CN1793179A - Optimizing fusion protein containing VEGF recerver segment and medical application thereof - Google Patents

Abstract

The invention supplies a series of VEGF acceptor section and human immunoglobulin Fc optimizing interfusing albumens that contain a series elastic structure peptide connection section and has the function of curing kinds of cancer and retinal vasculopathy AMD that relatives to VEGF diseases. The invention includes the optimized albumen, coding rebuilt DNA sequence and expression carrier, the drug use of the rebuilt albumen and the drug and the agent of the rebuilt albumen, and the gene curing method.

Description

Comprise segmental optimization fused protein of vegf receptor and medical applications thereof

Technical field

The present invention relates to genetically engineered and protein engineering field, specifically, relate to dna sequence dna, its coded fusion rotein, the pharmaceutical use of this fusion rotein, the medicine that contains this fusion rotein and preparation thereof that coding comprises the segmental optimization recombination fusion protein of vegf receptor, and delivery and express the gene therapy method of this fusion rotein.

Background technology

Tumour fast an essential condition of growth be angiogenesis (angiogenesis) (Hanahan et al, Cell, 1996,86:353-364).A large amount of animal models and human clinical trial are verified, and the formation of new vessel can stop the death of growth of tumor and triggering tumor cell effectively in the blocking-up tumour, thereby reaches the tumor treatment effect.Therefore, angiogenesis inhibiting (anti-angiogenesis) is the focus and the most promising field of present anti-cancer agent research, a listing approval that has obtained U.S. FDA has been arranged in this type of biological new drug, also had tens kinds carrying out the various clinical test simultaneously.In addition, for senile retinal vasculopathy (Age-related macular degeneration, abbreviate the multiple diseases relevant such as AMD, diabetic retinopathy, sacroiliitis as with angiogenesis, angiogenesis inhibiting treatment new drug also has extensive and important effect (Folkman, Nature Medicine, 1995,1:27-31; Ferrara, Seminars in Oncology, 2002,29 (6sumppl): 10-14).

Angiogenesis is a complex process that has the various active biotic factor to regulate and control, thereby one of them key link is an endothelial cell surface receptor to be combined by multiple somatomedin and is activated, control the endotheliocyte activity through the signal transduction system of intracellular tyrosine phosphorylation then, thereby impel angiogenesis.In the multiple somatomedin, vascular endothelial growth factor (Vascular endothelial cell growth factor, abbreviate VEGF as) be the most important factor (Ferrara of control angiogenesis, Endocrine Reviews, 2004,25 (4): 581-611), its overexpression all in nearly all human tumor is an important molecule target of anticancer research.VEGF has multiple acceptor on the vascular endothelial cell surface, comprising VEGFR-1 (be called not only Flt-1) and VEGFR-2 (but also being called KDR or Flk-1), their extracellular part is formed by seven immunoglobulin-like zones (D1-D7) that can combine with VEGF, and intracellular portion includes the Tyrosylprotein kinase group.After acceptor is activated by VEGF, intracellular Tyrosylprotein kinase group generation phosphorylation, and cause a series of signal transmission, finally cause angiogenesis.

Because the VEGF signal transmits the importance to angiogenesis, thereby blocking VEGF or vegf receptor reach angiogenesis inhibiting has important antitumous effect, also other is comprised that simultaneously the disease relevant with angiogenesis such as retinal vasculopathy has important therapeutic action.Unique cancer therapy drug at angiogenesis by drugs approved by FDA is exactly the monoclonal antibody of specificity in conjunction with VEGF-A at present, its mechanism is exactly by the purpose that reaches blocking VEGF and its receptors bind in conjunction with VEGF (Ferrara et al, Nature ReviewsDrug Discovery, 2004,3:391-400).The VEGF blocker of another kind of possibility better effects if is the extracellular fragment of vegf receptor, it has the natural high specific at VEGF (Kuo et al, Proceedings of the National Academy of Sciences, 2001,98:4605-4610).In order to be increased in transformation period and the serum-concentration in the serum, the extracellular fragment of vegf receptor often partly is connected to fusion rotein with the Fc of immunoglobulin (Ig), and this fusion rotein is dimerization in vivo, and has and the similar excellent pharmaco-kinetic properties of immunoglobulin (Ig).Studies show that, this fusion rotein external can neutralize VEGF, thereby the blocking VEGF signal transmits and the hyperplasia of vascular endothelial cell, but not ideal enough (the Ferrar et al of the angiogenesis inhibiting effect in animal experiment, Nature Medicine, 1998,4:336-340; Gerberet al, Nature Medicine, 1999,5:623-628; Gerber et al, Cancer Reserch, 2000,60:6253-6258), its reason may be that the structure of this kind fusion rotein is stable inadequately, thereby influences their serum-concentration and to the affinity of VEGF, cause the incomplete blocking-up to VEGF.

For the extracellular fragment of improving above-mentioned vegf receptor and the fusion rotein of Fc, the invention provides a method of optimizing fusion rotein, the stability and the biological activity of fusion rotein have been improved greatly, make its more effectively combination and neutralize VEGF in external and animal, the blocking-up angiogenesis suppresses tumor development.

Summary of the invention

One of the object of the invention provides the fusion rotein that comprises vegf receptor extracellular fragment and human normal immunoglobulin Fc of optimization, it all has advantages of excellent stability and biological activity in vivo and in vitro, and can transmit by the blocking VEGF signal, thereby angiogenesis inhibiting and tumor development.

Two of the object of the invention provides the nucleotide sequence of encoding said fusion protein.

Three of the object of the invention provides the nucleotide sequence that contains encoding said fusion protein, its expression vector and the recombinant chou that is transformed by this carrier, and express cell.

Four of purpose of the present invention provides medicine the application in relative diseases such as treatment tumour and retinal vasculopathy of this fusion rotein as the blocking-up angiogenesis, comprise the pharmaceutical composition of above-mentioned fusion rotein and pharmaceutically acceptable carrier in addition and treating relative disease, particularly antineoplastic application.

Main points of the present invention are the vegf receptor extracellular fragment of attempting according to forefathers and the structural unstable characteristics of fusion rotein of Fc, design and built series of optimum peptide linkage section (peptide linker) insert between the extracellular fragment and Fc of vegf receptor, make and have sufficiently high structural elasticity and plasticity-between the extracellular fragment of vegf receptor and the Fc, thereby increased the stability of fusion rotein greatly, the more important thing is, significantly improved and multiple VEGF bonded avidity.This optimization fusion rotein that comprises the peptide linkage section is compared with the fusion rotein that does not comprise this peptide linkage section, has higher biological activity and longer serum half-life, thus more effectively neutralize VEGF, angiogenesis inhibiting and tumor development.

Studies show that, second particularly important of effect immunoglobulin-like region D 2 has to(for) the affinity of VEGF of N end of VEGFR-1, the multiple vegf receptor fusion rotein that does not comprise D2 has lost most VEGF avidity, the multiple vegf receptor fusion rotein that comprises D2 has then been preserved the avidity to VEGF, therefore, D2 is considered to in conjunction with the VEGF decisive role.Nature, the fusion rotein of D2 and Fc is the first-selection as the VEGF blocker, but this trial has been failed, independent D2 fragment and the fusion rotein of Fc have been lost most of activity (Davis-Smyth et al, United States Patent Application 0030092604) in conjunction with VEGF.In the D2-Fc of this failure fusion rotein, have only a linkage section that comprises two amino acid (FE) between D2 fragment and Fc, this may be exactly the reason of its failure.D2 and Fc are two zones that independently have difference in functionality, all must form the activated three-D space structure of each self stabilization, particularly behind two Fc zone dimerizations, two D2 zones must keep its structural stability and biological activity, thereby guarantee its avidity to VEGF.And in the D2-Fc of this failure fusion rotein, the structure space that two amino acid between D2 and Fc are not enough to provide enough probably satisfies the needs of D2 and Fc rock steady structure, make D2 or Fc or the two all can not form stable structure, finally influence the avidity of D2-Fc fusion rotein greatly VEGF.The present invention then provides the optimization peptide linkage section of a series of whippy structures to connect D2 and Fc, make D2 and Fc all have enough spaces to form stable structure, thereby guaranteed avidity and the biological activity of the D2-Fc fusion rotein of this optimization, improved about thousand times VEGF avidity than original D2-Fc fusion rotein VEGF.

The optimization design of this whippy structure peptide linkage section also can be used for other vegf receptor extracellular fragment and the fusion rotein of Fc, with stability and the biological activity that improves them.The 3rd the immunoglobulin-like region D 3 of second immunoglobulin-like region D 2 that a kind of known good VEGF blocker is VEGFR-1, VEGFR-2 and the fusion rotein of Fc, its to VEGF in external avidity up to～1pM (Holashet al, Proceedings of the National Academy of Sciences, 2002,99 (17): 11393-11398).The present invention has utilized same optimization design, the optimization peptide linkage section that has inserted whippy structure between D2 in this fusion rotein, D3 and the Fc finds that with after replacing original three amino acid (GPG) its avidity to VEGF has improved 10 times than original fusion rotein.

Vegf receptor extracellular fragment that the present invention describes and the optimization fusion rotein of Fc are to build by the gene recombination technology of routine, concrete experimental procedure is put down in writing as " molecular cloning " third edition (Joseph Sambrook, Science Press) and similar laboratory manual.Used vegf receptor extracellular fragment and Fc are:

VEGFR-1 the 2nd immunoglobulin-like zone, be expressed as D2, aminoacid sequence is as described in the SEQ ID NO.1 in the sequence table.

VEGFR-2 the 3rd immunoglobulin-like zone, be expressed as D3, aminoacid sequence is as described in the SEQ ID NO.2 in the sequence table.

3. human normal immunoglobulin Fc comes from IgG1, is expressed as Fc, and aminoacid sequence is as described in the SEQ IDNO.3 in the sequence table.

Peptide linkage section used in the fusion rotein of optimization of the present invention is:

1. G9, aminoacid sequence is as described in the SEQ ID NO.4 in the sequence table.

2. G12, aminoacid sequence is as described in the SEQ ID NO.5 in the sequence table.

3. GS, aminoacid sequence is as described in the SEQ ID NO.6 in the sequence table.

As shown in Figure 1, YY-001 and YY-005 are the fusion roteins that document had been put down in writing.YY-001 by VEGFR-1 the 2nd immunoglobulin-like zone and human normal immunoglobulin Fc constituted, be expressed as D2-Fc.YY-005 by VEGFR-1 the 2nd immunoglobulin-like zone, VEGFR-2 the 3rd immunoglobulin-like zone and human normal immunoglobulin Fc constituted, be expressed as D2D3-Fc.Optimization fusion rotein provided by the invention then is to have inserted the peptide linkage section that increases structural elasticity before the Fc in YY-001 and YY-005:

1. YY-002：D2-G9-Fc。

2. YY-003：D2-G12-Fc。

3. YY-004：D2-GS-Fc。

4. YY-006：D2D3-G9-Fc。

5. YY-007：D2D3-G12-Fc。

6. YY-008：D2D3-GS-Fc。

Wherein YY-001 sees SEQ ID NO.7-14 in the sequence table to the DNA sequences encoding of YY-008.

Above-mentioned optimization fusion rotein and coding DNA thereof can obtain by conventional gene recombination technology.The dna sequence dna of required coding VEGFR-1, VEGFR-2 and Fc is by after obtaining among the GenBank of NCBI (National Center forBiotechnology Information), the dna sequence dna of the above-mentioned optimization fusion rotein of coding is cloned into respectively in the carrier after synthetic by PCR, and used carrier can be plasmid, virus or the dna fragmentation that molecular biology is used always.Before the end of dna sequence dna of the above-mentioned optimization fusion rotein of coding, add the protein excretion signal sequence, from cell, secrete to guarantee protein.Comprise the promotor, protein translation initial sum termination signal and polyadenylic acid (PolyA) sequence that are used to drive genetic expression in the carrier sequence.Antibiotic resistance genes is arranged in the carrier, be beneficial to carrier, as breeding in the bacterium at host cell.In addition, also comprise the eukaryotic cell selected gene in the carrier, be used for the selection of stable transfection host cell strain.

Owing to do not have absolute boundary between the aminoacid sequence in each immunoglobulin-like zone among VEGFR-1 and the VEGFR-2, so the length of the aminoacid sequence in each immunoglobulin-like zone can have certain variation.So the aminoacid sequence of optimization fused protein involved in the present invention also can have certain variation, they all belong to scope of the present invention.

Fc in the above-mentioned optimization fusion rotein is from human normal immunoglobulin IgG1, also can be hypotype IgG2, IgG3, IgG4 or human normal immunoglobulin IgM and IgA, this immunoglobulin Fc fragment can be Fc total length or part Fc sequence, as being selected from CH2 segment, CH3 segment or stranded regional fragment, they all belong to scope of the present invention.

Peptide linkage section purpose in the above-mentioned optimization fusion rotein is to provide better elasticity and plasticity-, therefore its aminoacid sequence and length can have certain variation, also can be got by screening in the peptide chain library of a completely random, they all belong to scope of the present invention.

After the plasmid construction of finishing the dna sequence dna that contains the above-mentioned optimization fusion rotein of encoding, promptly available this recombinant vectors transfection or transformed host cell are expressed corresponding fusion proteins matter.Can be used in and express these Expression of Fusion Protein systems and have multiplely, can be that eukaryotic cell also can be a prokaryotic cell prokaryocyte, and they include, but is not limited to mammalian cell, bacterium, yeast, insect cell etc.Because comprising in the aminoacid sequence of optimization fused protein of the present invention can glycosylated amino acid, therefore, mammalian cell is to express these proteinic optimizer systems.Can be used for the extensive mammalian cell of expressing of protein has multiple, for example 293 cells, Chinese hamster ovary celI, SP20 cell, NS0 cell, COS cell, bhk cell or PerC6 cell etc., therefore many other cells also can be used for these protein expressions and production, all are included in the row of the cell that the present invention can use.The recombinant plasmid that contains the above-mentioned optimization fusion rotein of encoding can enter host cell through transfection, and the method for transfectional cell has multiple, comprising but be not limited to: electric drilling method (electroporation), liposome mediated-method, calcium mediated method etc.

A kind of preferable expression method is in the host cell of stable transfection recombinant vectors to be carried out gene amplification, to improve the expression amount of corresponding recombination fusion protein, after for example lacking the host cell of DHFR with the recombinant vectors stable transfection that contains Tetrahydrofolate dehydrogenase (DHFR), the concentration that can increase methotrexate (MTX) in cell culture fluid is with the number of copies of amplification recombinant vectors in host cell; Again for example to expressing the stable transfection host cell of glutamine synthetase (GS), utilize can the increase number of copies of recombinant vectors of the concentration that increases methionine sulfoxide (MSX) in the nutrient solution.

Other expression systems beyond the mammalian cell, for example bacterium, yeast or insect cell etc. also can be used to express these and optimize fusion rotein, and they are also included within the row of the cell that the present invention can use.The protein output of these expression systems is high than mammalian cell, and still, expressed potein deficiency glycosylation or formed sugar chain are different with mammalian cell.

After optimizing the fused protein expression, available enzyme linked immunosorbent adsorption test (ELISA) or additive method are measured the concentration of fused protein in the cell culture fluid.Comprise immunoglobulin Fc because these optimize fused proteins, therefore available albumin A affinity chromatography purifies the optimization fused protein expressed.

From the recombinant chou nutrient solution, obtain corresponding optimize fused protein after, can utilize that VEGF is external to be detected and compare the avidity of range protein to VEGF in conjunction with experiment.Experimental result proves, compares with the YY-005 fusion rotein with the disclosed YY-001 of prior art, and the constructed optimization fused protein of the present invention has improved the avidity (see figure 2) to VEGF greatly.Therefore, the constructed optimization fusion rotein of the present invention has better blocking effect to VEGF, thereby angiogenesis inhibiting more effectively, the disease that treatment is relevant with angiogenesis or VEGF, they include but not limited to various tumours, retinal vasculopathy AMD, diabetic retinopathy, sacroiliitis, anaemia or endometrial hyperplasia etc.

The present invention also provides experimentation on animals to prove optimization fused protein provided by the invention anti-angiogenic rebirth effect in vivo.Experimental results show that, in the mouse model of HCC Hep3B liver cancer and Lovo large bowel cancer, fusion rotein provided by the invention has all fully suppressed growth of tumor, and effect is significantly better than the YY-001 and the YY-005 fusion rotein of prior art, therefore, the constructed fusion rotein of the present invention has anti-cancer ability efficiently.In addition, at laser induced choroidal neovascularization (choroidal neovascularization, abbreviate CNV as) in the mouse model, optimization fusion rotein YY-007 provided by the invention is by the expression of adeno-associated virus AAV2 carrier, suppress and prevented the generation of CNV effectively, therefore, retinal vasculopathy is had good medical prospect.

The present invention also provides with virus vector and has delivered and expressed the method that these optimize fusion rotein.These carriers comprise adeno-associated virus AAV, comprising but be not limited to serotype A AV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9, with other virus vector, include but not limited to adenovirus carrier (adenoviral vectors), retrovirus vector (retroviral vectors), herpes simplex virus vector (herpes simplex virus-based vectors, HSV) and lentiviral vectors (lentiviralvectors).

The present invention also provides the pharmaceutical composition that contains fusion rotein of the present invention and pharmaceutical carrier.This pharmaceutical composition can be made various forms of pharmaceutical preparations according to the technology of pharmaceutics routine techniques, preferably injection, most preferably freeze drying injection.

Pharmaceutical composition of the present invention, wherein also comprise any or several other medicines with synergistic angiogenesis inhibiting, described composition can be treated relevant diseases of angiogenesis together with the other treatment method, and described other treatment method is selected from chemotherapy, reflexotherapy, gene therapy.

The subordinate list explanation

Table 1 has been enumerated the external avidity in conjunction with VEGF of multiple optimization fusion rotein.

Table 2 has shown that the YY-007 of adeno-associated virus AAV2 expression prevents the positive effect of CNV in laser induced CNV mouse model.

The VEGF avidity of each fusion rotein of table 1.

	YY-002	YY-003	YY-004	YY-005	YY-006	YY-007	YY-008
								Avidity (pM)	0.99	0.97	0.87	0.91	0.13	0.09	0.10
Standard deviation (pM)	0.049	0.060	0.059	0.049	0.048	0.023	0.030

The YY-007 that table 2. adeno-associated virus AAV2 expresses can prevent the generation of CNV at the mouse intraocular

	The eyes sum	The calcination sum	The CNV sum	The CNV ratio
					Left eye	11	54	37	68.5％
Whole right eyes	9	42	5	11.9％
					Right eye-do not comprise major injury	7	35	5	14.3％
The whole damages of right eye-do not comprise	3	20	5	20％

Description of drawings

Fig. 1 is the fusion rotein synoptic diagram

The 2nd immunoglobulin-like region representation of VEGFR-1 is D2, and the 3rd immunoglobulin-like region representation of VEGFR-2 is D3, and human normal immunoglobulin Fc region representation is Fc, optimizes the peptide linkage section for three kinds and is expressed as G9, G12 and GS.

Fig. 1 has described vegf receptor extracellular fragment and the fusion rotein YY-001 of Fc and the albumen composition structure of YY-005 that document had been put down in writing, and comprises the composition structure of the optimization fusion rotein of peptide linkage section accordingly.

Fig. 2 is that various fusion roteins are in external comparison in conjunction with VEGF

The VEGF avidity of Fig. 2-A.YY-002, YY-003 and YY-004 has improved at least thousand times than YY-001.

Fig. 2-B.YY-006, the VEGF avidity of YY-007 and YY-008 has improved about 10 times up to 0.1pM than YY-005, and wherein YY-007 and YY-008 are than the avidity high more slightly 10% of YY-006.

Fig. 2 has shown the fusion rotein of multiple vegf receptor extracellular fragment and Fc in a preferred embodiment of the present invention and VEGF in external bonded test-results, and wherein X-axis is represented the concentration (pM) of fusion rotein, and Y-axis is represented the concentration of VEGF (pM) freely.The result shows that three kinds of optimization fusion roteins that comprise the peptide linkage section have all improved its avidity to VEGF greatly, then not obviously difference between the optimization fusion rotein of three kinds of different peptide linkage sections.YY-002 wherein, YY-003, YY-004 has improved at least thousand times than the VEGF avidity of YY-001, and YY-006, YY-007, YY-008 has improved about 10 times than YY-005.

Fig. 3 suppresses HCC Hep3B liver cancer in mouse intravital growth YY-001 and the Fc not influence of growth to HCC Hep3B liver cancer cell effectively for optimizing fusion rotein, the effect of YY-003 and YY-005 is close, growth to liver cancer cell has significant inhibitory effect, and antitumous effect the best of YY-007 has been contained the growth of liver cancer cell fully.

Fig. 3 has shown that the optimization fusion rotein that comprises the peptide linkage section can more effectively suppress HCC Hep3B liver cancer in the intravital growth of mouse.

Fig. 4 suppresses the Lovo large bowel cancer in mouse intravital growth YY-001 and the Fc not influence of growth to the Lovo colorectal cancer cells effectively for optimizing fusion rotein, YY-003 and YY-005 have the obvious suppression effect to the growth of liver cancer cell, and antitumous effect the best of YY-007 has been contained the growth of Lovo colorectal cancer cells fully.

Fig. 4 has shown that the optimization fusion rotein that comprises the peptide linkage section can more effectively suppress the Lovo large bowel cancer in the intravital growth of mouse.

Fig. 5 is on average got by all laser calcination points at the area that the mouse intraocular suppresses the generation CNV of CNV for the YY-007 that adeno-associated virus AAV2 expresses, the eyes sum that the digitized representation in each row detected.

Fig. 5 has shown that the YY-007 of adeno-associated virus AAV2 expression can suppress the generation of CNV in laser induced choroidal neovascularization CNV mouse model.

Embodiment

Following examples make up, test optimization fusion rotein involved in the present invention and should be used as detailed description.But content of the present invention and purposes are not restricted to the scope of example.

Embodiment 1: clones coding is optimized the dna sequence dna of fusion rotein and is made up recombinant vectors

The coding DNA of multiple optimization fusion rotein is to get with different primer amplifications by the cDNA of polymerase enzyme chain reaction (PCR) by VEGFR-1, VEGFR-2 and Immunoglobulin IgG1 Fc among the present invention.

The structure of eight kinds of fusion roteins that this preferred embodiment is constructed is seen accompanying drawing 1.

Example 1 makes up YY-001 gene and recombinant vectors

YY-001 by VEGFR-1 the 2nd immunoglobulin-like zone (D2) and human normal immunoglobulin Fc merge and to form, the signal peptide that its N end has added VEGFR-1 to be to guarantee its extracellular secretion, and a linkage section that comprises two amino acid (FE) is arranged between D2 and Fc.

Wherein the PCR fragment (140bp) of signal peptide is to be that template and following primer amplification get with plasmid pBLAST-hFLT-1 (Invivogen company):

Primer 1:5 '-GAGCTCGAATTCGCAACCACCATGGTCAGCTACTGGGAC-3 '

Primer 2: 5 '-ATAAATATATAGATAGTCAGATCTGGATCTTTTAATTTTGATCC

GGAACTAGATCCTGTG-3’

Wherein the PCR fragment (340bp) of D2 is to be that template and following primer amplification get with plasmid pBLAST-hFLT-1 (Invivogen company):

Primer 3:5 '-CTGACTATCTATATATTTATTAGTGATACCGGTAGACCTTTT

GTAGAGATGTAC-3’

Primer 4:5 '-GTGGGCATGTGTGAGTTTTGTCTTCGAATTGTCGATGTGT

GAGATAG-3’

Wherein the PCR fragment (724bp) of Fc is to get for template and following primer amplification from people's lymphoglandula cDNA (BD Clontech company):

Primer 5:5 '-CTATCTCACACATCGACAATTCGAAGACAAAACTCACA

CATGCCCAC-3’

Primer 6:5 '-AAGGGAATCTAGAGCGGCCGCTCATTTACCCGGAGACA

GGGAG-3’

The fusion fragment (1017bp) of D2-Fc is that the PCR fragment with above-mentioned D2 and Fc is a template then, by primer 3 and primer 6 splicing pcr amplification gained.The fusion fragment (1137bp) that comprises the D2-Fc of signal peptide at last then is that the fusion fragment with the PCR fragment of above-mentioned signal peptide and D2-Fc is a template, by primer 1 and primer 6 splicing pcr amplification gained.

The dna clone of coding YY-001 merges segmental EcoRI by the above-mentioned D2-Fc that comprises signal peptide and the NotI enzyme is cut EcoRI and the NotI enzyme point of contact gained that product (1117bp) is inserted into vector plasmid pCI-neo (Promega company).This recombinant plasmid utilizes the CMV promotor to express YY-001, and comprises the expression amount of polyadenylic acid (PolyA) sequence to guarantee that it is best of SV40.This recombinant plasmid also comprises penbritin (Ampicillin) resistant gene and is beneficial to breeding in bacterium, and Xin Meisu (Neomycin) resistant gene is to be used for the selection of stable transfection mammalian cell.Recombinant plasmid transfection E.coli (DH5 α) the back adding LB culture medium culturing of coding YY-001 is spent the night, extracted by the Qiagen plasmid extraction kit and to carry out the enzyme evaluation of cutting and check order behind the plasmid, the dna sequence dna of the coding YY-001 that is obtained is as described in the SEQ ID NO.7.

Example 2 makes up YY-002, YY-003 and YY-004 gene and recombinant vectors

YY-002, YY-003 and YY-004 are similar to YY-001, also be to merge, but they are the optimization fusion roteins that comprised G9, G12 and GS peptide linkage section before Fc respectively by VEGFR-1 the 2nd immunoglobulin-like zone (D2) and human normal immunoglobulin Fc.Their construction is to be template with the YY-001 plasmid, and the method construct by splicing PCR (Bridge PCR) gets.

The construction of YY-002 coding DNA is at first with primer 1 and 7, and primer 8 and 9 respectively pcr amplification YY-001 plasmid obtain two PCR products (460bp and 244bp), be template with them then, splice PCR gained (682bp) with primer 1 and 9.

Primer 7:5 '-CTCCACCTCCGCCACCTCCGCCTCCACCTTGTCGATGTGT

GAGATAG-3’

Primer 8:

5’-GGCGGAGGTGGCGGAGGTGGAGACAAAACTCACACATGCC-3’

Primer 9:5 '-GCTCCTCCCGCGGCTTTGTCTTGGC-3 '

AccIII and SacII enzyme are cut back (579bp) and are inserted into the AccIII and the SacII enzyme point of contact of YY-001 plasmid subsequently, the recombinant plasmid of the YY-002 that obtains encoding.Transfection E.coli (DH5 α) back obtains the recombinant plasmid of coding YY-002, and the dna sequence dna of the coding YY-002 that is obtained is as described in the SEQ ID NO.8.

Construction and the YY-002 of YY-003 are similar, just with primer 1 and 7, and behind primer 10 and the 9 difference pcr amplification YY-005 plasmids (460bp and 253bp), are template with them again, splice PCR gained (691bp) with primer 1 and 9.

Primer 10:

5’-GGCGGAGGTGGCGGAGGTGGAGGCGGTGGTGACAAAACTCA

CACATGCC-3’

AccIII and SacII enzyme are cut back (588bp) and are inserted into the AccIII and the SacII enzyme point of contact of YY-001 plasmid, the recombinant plasmid of the YY-003 that obtains encoding, and the dna sequence dna of the coding YY-003 that is obtained is as described in the SEQ ID NO.9.

Construction and the YY-002 of YY-004 are similar, just with primer 1 and 11, and behind primer 12 and the 9 difference pcr amplification YY-005 plasmids (464bp and 256bp), are template with them again, splice PCR gained (700bp) with primer 1 and 9.

Primer 11:5 '-CCGCTGCCACCGCCACCGGAGCCACCTCCACCTTGTC

GATGTGTGAGATAG-3’

Primer 12:

5’-TCCGGTGGCGGTGGCAGCGGCGGTGGCGGATCTGACAAAAC

TCACACATGCC-3’

AccIII and SacII enzyme are cut back (597bp) and are inserted into the AccIII and the SacII enzyme point of contact of YY-001 plasmid, the recombinant plasmid of the YY-004 that obtains encoding, and the dna sequence dna of the coding YY-004 that is obtained is as described in the SEQ ID NO.10.

Example 3 makes up YY-005 gene and recombinant vectors

YY-005 by the 2nd immunoglobulin-like zone (D2) of VEGFR-1, VEGFR-2 the 3rd immunoglobulin-like zone (D3) and human normal immunoglobulin Fc merge and form, the signal peptide that its N end has added VEGFR-1 to be to guarantee its extracellular secretion, and a linkage section that comprises three amino acid (GPG) is arranged between D2D3 and Fc.The DNA of coding YY-005 is that the independent PCR fragment with D2, D3 and Fc is a template, and the method construct by splicing PCR (Bridge PCR) gets.

Wherein the PCR fragment (325bp) of D2 is to be that template and following primer amplification get with plasmid pBLAST-hFLT-1 (Invivogen company):

Primer 13:5 '-CTCGAGAATTCGCAACCTCCGGAGGTAGACCTTT

CGTAGAGATG-3’

Primer 14:5 '-ACATCTATGATTGTATTGGTTTGTCGATGTGTGAGATAG-3 '

Wherein the PCR fragment (340bp) of D3 is to be that template and following primer amplification get with plasmid pBLAST-hFlk-1 (Invivogen company):

Primer 15:5 '-ACCAATACAATCATAGATGTGGTTCTGAGTCCGTCTCATG-3 '

Primer 16:5 '-TTTTGTCGCCCGGGCCCTTTTCATGGACCCTGACAAATG-3 '

Wherein the PCR fragment (712bp) of Fc be from people's lymphoglandula cDNA (BD Clontech company) with primer 17 and 6 the amplification and get:

Primer 17:

5’-AAAGGGCCCGGGCGACAAAACTCACACATGCCCACTGTGC-3’

The fusion fragment (645bp) of D2D3 is that the PCR fragment with above-mentioned D2 and D3 is a template then, by primer 13 and primer 16 splicing pcr amplification gained.The fusion fragment (1337bp) of D2D3-Fc then is that the PCR fragment with the fusion fragment of above-mentioned D2D3 and Fc is a template, by primer 13 and primer 6 splicing pcr amplification gained.

The dna clone of coding YY-005 merges segmental EcoRI by above-mentioned D2D3-Fc and the NotI enzyme is cut EcoRI and the NotI enzyme point of contact gained that product (1318bp) is inserted into vector plasmid pCI-neo (Promega company).At last following synthetic oligonucleotide directly is inserted into EcoRI and AccIII enzyme point of contact to add the signal peptide of VEGFR-1:

The forward chain:

5’-AATTCGCAACCACCATGGTCAGCTACTGGGACACCGGGGTCCT

GCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTAGTT

-3’

Reverse strand:

5’-CCGGAACTAGATCCTGTGAGAAGCAGACAGCTGAGCAGCGCGC

ACAGCAGGACCCCGGTGTCCCAGTAGCTGACCATGGTGGTTGCG-3’

Recombinant plasmid transfection E.coli (DH5 α) the back adding LB culture medium culturing of coding YY-005 is spent the night, extracted by the Qiagen plasmid extraction kit and to carry out the enzyme evaluation of cutting and check order behind the plasmid, the dna sequence dna of the coding YY-005 that is obtained is as described in the SEQ ID NO.11.

Example 4 makes up YY-006, YY-007 and YY-008 gene and recombinant vectors

YY-006, YY-007 and YY-008 are similar to YY--005, also be the 3rd immunoglobulin-like zone (D3) and human normal immunoglobulin Fc fusion, but they are the optimization fusion roteins that comprised G9, G12 and GS peptide linkage section before Fc respectively by VEGFR-1 the 2nd immunoglobulin-like zone (D2), VEGFR-2.Their construction is to be template with the YY-005 plasmid, and the method construct by splicing PCR (BridgePCR) gets.

The construction of YY-006 coding DNA is at first with primer 13 and 18, and primer 8 and 9 respectively pcr amplification YY-001 plasmid obtain two PCR products (657bp and 244bp), be template with them then, splice PCR gained (879bp) with primer 13 and 9.AccIII and SacII enzyme are cut back (852bp) and are inserted into the AccIII and the SacII enzyme point of contact of YY-005 plasmid subsequently, the recombinant plasmid of the YY-006 that obtains encoding, and the dna sequence dna of the coding YY-006 that is obtained is as described in the SEQ ID NO.12.

Primer 18:5 '-CTCCACCTCCGCCACCTCCGCCTCCACCCTTTTCATGGA

CCCTGAC-3’

Construction and the YY-006 of YY-007 are similar, just with primer 13 and 18, and behind primer 10 and the 9 difference pcr amplification YY-001 plasmids (657bp and 253bp), are template with them again, splice PCR gained (888bp) with primer 13 and 9.AccIII and SacII enzyme are cut back (861bp) and are inserted into the AccIII and the SacII enzyme point of contact of YY-005 plasmid, the recombinant plasmid of the YY-007 that obtains encoding, and the dna sequence dna of the coding YY-007 that is obtained is as described in the SEQ ID NO.13.

Construction and the YY-006 of YY-008 are similar, just with primer 13 and 19, and behind primer 12 and the 9 difference pcr amplification YY-001 plasmids (661bp and 256bp), are template with them again, splice PCR gained (897bp) with primer 13 and 9.AccIII and SacII enzyme are cut back (870bp) and are inserted into the AccIII and the SacII enzyme point of contact of YY-005 plasmid, the recombinant plasmid of the YY-008 that obtains encoding, and the dna sequence dna of the coding YY-008 that is obtained is as described in the SEQ ID NO.14.

Primer 19:

5’-CCGCTGCCACCGCCACCGGAGCCACCTCCACCCTTTTCATGG

ACCCTGAC-3’

Embodiment 2 optimizes expression and the purification of fusion rotein in cell

Multiple optimization fusion rotein is expressed justacrine in nutrient solution among the present invention in 293 cells and CHOS cell, and utilizes the method purifying gained of staphylococcal protein A,SPA affinity chromatography.

The transient expression of example 5 fusion roteins in the 293T cell

After YY-001 among the present invention builds to the YY-008 recombinant plasmid, extract the high purity plasmid DNA by the medicine box of purifying with plasmid DNA (QIAGEN company), utilize Lipofectamine 2000 plasmid transfection medicine boxs (INVITROGEN company) that this recombinant plasmid dna is imported in 293 cells (ATCC mechanism) then, in serum-free medium 293SFMII (INVITROGEN company), cultivate to collect after three days to comprise and expressed the supernatant liquor of optimizing fusion rotein.This method can be used for obtaining a spot of optimization fusion rotein, and its concentration can be determined with the ELISA standard measure.

The stably express of example 6 fusion roteins in the CHOS cell

Coding YY-001 is utilized in Lipofectamine 2000 plasmid transfection medicine boxs (Invitrogen company) the importing CHOS cells (Invitrogen company) to the reorganization high purity plasmid of YY-008, in serum-free medium CHOS SFMII (Invitrogen company), cultivate and add new enzyme element after two days, clone cultivation with limited density dilution method, the plain resistance clone of the new enzyme of picking carries out the enlarged culturing of cell and freezing collection in liquid nitrogen after about 14 days.CHOS cell behind the stable transfection can be cultivated to produce in the rotary drum culturing bottle and optimize fusion rotein, and this method can be used for obtaining a large amount of optimization fusion roteins, and its concentration can be determined with the ELISA standard measure.

The purifying of example 7 fusion roteins

The cell culture fluid that comprises fusion rotein can take the method for staphylococcal protein A,SPA affinity chromatography to carry out purifying.Albumin A-Sepharose chromatography column is washed with after the balance with the PBS damping fluid, sample on the nutrient solution that ultra-fine filter was concentrated with the speed of 2 ml/min, wash until unconjugated albumen entirely by wash-out (monitoring) with the PBS damping fluid with A280, use citric acid (PH 3) the elution of bound albumen of 100mM then, neutralize with abundant 2MIris at once.Fusion rotein behind the purifying can be determined concentration with ELISA method or A280 absorption process, and can put-20 ℃ of storages.

Embodiment 3 fusion roteins and VEGF external combines experiment

The present invention utilizes high specific and highly sensitive VEGF to detect medicine box (R﹠amp; D Systems company) determines the avidity of each fusion rotein and VEGF.In the experiment, with the YY-001 of different concns (0 to 1000pM) people VEGF to YY-008 fusion rotein and 10pM ₁₆₅(Sigma company) at room temperature mixed mutually, detects medicine box with VEGF after the overnight incubation and detects not by the free VEGF concentration of fusion rotein bonded.Original experimental result shows that at Fig. 2 resulting avidity and standard deviation thereof are displayed in Table 1 behind Prism4 software (GraphPad company) Sigmoidal nonlinear fitting.

Wherein do not have under this experiment condition can detected avidity for the disclosed fusion rotein YY-001 of prior art, and the optimization fusion rotein YY-002, YY-003, the YY-004 that comprise peptide linkage section G9, G12 and GS have shown good VEGF avidity, be approximately 1pM, improved more than at least thousand times than YY-001.In addition, the avidity of the disclosed fusion rotein YY-005 of prior art also is about 1pM, similarly comprise the optimization fusion rotein YY-006 of peptide linkage section G9, G12 and GS with it, the VEGF avidity of YY-007, YY-008 has then improved about ten times, up to 0.1pM, wherein YY-007 and YY-008 are higher especially by about 10% than the VEGF avidity of YY-006.

Embodiment 4 optimizes the growth that fusion rotein can suppress mouse tumor effectively

Xenotransplantation (Xenograft) model that the human tumor cell grows in BALB/C nude mice (Simonsen Labratories company) body is the animal tumor model the most approaching with human tumor.The present invention has utilized two kinds of human tumor cells, and HCC Hep3B liver cancer and Lovo colorectal cancer cells have been set up tumor model in nude mouse, and is used for suppressing growth of tumor with the optimization fusion rotein among the present invention as the VEGF blocker.

Example 8 preparations contain the injection of fusion rotein

The injection of optimizing fusion rotein can be prepared from by multiple formulations, usually the concentration of this fusion rotein be 0.1mg/ml to 100mg/ml, other composition includes but not limited to various carrier proteinss, buffer reagent, electrolyte ion and sustained release dosage.Wherein a kind of preferable prescription is: the optimization fusion rotein of 20mg/ml, 5mM Phosphate, 5mM Citrate, 100mM NaCl, 0.1%Tween 20,20%Sucrose, pH6.0, make according to the conventional preparation method of injection.

Example 9 is optimized fusion rotein can suppress the growth of HCC Hep3B liver cancer in nude mice effectively

Select well-grown BALB/C nude mice in this experiment, every in back subcutaneous injection 2 * 10 ⁶HCC Hep3B liver cancer cell, 0.05ml carries various fusion roteins at tail vein injection, and consumption is 2mg/kg, and twice until the 7th week weekly, the human normal immunoglobulin Fc of control group injection same dose.Tumor size is pressed W * (L) weekly ²/ 2 method is measured once, and experimental result as shown in Figure 3.The disclosed YY-001 of prior art is the same with the Fc control group not to have restraining effect to growth of tumor, and this result does not have observable VEGF avidity consistent with YY-001.The optimization fusion rotein YY-003 that is similar to YY-001 has then shown significant antitumous effect, simultaneously, prior art is disclosed, the YY-005 that has close VEGF avidity with YY-003 has also shown similar anti-cancer function, has proved that further these fusion roteins are directly proportional with their avidity to VEGF to the restraining effect of growth of tumour cell.Simultaneously, the optimization fusion rotein YY-007 that VEGF avidity provided by the present invention is the highest has shown remarkable antitumous effect, does not almost observe growth of tumor in their mouse of injection in seven weeks.

Example 10 is optimized fusion rotein can suppress the growth of Lovo large bowel cancer in nude mice effectively

This experimentation is as the same example, just at every mouse bare subcutaneous injection 5 * 10 ⁶The Lovo colorectal cancer cells.Carry various fusion roteins at tail vein injection equally, consumption is 2mg/kg, and twice until the 7th week weekly, the human normal immunoglobulin Fc of control group injection same dose.Tumor size is measured weekly once, and experimental result as shown in Figure 4.The result of the same example is similar, and these fusion roteins are directly proportional with their avidity to VEGF to the restraining effect of growth of tumour cell.YY-001 and Fc control group do not have antitumous effect, and YY-003 and YY-005 have shown good anticancer effect, and YY-007 has then shown optimum anticancer effect, have suppressed tumour cell fully in the intravital growth of mouse.

Embodiment 5 expresses with adeno-associated virus AAV and optimizes fusion rotein and medical applications thereof

Adeno-associated virus AAV in vivo itself not reproducible, can infect many in human body cells, and long-term expression recombinant protein in vivo, therefore purposes is widely arranged in gene therapy.The adeno-associated virus of superiorization of application table fusion rotein of the present invention has been obtained good therapeutic action in the mouse model CNV of retinal vasculopathy AMD.

The structure of example 11 reorganization AAV carriers

Expressing the adeno-associated virus AAV2 that optimizes fusion rotein YY-007 is to utilize AAV Helper-free medicine box (Invitrogen company) to build and get, this medicine box comprises three kinds of plasmids, pAAV-MCS, pAAV-RC and pHelper, and the AAV-293 cell, it by these three kinds of plasmids of while transfection to the AAV2 virus that obtains to recombinate of the method in the AAV-293 cell.The recombinant protein of need expressing must be cloned between polyadenylic acid (PolyA) sequence of CMV promotor among the pAAV-MCS and hGH to guarantee the expression of its best.

Behind the recombinant plasmid with EcoRI and XbaI enzyme cutting expression YY-007 among the present invention, with obtain～the 1.4kb dna fragmentation is cloned into EcoRI and the XbaI enzyme cutting site of pAAV-MCS, to obtain recombinant plasmid pAAV-YY-007, then itself and pAAV-RC, pHelper transfection have together been obtained to express the reorganization AAV2 virus of YY-007 in the AAV-293 cell.

The YY-007 that example 12 reorganization AAV2 express can suppress the generation of CNV in mouse

This experiment mouse by laser induced choroidal neovascularization CNV to set up the animal model of retinal vasculopathy AMD.After mouse is anaesthetized by vetatar (100mg/kg), with 1% mydriacyl mydriasis, then as document (Tobe et al, American Journal of Pathology, 1998,153:1641-1646) described, destroy the Bruch film with fixed attention in three different positions laser light of every eyes.Laser light coagulate (laser power 120mW, time shutter 0.1s, spot diameter 75 μ m) by slit lamp and portable condensing lens calcination to 9 points, 12 points and 3 o ' clock positions apart from an optic nerve 2-3 disc diameter DD.The bubble that the Bruch film rupture produces is an important indicator of bringing out choroidal neovascularization, so this experiment has only comprised the calcination point that produces bubble.After laser light was coagulated, the right eye of mouse was injected 5 microlitres totally 5 * 10 by vitreous space ⁹Individual AAV2-YY-007 virosome, left eye are organized in contrast and are not accepted any processing.After two weeks, mouse is condemned to death also as document (Mori et al, Investigative Ophthalomology ﹠amp; Visual Science, 2002,43 (6): 1994-2000) described method detects the degree of choroidal neovascularization, and the result is as shown in Fig. 5, and YY-007 can suppress the generation of laser induced CNV.

The YY-007 that example 13 reorganization AAV2 express can prevent the generation of CNV in mouse

The AAV2 that passes through in this experiment to recombinate expresses YY-007 in the mouse eyes after, accept laser induced choroidal neovascularization CNV again, to detect the preventive effect of YY-007 to CNV.The right eye of mouse is injected 5 microlitres totally 5 * 10 by vitreous space ⁹Individual AAV2-YY-007 virosome, left eye are organized in contrast and are not accepted any processing.After two months, as example 12, laser light is destroyed the Bruch film detects CNV after two weeks generation with fixed attention.The result is as shown in the table 2, and YY-007 has the effect of tangible prevention CNV.

In sum, the optimization fused protein that comprises the peptide linkage section provided by the present invention has improved stability of structure and greatly to the affinity of VEGF, and can block angiogenesis in vivo.In animal model, these optimize fusion rotein can suppress growth of tumor efficiently, can be used for the treatment of kinds of tumors, also can effectively treat retinal vasculopathy AMD, is possible be used for the treatment of multiple and the colleges and universities' angiogenesis inhibitors VEGF relative disease.

Sequence table

＜110〉repercussions, Tong Liqin

＜120〉comprise segmental optimization fused protein of vegf receptor and medical applications thereof

<160>14

<210>1

<211>93

<212>PRT

＜213〉artificial sequence

<400>1

Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr

1 5 10 15

Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile

20 25 30

Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly

35 40 45

Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala

50 55 60

Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly

65 70 75 80

His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr

85 90 93

<210>2

<211>102

<212>PRT

＜213〉artificial sequence

<400>2

Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys

1 5 10 15

Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp

20 25 30

Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val

35 40 45

Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu

50 55 60

Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr

65 70 75 80

Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe

85 90 95

Val Arg Val His Glu Lys

100 102

<210>3

<211>227

<212>PRT

＜213〉artificial sequence

<400>3

Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

2l0 215 220

Pro Gly Lys

225 227

<210>4

<211>9

<212>PRT

＜213〉artificial sequence

<400>4

Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 9

<210>5

<211>12

<212>PRT

＜213〉artificial sequence

<400>5

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10 12

<210>6

<211>15

<212>PRT

＜213〉artificial sequence

<400>6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>7

<211>1029

<212>DNA

＜213〉artificial sequence

<400>7

1 tccggatcaa aattaaaaga tccagatctg actatctata tatttattag tgataccggt

61 agaccttttg tagagatgta cagtgaaatc cccgaaatta tacacatgac tgaaggaagg

121 gagctcgtca ttccctgccg ggttacgtca cctaacatca ctgttacttt aaaaaagttt

181 ccacttgaca ctttgatccc tgatggaaaa cgcataatct gggacagtag aaagggcttc

241 atcatatcaa atgcaacgta caaagaaata gggcttctga cctgtgaagc aacagtcaat

301 gggcatttgt ataagacaaa ctatctcaca catcgacaat tcgaagacaa aactcacaca

361 tgcccactgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca

421 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac

481 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat

541 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc

601 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac

661 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa

721 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg

781 acctgcctag tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg

841 cagccggaga acaactacaa ggccacgcct cccgtgctgg actccgacgg ctccttcttc

901 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc

961 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg

1021 ggtaaatga

<210>8

<211>1050

<212>DNA

＜213〉artificial sequence

<400>8

1 tccggatcaa aattaaaaga tccagatctg actatctata tatttattag tgataccggt

61 agaccttttg tagagatgta cagtgaaatc cccgaaatta tacacatgac tgaaggaagg

121 gagctcgtca ttccctgccg ggttacgtca cctaacatca ctgttacttt aaaaaagttt

181 ccacttgaca ctttgatccc tgatggaaaa cgcataatct gggacagtag aaagggcttc

241 atcatatcaa atgcaacgta caaagaaata gggcttctga cctgtgaagc aacagtcaat

301 gggcatttgt ataagacaaa ctatctcaca catcgacaag gtggaggcgg aggtggcgga

361 ggtggagaca aaactcacac atgcccactg tgcccagcac ctgaactcct ggggggaccg

421 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag

481 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac

541 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc

601 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag

661 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa

721 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg

781 accaagaacc aggtcagcct gacctgccta gtcaaaggct tctatcccag cgacatcgcc

841 gtggagtggg agagcaatgg gcagccggag aacaactaca aggccacgcc tcccgtgctg

901 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag

961 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag

1021 aagagcctct ccctgtctcc gggtaaatga

<210>9

<211>1059

<212>DNA

＜213〉artificial sequence

<400>9

1 tccggatcaa aattaaaaga tccagatctg actatctata tatttattag tgataccggt

61 agaccttttg tagagatgta cagtgaaatc cccgaaatta tacacatgac tgaaggaagg

121 gagctcgtca ttccctgccg ggttacgtca cctaacatca ctgttacttt aaaaaagttt

181 ccacttgaca ctttgatccc tgatggaaaa cgcataatct gggacagtag aaagggcttc

241 atcatatcaa atgcaacgta caaagaaata gggcttctga cctgtgaagc aacagtcaat

301 gggcatttgt ataagacaaa ctatctcaca catcgacaag gtggaggcgg aggtggcgga

361 ggtggaggcg gtggtgacaa aactcacaca tgcccactgt gcccagcacc tgaactcctg

421 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg

481 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc

541 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag

601 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat

661 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc

721 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg

781 gatgagctga ccaagaacca ggtcagcctg acctgcctag tcaaaggctt ctatcccagc

841 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa ggccacgcct

901 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc

961 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac

1021 tacacgcaga agagcctctc cctgtctccg ggtaaatga

<210>10

<211>1068

<212>DNA

＜213〉artificial sequence

<400>10

1 tccggatcaa aattaaaaga tccagatctg actatctata tatttattag tgataccggt

61 agaccttttg tagagatgta cagtgaaatc cccgaaatta tacacatgac tgaaggaagg

121 gagctcgtca ttccctgccg ggttacgtca cctaacatca ctgttacttt aaaaaagttt

181 ccacttgaca ctttgatccc tgatggaaaa cgcataatct gggacagtag aaagggcttc

241 atcatatcaa atgcaacgta caaagaaata gggcttctga cctgtgaagc aacagtcaat

301 gggcatttgt ataagacaaa ctatctcaca catcgacaag gtggaggtgg ctccggtggc

361 ggtggcagcg gcggtggcgg atctgacaaa actcacacat gcccactgtg cccagcacct

421 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg

481 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag

541 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg

601 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac

661 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc

721 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc

781 ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctagt caaaggcttc

841 tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag

901 gccacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg

961 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg

1021 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga

<210>11

<211>1305

<212>DNA

＜213〉artificial sequence

<400>11

1 tccggaggta gacctttcgt agagatgtac agtgaaatcc ccgaaattat acacatgact

61 gaaggaaggg agctcgtcat tccctgccgg gttacgtcac ctaacatcac tgttacttta

121 aaaaagtttc cacttgacac tttgatccct gatggaaaac gcataatctg ggacagtaga

181 aagggcttca tcatatcaaa tgcaacgtac aaagaaatag ggcttctgac ctgtgaagca

241 acagtcaatg ggcatttgta taagacaaac tatctcacac atcgacaaac caatacaatc

301 atagatgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc

361 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct

421 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt

481 gagatgaaga aatttttgag caccttaact atagatggtg taacccggag tgaccaagga

541 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg

601 gtccatgaaa agggcccggg cgacaaaact cacacatgcc cactgtgccc agcacctgaa

661 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc

721 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc

781 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag

841 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg

901 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag

961 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca

1021 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctagtcaa aggcttctat

1081 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaaggcc

1141 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac

1201 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac

1261 aaccactaca cgcagaagag cctctccctg tctccgggta aatga

<210>12

<211>1321

<212>DNA

＜213〉artificial sequence

<400>12

1 tccggaggta gacctttcgt agagatgtac agtgaaatcc ccgaaattat acacatgact

61 gaaggaaggg agctcgtcat tccctgccgg gttacgtcac ctaacatcac tgttacttta

121 aaaaagtttc cacttgacac tttgatccct gatggaaaac gcataatctg ggacagtaga

181 aagggcttca tcatatcaaa tgcaacgtac aaagaaatag ggcttctgac ctgtgaagca

241 acagtcaatg ggcatttgta taagacaaac tatctcacac atcgacaaac caatacaatc

301 atagatgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc

361 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct

421 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt

481 gagatgaaga aatttttgag caccttaact atagatggtg taacccggag tgaccaagga

541 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg

601 gtccatgaaa agggtggagg cggaggtggc ggaggtggag acaaaactca cacatgccca

661 ctgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc

721 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc

781 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc

841 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc

901 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc

961 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag

1021 gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc

1081 ctagtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg

1141 gagaacaact acaaggccac gcctcccgtg ctggactccg acggctcctt cttcctctac

1201 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg

1261 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa

1321 tga

<210>13

<211>1332

<212>DNA

＜213〉artificial sequence

<400>13

1 tccggaggta gacctttcgt agagatgtac agtgaaatcc ccgaaattat acacatgact

61 gaaggaaggg agctcgtcat tccctgccgg gttacgtcac ctaacatcac tgttacttta

121 aaaaagtttc cacttgacac tttgatccct gatggaaaac gcataatctg ggacagtaga

181 aagggcttca tcatatcaaa tgcaacgtac aaagaaatag ggcttctgac ctgtgaagca

241 acagtcaatg ggcatttgta taagacaaac tatctcacac atcgacaaac caatacaatc

301 atagatgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc

361 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct

421 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt

481 gagatgaaga aatttttgag caccttaact atagatggtg taacccggag tgaccaagga

541 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg

601 gtccatgaaa agggtggagg cggaggtggc ggaggtggag gcggtggtga caaaactcac

661 acatgcccac tgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc

721 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg

781 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg

841 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc

901 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc

961 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga

1021 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc

1081 ctgacctgcc tagtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat

1141 gggcagccgg agaacaacta caaggccacg cctcccgtgc tggactccga cggctccttc

1201 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca

1261 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct

1321 ccgggtaaat ga

<210>14

<211>1341

<212>DNA

＜213〉artificial sequence

<400>14

1 tccggaggta gacctttcgt agagatgtac agtgaaatcc ccgaaattat acacatgact

61 gaaggaaggg agctcgtcat tccctgccgg gttacgtcac ctaacatcac tgttacttta

121 aaaaagtttc cacttgacac tttgatccct gatggaaaac gcataatctg ggacagtaga

181 aagggcttca tcatatcaaa tgcaacgtac aaagaaatag ggcttctgac ctgtgaagca

241 acagtcaatg ggcatttgta taagacaaac tatctcacac atcgacaaac caatacaatc

301 atagatgtgg ttctgagtcc gtctcatgga attgaactat ctgttggaga aaagcttgtc

361 ttaaattgta cagcaagaac tgaactaaat gtggggattg acttcaactg ggaataccct

421 tcttcgaagc atcagcataa gaaacttgta aaccgagacc taaaaaccca gtctgggagt

481 gagatgaaga aatttttgag caccttaact atagatggtg taacccggag tgaccaagga

541 ttgtacacct gtgcagcatc cagtgggctg atgaccaaga agaacagcac atttgtcagg

601 gtccatgaaa agggtggagg tggctccggt ggcggtggca gcggcggtgg cggatctgac

661 aaaactcaca catgcccact gtgcccagca cctgaactcc tggggggacc gtcagtcttc

721 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc

781 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc

841 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt

901 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc

961 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg

1021 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac

1081 caggtcagcc tgacctgcct agtcaaaggc ttctatccca gcgacatcgc cgtggagtgg

1141 gagagcaatg ggcagccgga gaacaactac aaggccacgc ctcccgtgct ggactccgac

1201 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac

1261 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc

1321 tccctgtctc cgggtaaatg a

Claims (13)

1. a fused protein is made of different fragments and human normal immunoglobulin Fc from vegf receptor, it is characterized in that, optimizes the peptide linkage section by one between receptor fragments and the Fc and links to each other, and this peptide linkage section is selected from following group:

A.Gly Gly Gly Gly Gly Gly Gly Gly Gly is expressed as G9;

B.Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly is expressed as G12;

C.Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser is expressed as GS;

D. any one length is 4 to 30 aminoacid sequence, structural stability and biological activity that it has whippy structure and can increase fusion rotein.

2. fused protein as claimed in claim 1, wherein the receptor fragments of VEGF is selected from following group:

A.VEGFR-1 the 2nd immunoglobulin-like zone, be expressed as D2, aminoacid sequence is as described in the SEQ ID NO.1 in the sequence table;

B.VEGFR-2 the 3rd immunoglobulin-like zone, be expressed as D3, aminoacid sequence is as described in the SEQ ID NO.2 in the sequence table;

C. the immunoglobulin-like zone, extracellular that comprises all vegf receptors of VEGFR-1, VEGFR-2, VEGFR-3 and VEGFR-4.

3. fused protein as claimed in claim 1, wherein human normal immunoglobulin Fc: come from IgG1, aminoacid sequence is as described in the SEQ ID NO.3 in the sequence table.

4. fused protein as claimed in claim 1 is expressed as

YY-002：D2-G9-Fc

YY-003：D2-G12-Fc

YY-004：D2-GS-Fc

YY-006：D2D3-G9-Fc

YY-007：D2D3-G12-Fc

YY-008：D2D3-GS-Fc

Wherein

The aminoacid sequence of D2 is as described in the SEQ ID NO.1 in the sequence table;

The aminoacid sequence of D3 is as described in the SEQ ID NO.2 in the sequence table;

The aminoacid sequence of Fc is as described in the SEQ ID NO.3 in the sequence table;

G9, G12, the aminoacid sequence of GS is according to claim 1.

5. fused protein as claimed in claim 3, wherein this immunoglobulin Fc fragment is coding Fc full length sequence or part Fc sequence.

6. the recombinant DNA of the fused protein of coding claim 1-5, its dna sequence dna is seen the SEQID NO.8 in the sequence table, 9,10,12,13,14.

7. the recombinant vectors that comprises the described recombinant DNA of claim 6, this carrier is selected from plasmid, virus or dna fragmentation.

8. as the recombinant vectors in the claim 7, wherein said virus is adeno-associated virus, comprises serotype 1,2,5,6,7 and 8.

9. contain the recombinant chou that right requires 7 described recombinant vectorss, wherein host cell is eukaryotic cell or prokaryotic cell prokaryocyte, comprises the host cell of having used DHFR/MTX or GS/MSX gene amplification system.

10. the application of each described fused protein in the medicine of preparation angiogenesis inhibiting among the claim 1-5.

11. application as claim 10, the medicine of wherein said angiogenesis inhibiting is the medicine of treatment relevant diseases of angiogenesis, and described relevant diseases of angiogenesis comprises tumour, retinal vasculopathy, diabetic retinopathy, sacroiliitis, anaemia or endometrial hyperplasia.

12. a pharmaceutical composition comprises that described composition is injection, powder injection, lyophilized preparation or nasal mist as fused protein and pharmaceutically acceptable carrier as described in each among the claim 1-5.

13. pharmaceutical composition as claimed in claim 12, it is characterized in that, also comprise any or several other medicines with synergistic angiogenesis inhibiting, described composition can be treated relevant diseases of angiogenesis together with the other treatment method, described other treatment method is selected from chemotherapy, reflexotherapy, gene therapy, and described angiogenesis disease comprises tumour, retinal vasculopathy, diabetic retinopathy, sacroiliitis, anaemia or endometrial hyperplasia.

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