically, stimulating endothelial cell mitotic division, increases vascular permeability with VEGFR-2.

The VEGF of all kinds of hypotype combines with three kinds of vascular endothelial growth factor receptor specific combination.The VEGFR of current discovery mainly contains VEGFR-1 (fms-like tyrosine kinase, Flt-1), VEGFR-2 (kinaseinsert domain containing receptor, and VEGFR-3 (Flt-4) KDR/Flk-1), wherein the production Methods of VEGFR-1, VEGFR-2 and blood vessel is close, and VEGF-C/D VEGFR-3 then generates closely related with lymphatic vessel.VEGFR all belongs to tyrosine kinase receptor superfamily, 7 Ig-like domain (immunoglobulin-like domains) are had at extracellular fragment, wherein 1-3 GeIgYang district and VEGF are in conjunction with relevant, and the activity form of homodimer between two acceptors, is formed by the 4th GeIgYang district, 5-7 GeIgYang district is relevant with kinase activity in VEGFR1, acts synergistically relevant in VEGFR2 with heparin.That be combined with VEGFR-1 is VEGF-A, VEGF-B and PLGF, and that be combined with VEGF-2 is VEGF-A, VEGF-C, VEGF-D, VEGF-E and PLGF, and that be combined with VEGF3 is VEGF-C, VEGF-D.VEGFR-1 and VEGFR-2 is the acceptor of current most study, is positioned at Surface of Vascular Endothelial Cells.VEGFR-1 and VEGF has higher avidity, and activating this receptor can promote cell migration, but inducing cell proliferation differentiation effect is not as VEGFR-2 (Barleon, B. etc., Blood, 1996,87,3336-3343; Seetharam, L., etc., Oncogene, 1995,10,135-147).VEGFR-3 specifically expressing is on lymphatic endothelium surface, substantially do not express in blood vessel endothelium, with the transfer of the vasculolymphatic hyperplasia of induced tumor and lymphoglandula after the combination of VEGF-C and VEGF-D (Neufeld G etc., FASEB J.1999,13:9-22).VEGFs sends signal by being combined with 3 kinds of tyrosine kinase receptors VEGF-1, VEGF-2 and VEGF-3.The intracellular signalling pathway of VEGFR also has many arguements.Different investigators observes different results, but it is believed that the receptor tyrosine kinase signal paths such as Ras-MAPK, PLC-γ-PK and PI3K all may play the part of certain role, plays certain effect, the different physiological roles of mediation VEGFs.The principal biological function of VEGFs is: (1) selectivity promotes vascular endothelial cell mitotic division, and stimulating endothelial cell is bred and promoted vascularization; (2) raise the permeability of blood vessel especially tiny blood vessels, make blood plasma macromolecular extravasation be deposited in EV matrix, for the growth of tumour cell and the foundation of new capillary vessel net provide nutrition; (3) transfer of tumour is promoted, the propagation of tumour and transfer rely on vasculogenesis VEGFs makes vascular endothelial cell secrete collagenase and Profibrinolysin, so as to degraded basement membrane of blood vessel, simultaneously, the inner new microvascular basement membrane imperfection formed of tumor tissues, this character makes tumour be easy to enter circulation of blood; (4) other effect: VEGFs can induce epithelial cell gap to occur and phenomenon of windowing, can the kytoplasm vesicle of activated epithelial and organoid.VEGFs direct stimulating endothelial cell release proteolytic ferment, matrix degradation, discharges more VEGFs, accelerates the development of tumour, and extracellular protease again can the release of associativity VEGFs of activating cells epimatrix.VEGFs makes plasma proteins comprise Fibrinogen release by increasing vascular permeability, forms Mierocrystalline cellulose network, for tumor growth, development and transfer provide good matrix.(5) VEGFs may suppress the immune response of body, promotes infiltration and the transfer of malignant tumour.Early stage research is thought, tumour itself does not form newborn lymphatic capillary, and tumour peripheral lymphoid pipe is only the remaining lymphatic vessel in healthy tissues, there is no direct evidence and shows that tumor tissues can cause lymphatic vessel hyperplasia.The formation of this understanding is technical limitation on the one hand, and blood vessel and lymphatic vessel cannot be separated, may be it is believed that lymphatic metastasis is also arrive lymph again by blood vessel on the other hand.Increasing research shows that lymphatic vessel generation is the key step of transfer process, and direct dependency has been sent out in the transfer that the lymphatic vessel that human tumor is expressed generates factor Ⅴ EGF-C and VEGF-D and tumour.

Clinical studies show utilizes the combination of monoclonal antibody or soluble VEGFR s blocking VEGF s and its acceptor, hinders the conduction of VEGFs signal path to be one of effective ways for the treatment of tumour at present.Genentech Bevacizumab that company researches and develops (trade(brand)name, Avastin) be a kind of human mouse chimeric antibody of restructuring, block it by closed VEGF-A to be combined with vascular endothelial growth factor receptor, make VEGFR cannot activate and play the effect of angiogenesis inhibitor.The effect of Bevacizumab can be divided into 3 stages: in the early stage, when drug effect is in tumour, can causes tumor vascular degeneration, make tumor mass reduction; Then can make the tumor vessel trend normalizing that the form of survival is disorderly, tube wall permeability is high, chemotherapeutics more effectively be transmitted in tumor tissues, strengthen tumour cell to the sensitivity of cellulotoxic chemotherapeutics; In the later stage, Bevacizumab can suppress the angiogenesis relevant to tumour and regeneration (Jain RK..N Engl J Med.2009,360 (25): 2669-71.) further.This product is at present for first-line treatment metastatic colorectal carcinoma, and future is likely for the treatment of the diseases such as pulmonary metastasis, breast cancer, cancer of pancreas, renal cancer.This product is also develop comparatively successfully one of antibody drug, and within 2010, global marketing volume reaches 6,900,000,000 dollars, the best-selling medicine in the row world the 6th.Because soluble VEGFR does not possess cross-film district and intracellular tyrosine kinase structural domain, therefore it can not produce intracellular signaling after being combined with VEGF, use sVEGFR competitive binding circulation VEGFs, consume the VEGFs that tumour cell produces, the signal transduction of blocking VEGF s functional receptor, thus the biological function suppressing VEGFs induction.The part extracellular regions of VEGFR1 and VEGFR2 and the human IgG constant domain albumen (VEGFR1-Fc and VEGFR2-Fc) of expressing not only can in and VEGF, and have the longer transformation period.The avidity of VEGFR1-Fc and VEGFs exceeds hundreds of times of (Kuo than VEGFR2-Fc, C.etal.PNAS, 2001 98,4605-4610), but VEGFR1-Fc contains more basic aminoacids, and iso-electric point is greater than 9.5, comparatively easily with in acid matrix be combined in vivo, reduce its transformation period (2000, Cancer Res.60,6253-6258.) in blood.The VEGF-Trap (aflibercept) of investigator's developments such as the Jocelyn H of Sanofi-aventis company and Regeneron company is that the IgG1 constant region of the 3rd structural domain outside outer for VEGFR1 born of the same parents the 2nd structural domain and VEGFR2 born of the same parents and people is merged.Experimental result shows that VEGF-Trap can the growth of Tumor suppression and angiogenesis effectively, and has stronger antitumor action (WulffC etc., J Clin Endocrinol Metab.2001,86 (7): 3377-86 than Avastin; Holash J etc., PNAS 2002,17:11393-98).This fusion rotein can in conjunction with VEGF-A, VEGF-B and PIGF but can not in conjunction with VEGF-C and VEGF-D (Cursiefen C, et al:J Clin Invest.2004,113 (7): 1040-50.).To be Bevacizumab or aflibercept be all by blocking VEGF-A or and the VEGF-B biological function of inducing, namely the vasculogenesis of Tumor suppression plays antitumor action.They all can not combine with VEGF-C or VEGF-D, and VEGF-C and VEGF-D can generate by induced tumor ductus endolymphaticus, and then promote that tumor lympha is carried down and moved.Adopt molecular biology method (the Nat Med.2001Feb such as T; 7 (2): 199-205) the Fc coupling VEGF expression R-3-Ig immune fusion protein of VEGFR-3 ligand binding domain at interior extracellular region and IgG, VEGFR-3-Ig can be combined with VEGF-C or VEGF-D, make VEGF-C or VEGF-D cannot carry out the intracellular signaling (being also referred to as VEGF-C/VEGF-D Trap) in downstream, thus reach the effect suppressing lymphatic vessel to generate.Found that VEGFR-3-Ig can suppress lymphatic vessel to generate.Karpanen etc. (Cancer Res, 2001,61 (5): 1786 ~ 1790) also observe on MCF7 breast cancer orthotopic transplantation model soluble VEGFR-3-Ig albumen can Tumor suppression lymphatic vessel generate phenomenon.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art part, a kind of Ultra-VEGF-trap immune fusion protein, its preparation method and application thereof are provided.

Ultra-VEGF-trap immune fusion protein of the present invention is 1) or 2) in albumen:

1) protein of the aminoacid sequence composition as shown in SEQ ID.1;

2) aminoacid sequence as shown in SEQ ID.1 through replacement and/or disappearance and/or add one or several amino acid and antitumor relevant by 1) derivative protein.

The construction process of Ultra-VEGF-trap immune fusion protein of the present invention comprises the steps:

1) be connected with the IgG1 constant region fc encoding gene of people by the 3rd functional domain encoding gene outside outer for VEGFR1 born of the same parents the 2nd functional domain encoding gene, VEGFR2 born of the same parents, obtain AmFc1 gene, its nucleotide sequence is as shown in SEQ ID.2; The aminoacid sequence of the AmFc1 fusion rotein of expressing, as shown in SEQ ID.3, can be combined with VEGF-A or VEGF-B high-affinity; The Fc of the human IgG constant region in described fusion rotein AmFc1 has carried out two amino acid whose sudden changes, and the E of the 357th sports K, and the D of the 399th sports K; (Fc numbering amino acid residues presses Kabat database);

2) outer for VEGFR3 born of the same parents the 1st functional domain is connected to the 3rd functional domain encoding gene with the IgG1 constant region fc encoding gene of people, obtain the BmFc2 gene of nucleotide sequence as shown in SEQ ID.4, the aminoacid sequence of the BmFc2 fusion rotein of expressing, as shown in SEQ ID.5, can be combined with VEGF-C or VEGF-D high-affinity; In described fusion rotein BmFc2, the Fc of the constant region of human IgG1 has carried out two amino acid whose sudden changes, and the K of the 392nd sports D, and the K of the 409th sports D; (Fc numbering amino acid residues presses Kabat database);

3) these two kinds of immune fusion proteins of AmFc1 and BmFc2 are assembled into heterodimer in engineering cell.

The charge polarity of AmFc1 and BmFc2 immune fusion protein contact surface is changed by sudden change, the expression product of formation is made to be the heterodimer Ultra-VEGF-trap (being called for short UVT) of AmFc1 and BmFc2, but not the homodimer of AmFc1 or BmFc2, also non-part is the heterodimer of AmFc3 and BmFc4.

Said engineering cell is selected from CHO, 293 or NS0 cell.

Ultra-VEGF-trap immune fusion protein of the present invention, has the purposes for the preparation of antitumor drug.

The functional zone that Ultra-VEGF-trap fusion rotein of the present invention has and VEGF-A ,-B ,-C ,-D combine, can simultaneously in conjunction with the multiple vascular endothelial growth factor such as VEGF-A ,-B ,-C ,-D.If immune fusion protein Ultra-VEGF-trap enters in body can hinder VEGF-A, VEGF-B, VEGF-C and VEGF-D and their acceptor VEGFR-1 (fms-like tyrosine kinase, Flt-1), VEGFR-2 (kinase insertdomain containing receptor, and the combination of VEGFR-3 (Flt-4) KDR/Flk-1), Tumor suppression organizes new vessel and vasculolymphatic generation, reach Tumor suppression growth and by the object that lymphsystem shifts, there is the value of huge, potential treatment Several Kinds of Malignancy.Specifically, Ultra-VEGF-trap fusion rotein has following function: on the one hand it can the activation of vascular endothelial cell that produces of blocking VEGF-A and VEGF-B, propagation, differentiation and migration, block the formation of new vessel in tumour, reduce blood supply and the oxygen supply of tumour, the growth of restriction tumour, can vascular permeability be reduced again, reduce the chance that transfer occurs by blood vessel tumour; On the other hand, the activation of its energy blocking VEGF-C and VEGF-D generation lymphatic endothelium, Tumor suppression organizes the formation of Lymphangiogenesis, hinders tumour cell lymphatic vessel route of metastasis.Ultra-VEGF-trap has almost all blocked VEGFs/VEGFRs pathway, suppresses the formation of new vessel, reduces vascular permeability, reduces blood supply and the oxygen supply of tumour, can block tumour ductus endolymphaticus simultaneously and generate, the lymphatic vessel transfer of Tumor suppression.In addition, Ultra-VEGF-trap fusion rotein has very high molecular weight (140kDa), and thus its transformation period obviously extends.Therefore, Ultra-VEGF-trap immune fusion protein, to having in the treatment of malignant tumour than going on the market Bevacizumab and be about to the more advantage of listing aflibercept, has the value of potential treatment Several Kinds of Malignancy.

Accompanying drawing explanation

Fig. 1 is fusion rotein Ultra-VEGF-trap structural representation;

Fig. 2 is PBHL-UVT expression vector structural representation.Wherein, Amp: amicillin resistance, CMVLE: human macrophage virus early promoter, SV40late Poly A:SV40 late promoter and polyadenylation signal, GS: glutamine synthetase.

Fig. 3 is expression vector restriction endonuclease qualification electrophorogram, and wherein, 1 is molecular weight standard DL2000; 2 is pBHL-AmFc1; 3 is pBHL-UVT.

Fig. 4 is the non-reduced SDS-PAGE electrophorogram of Ultra-VEGF-trap of purifying, and wherein 1 is Protein Marker; 2 is immune fusion protein Ultra-VEGF-trap.

Fig. 5 is the Ultra-VEGF-trap reduction SDS-PAGE electrophorogram of purifying, and wherein 1 is immune fusion protein Ultra-VEGF-trap; 2 is Protein Marker.

Fig. 6 be ELISA detect Ultra-VEGF-trap and VEGFs in conjunction with result figure.

Embodiment

Embodiment 1 builds the expression vector of immune fusion protein Ultra-VEGF-trap

1, the synthesis of AmFc1 and BmFc2 gene

Entrust Shanghai Jierui Biology Engineering Co., Ltd synthesis AmFc1 coding gene sequence, its nucleotide sequence is as shown in SEQ ID.2; The aminoacid sequence of AmFc1 albumen is as shown in SEQ ID.3, comprising the 3rd functional zone outside signal peptide sequence, outer 2nd district of VEGFR1 born of the same parents, VEGFR2 born of the same parents, and the constant region fc of human IgG1, wherein Fc has carried out two amino acid whose sudden changes, the E of the 357th sports K, the D of the 399th sports K, called after AmFc1.

Same trust Shanghai Jierui Biology Engineering Co., Ltd synthesis BmFc2 coding gene sequence, nucleotide sequence is as shown in SEQ ID.4; The aminoacid sequence of BmFc2 albumen is as shown in SEQ ID.5, comprising signal peptide sequence, VEGFR3 born of the same parents outer 1st district, the 2nd functional zone and the 3rd functional zone, and the constant region fc of human IgG1, wherein Fc has carried out two amino acid whose sudden changes, the K of the 392nd sports D, the K of the 409th sports D, called after BmFc2.

2, the expression vector of immune fusion protein Ultra-VEGF-trap is built

1) plasmid containing AmFc1 sequence is provided in the intestinal bacteria provided from JaRa biotechnology company limited, after HinDIII and EcoRI double digestion, reclaims digestion products.With the same expression vector pBHL processed of warp at T ₄after the lower 16 DEG C of connections of DNA ligase effect are spent the night, connect product and proceed in bacillus coli DH 5 alpha competence bacterium, screening and culturing in LB (ammonia benzyl enzyme element resistance) culture plate, picking positive colony.Extract plasmid after cultivation, HinDIII and EcoRI double digestion is identified, cuts out the fragment of expection size, called after pBHL-AmFc1.

2) substantially above-mentioned steps is repeated, the plasmid containing BmFc2 sequence JaRa biotechnology company limited synthesized is connected with after XhoI with XbaI double digestion respectively with pBHL-AmFc1 expression vector, build Ultra-VEGF-trap expression vector, as shown in Figure 2, fusion rotein Ultra-VEGF-trap structure as shown in Figure 1 for structure.With XhoI, XbaI, HinDIII and EcoRI 4 enzyme cut after cut out expection size fragment, the results are shown in Figure 3.

The Expression and purification of embodiment 2 immune fusion protein Ultra-VEGF-trap

293F is (purchased from Invitrogen company, Cat No.11625-019) cell suspension culture in serum-free CD293 nutrient solution (purchased from Invitrogen company, Cat No.11913-019) in, centrifugal replacing fresh culture before transfection, cell concn is adjusted to 1 × 106 cell/ml.For 100ml cell, DNA (pT1h-ONC) 150 μ g and PEI 300 μ g (Sigma, Cat.No:408727) is added in 10ml 293 nutrient solution mix respectively, leave standstill 5min.PEI/DNA suspension dropwise adds in shaking flask, mixes gently after placing 20min by room temperature, is placed in 5%CO2,37 DEG C of shaking tables are cultivated (115rpm) and collected culture supernatant after 5 days.

Transfectional cell, after 5 days, is collected supernatant and is done purifying.With PBS solution balance HiTrap MabSelectSuRe 1ml post (GE Healthcare Life Sciences product, Cat.No:11-0034-93) 10 bed volumes of pH 7.4, flow velocity is 0.5ml/min; By the 200ml supernatant liquor 0.45 μm of membrane filtration loading obtained through transfection, flow velocity is 0.5ml/min.Wash 5-10 bed volume again by the PBS solution of pH 7.4, flow velocity is 0.5ml/min; With 100mM citrate buffer solution (pH 4.0) wash-out, flow velocity is 0.5ml/min, collects elution peak.

The results are shown in Figure 4 and Fig. 5, immune fusion protein Ultra-VEGF-trap purity reaches more than 95%, and the molecular weight of molecular weight about about 140kDa, AmFc1 and the BmFc2 of immune fusion protein is respectively 80kDa and 60kDa, consistent with theory expectation.

The functional analysis of embodiment 3 immune fusion protein Ultra-VEGF-trap

Respectively by VEGF165 (Beijing Hong Yue Creative Technology Ltd., cat:100-20), VEGF-B (Beijing Hong Yue Creative Technology Ltd., cat:100-20B), VEGF-C (Beijing Hong Yue Creative Technology Ltd., cat:100-20C), VEGF-D (Beijing Hong Yue Creative Technology Ltd., cat:100-20D) 2 μ g/mL are diluted to 0.05mmol/L sodium carbonate, sodium bicarbonate buffer liquid (pH 9.6), 100 μ L/ holes, 4 DEG C of bags are spent the night; After 1%PBS-BSA room temperature closes 1h, add the fusion rotein of different concns, incubated at room 2h.After PBST (0.05%Tween-20) washs 3 times, add alkaline squama acid enzyme labelling anti-human igg (Zhong Shan Golden Bridge, cat:ZB2304) of 1: 30000 dilution, 100 μ L/ holes, incubated at room 45min; After PBST washs 3 times, add TMD substrate nitrite ion (health is century, cat:CW0050) 100 μ L/ hole, lucifuge develops the color; Add 2M vitriol oil termination reaction; Put in microplate reader and measure 450nm absorbance.Result as shown in Figure 6.

Result shows: immune fusion protein Ultra-VEGF-trap can combine in conjunction with the soluble vascular endothelial growth factor that VEGF-165, VEGF-B, VEGF-C, VEGF-D tetra-kinds is different simultaneously, and binding capacity increases with the increase of fusion rotein.

Organization Applicant

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<110> OrganizationName: Jiangsu Jian De Bioceuticals Inc.

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<120> Title: Ultra-VEGF-trap immune fusion protein, its preparation method and application thereof

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aagctgaccg tggacaagtc ccggtggcag cagggcaacg tgttcagctg cagcgtgatg 1320

cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgagccc cggaaagtaa 1380

gaattcc 1387

<212> Type : DNA

<211> Length : 1387

SequenceName : 2

SequenceDescription :

Sequence

--------

<213> OrganismName :

<400> PreSequenceString :

METDTLLLWV LLLWVPGSTG SDTGRPFVEM YSEIPEIIHM TEGRELVIPC RVTSPNITVT 60

LKKFPLDTLI PDGKRIIWDS RKGFIISNAT YKEIGLLTCE ATVNGHLYKT NYLTHRQTNT 120

IIDVVLSPSH GIELSVGEKL VLNCTARTEL NVGIDFNWEY PSSKHQHKKL VNRDLKTQSG 180

SEMKKFLSTL TIDGVTRSDQ GLYTCAASSG LMTKKNSTFV RVHEKGPGDK THTCPPCPAP 240

ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR 300

EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 360

PSRDKLTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLKSDG SFFLYSKLTV 420

DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKZ 456

<212> Type : PRT

<211> Length : 456

SequenceName : 3

SequenceDescription :

Sequence

--------

<213> OrganismName :

<400> PreSequenceString :

ctcgaggcca ccatggagac cgacaccctg ctgctctggg tgctgctgct ctgggtgccc 60

ggctccaccg gatactccat gacccctcct accctgaaca tcaccgagga gtcccacgtg 120

atcgacaccg gcgactccct gtccatctcc tgccgcggcc agcaccctct ggagtgggcc 180

tggcctggcg cccaggaggc ccctgccacc ggcgacaagg actccgagga caccggcgtg 240

gtgcgcgact gcgagggcac cgacgcccgc ccttactgca aggtgctgct gctgcacgag 300

gtgcacgcca acgacaccgg ctcctacgtg tgctactaca agtacatcaa ggcccgcatc 360

gagggcacca ccgccgcctc ctcctacgtg ttcgtgcgcg acttcgagca gcctttcatc 420

aacaagcctg acaccctgct ggtgaaccgc aaggacgcca tgtgggtgcc ttgcctggtg 480

tccatccctg gcctgaacgt gaccctgcgc tcccagtcct ccgtgctgtg gcctgacggc 540

caggaggtgg tgtgggacga ccgccgcggc atgctggtgt ccacccctct gctgcacgac 600

gccctgtacc tgcagtgcga gaccacctgg ggcgaccagg acttcctgtc caaccctttc 660

ctggtgcaca tcaccggcaa cgagctgtac gacatccagc tgctgcctcg caagtccctg 720

gagctgctgg tgggcgagaa gctggtgctg aactgcaccg tgtgggccga gttcaactcc 780

ggcgtgacct tcgactggga ctaccctggc aagcaggccg agcgcggcaa gtgggtgcct 840

gagcgccgct cccagcagac ccacaccgag ctgtcctcca tcctgaccat ccacaacgtg 900

tcccagcacg acctgggctc ctacgtgtgc aaggccaaca acggcatcca gcgcttccgc 960

gagtccaccg aggtgatcgt gcacgagaac cctttcatcg acaagaccca cacctgccct 1020

ccctgccccg cccccgagct gctgggcgga cccagcgtgt tcctgttccc tcccaagccc 1080

aaggacaccc tgatgatcag ccgcaccccc gaggtgacct gcgtggtggt ggacgtgagc 1140

cacgaggacc ccgaggtgaa gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 1200

aagaccaagc ctcgggagga gcagtacaac tccacctacc gcgtggtgag cgtgctgacc 1260

gtgctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtgag caacaaggcc 1320

ctgcccgctc ccatcgagaa gaccatcagc aaggccaagg gccagccccg ggagcctcag 1380

gtgtacaccc tgccccccag ccgcgacgag ctgaccaaga accaggtgag cctgacctgc 1440

ctggtgaagg gcttctaccc ctccgacatc gccgtggagt gggagagcaa cggccagcct 1500

gagaacaact acgacaccac ccctcccgtg ctggacagcg acggcagctt cttcctgtac 1560

agcgacctga ccgtggacaa gtcccggtgg cagcagggca acgtgttcag ctgcagcgtg 1620

atgcacgagg ccctgcacaa ccactacacc cagaagagcc tgagcctgag ccccggaaag 1680

taatctaga 1689

<212> Type : DNA

<211> Length : 1689

SequenceName : 4

SequenceDescription :

Sequence

--------

<213> OrganismName :

<400> PreSequenceString :

METDTLLLWV LLLWVPGSTG YSMTPPTLNI TEESHVIDTG DSLSISCRGQ HPLEWAWPGA 60

QEAPATGDKD SEDTGVVRDC EGTDARPYCK VLLLHEVHAN DTGSYVCYYK YIKARIEGTT 120

AASSYVFVRD FEQPFINKPD TLLVNRKDAM WVPCLVSIPG LNVTLRSQSS VLWPDGQEVV 180

WDDRRGMLVS TPLLHDALYL QCETTWGDQD FLSNPFLVHI TGNELYDIQL LPRKSLELLV 240

GEKLVLNCTV WAEFNSGVTF DWDYPGKQAE RGKWVPERRS QQTHTELSSI LTIHNVSQHD 300

LGSYVCKANN GIQRFRESTE VIVHENPFID KTHTCPPCPA PELLGGPSVF LFPPKPKDTL 360

MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ 420

DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG 480

FYPSDIAVEW ESNGQPENNY DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VFSCSVMHEA 540

LHNHYTQKSL SLSPGKZ 557

<212> Type : PRT

<211> Length : 557

SequenceName : 5

SequenceDescription :

Claims

1.Ultra-VEGF-trap immune fusion protein, it is characterized in that, described Ultra-VEGF-trap immune fusion protein is by the fusion rotein of aminoacid sequence as shown in SEQ ID NO.3 and the heterodimeric body protein linked together by the disulfide linkage of fusion rotein through hinge area of aminoacid sequence as shown in SEQ ID NO.5.

2. prepare the construction process of Ultra-VEGF-trap immune fusion protein as described in claim 1, it is characterized in that, comprise the steps:

1) be connected with the IgG1 constant region fc encoding gene of people by the 3rd functional domain encoding gene outside outer for VEGFR1 born of the same parents the 2nd functional domain encoding gene, VEGFR2 born of the same parents, obtain AmFc1 gene, its nucleotide sequence is as shown in SEQ ID NO.2; The aminoacid sequence of the AmFc1 fusion rotein of expressing is as shown in SEQ ID NO.3;

2) outer for VEGFR3 born of the same parents the 1st functional domain is connected to the 3rd functional domain encoding gene with the IgG1 constant region fc encoding gene of people, obtain the BmFc2 gene of nucleotide sequence as shown in SEQ ID NO.4, the aminoacid sequence of the BmFc2 fusion rotein of expression is as shown in SEQ ID NO.5;

3) these two kinds of fusion roteins of AmFc1 and BmFc2 are assembled in engineering cell the Ultra-VEGF-trap immune fusion protein of heterodimer.

3. construction process according to claim 2, is characterized in that, described engineering cell is selected from CHO, 293 or NSO cell.

4. Ultra-VEGF-trap immune fusion protein as described in claim 1, has the purposes for the preparation of antitumor drug.