CN101125888A - Expression production for recombination immunoglobulin capable of specifically identifying blood vessel endothelial cell growth factor and application thereof - Google Patents

Abstract

The invention relates to a treatment of depressing vascular endothelial growth factor, VEGE and the antineoplastic vascular proliferation of receptor thereof developing into an essential way to cure tumor at present. The invention chooses the vascular endothelial growth factor and the receptor thereof as targets, and a great deal of expression in vitro of genetic engineering and cell engineering are used to produce a recombine immunoglobin that can specifically identify and combine humanization of endothelial growth factor in human vein. The recombinant immunoglobin is a potential tumor treatment medicine for antagonizing vascular proliferation.

Description

But the expression production and the application thereof of the recombination immunoglobulin of specifically identifying blood vessel endothelial cell growth factor

Technical field

The invention belongs to biotechnology-recombination engineering field.More particularly, but the present invention relates to produce a kind of specific recognition and the humanized recombination immunoglobulin that combines human vascular endothelial growth factor with the recombination engineering and the method for cell cultures.Recombination immunoglobulin of the present invention maintains and VEGF high special bonded activity.This recombinant protein has purposes widely in the clinical diagnosis of the research of blood vessel hyperplasia and tumour and treatment.

Background technology

Blood vessel hyperplasia or new life (angiogenesis) be biologically, is meant that intravital already present blood vessel (as capillary vessel and Venule) is by sprouting or the splitted mode produces the process of new blood vessel.Blood vessel hyperplasia is grown in the many normal physiological process of keeping body such as pregnant and embryonic tissue, and the More of trauma wounds closes and repairs is useful for essential.But the recent new life who discovers blood vessel also plays an important role in generation, development and the transfer process of tumour.In in early days small solid tumor and the metastatic lesion below 2mm thereof, tumour can only depend on nutrition that the dispersion of surrounding tissue provides and oxygen and grow, and this phase generally need not angiogenesis.But when tumour further increased expansion, tumour then needed by setting up the blood circulation of himself by neonatal blood vessels, to obtain the essential nutrition of its growth; The tumor propagation of this phase quick and tool infiltration and transfer ability, and (summary sees Folkman J.Nat Med, 1995,1:27 for details often to cause tumour patient death; Carmeliet P.Nat Med, 2003,9:653; Ferrara N.Endocrine Reviews, 2004,25:581; Zhai Xin, Wang Yuya, the palace is flat: Chinese pharmaceutical chemistry magazine 2006,16:60).Because angiogenesis plays a part so important in generation, development and the transfer process of tumour, so be that " the anti-angiogenic prolotherapy " of purpose sent out and become traditional operation that continues, one of antineoplaston means of the forefront after radiotherapy and the chemotherapy at present to suppress neonate tumour blood vessel.The key that the antineoplastic vascular prolotherapy is achieved then is to develop the new medicinal preparation that can suppress outgrowth efficient, the low toxicity of tumor vessel specifically.And to successfully develop out this series antineoplastic medicament, then needing has a deep understanding to the molecular cytobiology basis of angiogenesis with principle.

Biologically, intravital blood vessel can be constantly newborn with the key that increases be that the vascular endothelial cell of its liner has continuous division and proliferation and directional migration is inserted the ability of existing vascular wall.Just (promptly stimulate blood vessel hyperplasia) in the hyperplasia acceptor of vascular endothelial cell, the regulation and control of negative (promptly suppressing blood vessel hyperplasia) two broad aspect factors (Cell such as Liotta LA., 1989,64:327; Nyberg P, Xie L and Kalluri R.Endogenous Inhibitors of Angiogenesis, Cancer Research, 2005,65:3967).The regulating vascular endothelial cell outgrowth factor in front has: vascular endothelial growth factor (vascularendothelial growth factor, VEGF), Prostatropin (basic fibroblast growthfactor, bFGF), Thr6 PDGF BB (platelet-derived growth factor, PDGF) and angiogenin (angiopoietin Ang1,2) etc.And the outgrowth factor of negative regulating vascular endothelial cell has angiostatin (angiostatin), interior chalone (endostatin) and PF4 etc.And in the outgrowth factor of these regulating vascular endothelial cells, the most important (the FerraraN.VascularEndothelial Growth Factor:Basic Science and Clinical Progress.Endocrine Reviews of effect with vascular endothelial growth factor (being VEGF), 2004,25:581).In fact, VEGF is the factor of at present known strong impulse vascular endothelial cell division and proliferation.The importance of VEGF in blood vessel hyperplasia also is proved in the research of VEGF gene knockout mice: as needing only after the portion in the VEGF gene is knocked out, blood vessel among the embryo promptly can't normally form and then cause embryo's death in the time of 11 to 12 days (Nature 1996 such as Carmeliet P, 380:435; Nature 1996 such as Ferrara N, 380:439).

The full length gene of people's VEGF is 14kb, and by 8 exons, 7 introns are formed.Wherein because the different montage of its mRNA, have 6 kinds of multi-form vegf proteins in the body at least: promptly VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206 (J Biol Chem 1991 such as Tischer E, 266:11947).These multi-form VEGF all mainly are present in extracellular matrix with solubility homodimer glycoprotein form.VEGF by be expressed in endothelial cellular membrane on the height of vegf receptor combine the activity that mediates out the mitotic division of stimulation vascular endothelial cell and promote blood vessel hyperplasia (Science 1989 such as Leung DW, 246:1306; Science such as Keck PJ, 1989,246:1309).Vascular endothelial growth factor receptor is at present known to mainly contain three kinds: i.e. VEGFR1 (fms-like tyrosine kinase, FLT-1) (Oncogene such as Shibuya M, 1990,5:519; Science 1992 such as De Vries, 255:989); VEGFR2 (Kinase-insert Domain Receptor, KDR; In mouse, be called Flk-1) (BiochemBiophys Res Commun 1992 such as Terman BI, 187:1579-1586) and VEGFR3 (FLT-4).These three kinds of acceptors are structurally very similar, are I type cross-cell membrane glycoprotein, and by an extracellular region that contains 7 immunoglobulin (Ig) spline structures, a film penetrating region and contains the film inner region of Tyrosylprotein kinase and forms.In these three kinds of acceptors, the most important and special with the effect of VEGFR2/KDR/Flk-1 in the biological effect of mediation VEGF.As finding in the research of gene knockout mice: after the KDR/Flk-1 gene was knocked out, the vascular endothelial cell in the mice embryonic can't hyperplasia, thus cause blood vessel island and blood vessel can't form and mice embryonic is being grown to 8.Dead in the time of 5 to 9 days (Nature such as Shalaby F, 1995,376:62).And in the mice embryonic that the FLT-1 gene is knocked out, but its vascular endothelial cell hyperplasia also, the process that to be new outgrowth vascular endothelial cell form vessel lumen in thereafter arrangement be subjected to inhibition (Nature 1995 such as Fong GH, 376:66).

VEGF and acceptor thereof also play a part very important in the angiogenesis of tumor tissues.Existing be reported in the genetic expression that (as cancer of the stomach, liver cancer, knot, the rectum cancer, ovarian cancer, mammary cancer etc.) in the multiple solid tumor tissue can detect VEGF and receptor KDR thereof, and its gene expression dose height becomes high-positive correlation (Folkman J.NatMed with growth of tumor with transfer and relapse, 1995,1:27; Carmeliet P.Nat Med, 2003,9:653; Ferrara N.Endocrine Reviews2004,25:581).Be the emphasis that the research of the inhibitor that generates of the antineoplastic vascular of target spot has become current international tumour medicine research field at present, only just entering clinical I-III phase experimental stage by experimentation on animals at just existing at present more than ten kind of the medicine of the U.S. in research and development with vascular endothelial growth factor and acceptor thereof.Medicament research and development field at the antineoplastic vascular hyperplastic inhibitory agent, the main two big means that exist: promptly 1) searching can directly suppress the material of intrinsic protein tyrosine kinase activity in the vegf receptor, thereby reach prevention by the effect of vegf receptor in the short vascular endothelial cell division and proliferation of mediation, if the drug main of these research and development various small molecules thing inhibitor such as PTK787/ZK 222584 (Cancer Res 2000 such as Wood JM, 60:2178); 2) by suppressing combining of VEGF and its acceptor, thereby reach the effect that stops VEGF and acceptor thereof to urge the effect system blood vessel hyperplasia of vascular endothelial cell division and proliferation in mediation, the medicine of these research and development comprises the various monoclonal antibodies of anti-VEGF or anti-vegf receptor, (Nature 1993 such as Kim KJ, 362:841 such as antisense oligonucleotide and micromolecular inhibitor; CancerRes such as Presta LG, 1997,57:4593; Clinical Cancer Research such as Posey J A, 2003,9:1323).Its kind by U.S. Genentech company research and development, produce and be exactly a kind of identification and the humanized monoclonal antibody that combines VEGF in the Bevacizumab of 2004 list marketings (trade(brand)name Avastin).Avastin is by neutralization or remove that VEGF reaches the inhibition tumor vascular growth in the body, and then the effect of containment growth of tumor and transfer (Cancer Res such as Presta LG, 1997,57:4593; N Engl J Med such as Hurwitz H, 2004; 350:2335).

Except the above-mentioned I type cross-cell membrane glycoprotein receptor that is expressed on the cytolemma, also have another kind in the body and wander about as a refugee in the vegf receptor of extracellular matrix with the soluble glycoprotein form.Such soluble VEGF-receptor remains with and VEGF height bonded biological activity, but owing to do not contain the tyrosine protein kinase district, so with after VEGF combines, can not mediate out activity (the Kendall RL and Thomas KA Proc Natl Acad Sci USA of division of stimulation vascular endothelial cell and angiogenesis, 1993,90:10705).The same with the acceptor on being expressed in cytolemma, in the vegf receptor of wandering about as a refugee, participate in key position in conjunction with the VEGF part and also be existing in the outer immunoglobulin-like structural area of its after birth (EMBO J such as Davis-Smyth T, 1996,15:4919; J Biol Chem such as Fuh G, 1998,273,11197).But because the vegf receptor gene expression dose of wandering about as a refugee is low in the body, so be difficult to extraction and the protein that separates q.s to do further research.The after birth outskirt gene that the present invention then chooses vegf receptor is for making up target spot, adopt the method for genetically engineered and cell cultures produce a kind of can with the recombination immunoglobulin of VEGF specific combination.The same with the endogenic vegf receptor of wandering about as a refugee, recombination immunoglobulin of the present invention can reach the effect that suppresses vascular endothelial proliferation by combining of vegf receptor on competitive inhibition VEGF and the cytolemma.This recombinant protein has purposes widely on the clinical treatment of the research of blood vessel hyperplasia and tumour.

Summary of the invention

The present invention be with genetically engineered/cell engineering be the means Development and Production go out a kind of can with the recombination immunoglobulin of VEGF specific combination.This recombination immunoglobulin (code name S2345) contains outer several immunoglobulin-like structural areas of after birth of vegf receptor.The same with the endogenic vegf receptor of wandering about as a refugee, recombination immunoglobulin of the present invention can reach the effect that suppresses vascular endothelial proliferation by combining of vegf receptor on competitive inhibition VEGF and the cytolemma.Recombinant protein of the present invention has purposes widely as antibody sample preparation in the clinical diagnosis of the research of blood vessel hyperplasia and tumour and treatment: as 1) can be used as research reagent and be used for the expression level that VEGF is detected in the inside and outside; 2) be used for the inside and outside with sorbent material in the conduct and separate absorption VEGF molecule and VEGF secreting, expressing positive cells and tissue; 3) be used to intervene angiogenesis and tumor proliferative and the transfer etc. that VEGF mediates as the blood-vessels target medicine.

Particular content of the present invention is as follows:

L. contain can with the expression vector establishment of the recombination immunoglobulin gene (code name S2345) of VEGF specific combination.

Structure contain can with the concrete technology and the working method of the recombination immunoglobulin expression carrier of VEGF specific combination can be referring to books such as " molecular clonings ".To have a molecular weight big in view of recombination immunoglobulin, contains disulfide linkage (S_S) and glycosyl, and it is ideal with the eukaryotic cell secretion expression that it expresses production strategy.The big advantage of another of eukaryotic cell secretion expression is that the synthetic immunoglobulin (Ig) can be collected in the cell culture fluid, is convenient to reclaim separate.

Guarantee that recombination immunoglobulin S2345 is to need segment signal peptides (signal peptide) guiding the penetrable mistake of new synthetic protein E.R in eukaryotic cell secretion expression's a big key factor, and finally be secreted into extracellular matrix.The coding of signal peptide source can be from immunoglobulin gene itself, or genes such as other foreign genes such as IL-2.Then adopt the secretion expression of the signal peptide (being CD24SP) of people CD24 genes encoding in the concrete enforcement of the present invention with the guiding fusion rotein.The constant section of immunoglobulin gene of the present invention can come from IgG, IgM, the arbitrary type of IgA or IgD.The present invention's its constant section in concrete enforcement comes from the heavy chain of IgG1.IgG1 and constant section fusion rotein thereof have as the long half time in serum (14 is big), are convenient to features such as detection, isolation and purification.

The structure of recombination immunoglobulin expression vector of the present invention can be divided into two portions: 1) clone human immunoglobulin gene's CH section, and its orientation is inserted into eukaryotic expression vector, acquisition contains the carrier of human immunoglobulin gene's CH fragment gene, 2) clone immunoglobulin-like structural area gene on the vegf receptor, and it is linked to each other with human immunoglobulin gene's CH fragment gene, thereby obtain can with the recombinant gene expression vector of the immunoglobulin (Ig) S2345 of VEGF specific combination.

1a. immune globulin is from the clonal expansion of constant region fragment gene and the structure of expression vector thereof

Human immunoglobulin CH fragment gene can be cDNA or genome shape.The cDNA that contains the immunoglobulin heavy chain constant region section can adopt RT-PCR method clonal expansion from healthy human peripheral blood monocyte (PBMCs) to obtain.The upstream and downstream primer that is used for PCR can be external synthetic with reference to the cDNA last of the twelve Earthly Branches glycosides sequence of having reported.Clone the hinge area (hinge) that the CH section cDNA that obtains can include immunoglobulin (Ig), CH1 district, CH2 district and CH3 district etc. with RT-PCR.The constant region cDNA that the clone obtains handles through digestion with restriction enzyme, with the T4 ligase enzyme it is linked to each other with the expression vector of cutting processing through corresponding enzyme again, experience bacterium through being transformed into, again through screening, amplification, extract plasmid DNA and enzyme and cut evaluation, promptly obtain to contain the expression vector of immunoglobulin heavy chain constant region fragment gene.The carrier that can be used for eukaryotic cell expression constant region gene comprises as pcDNA3.1 (Invitrogen), retroviral vector pLXSN (Clonetech) carrier, adenovirus (double-stranded DNA) carrier etc.The feature of such expression vector is to comprise sequences such as eukaryotic cell expression promotor/enhanser hCMV promotor, 5 ' LTR and polyA.

1b.VEGF the clonal expansion of the immunoglobulin-like structural area gene on the acceptor

Equally, adopt the clonal expansion acquisition from healthy human peripheral blood monocyte (PBMCs) of RT-PCR method to contain to discern with the VEGFR that combines people VEGF on the cDNA of immunoglobulin-like structural area gene.The upstream and downstream primer that is used for PCR can be external synthetic with reference to the VEGFR cDNA sequence of having reported.The clone obtains the cDNA gene and handles through digestion with restriction enzyme, with the T4 ligase enzyme its orientation is inserted in the above-mentioned expression vector that contains constant region for immunoglobulin gene section again, thus the carrier of acquisition band S2345 recombination.The reorganization the S2345 gene maintain unique open reading frame (open-reading frame, ORF).The coding of this ORF originates in the signal peptide of CD24S SP, ends at heavy chain immunoglobulin CH3 district.

2. the expression production of recombination immunoglobulin S2345 in mammalian cell

The expression production of recombination immunoglobulin S2345 is being ideal in mammalian cell.Mammalian cell commonly used has CHO, COS-7, Hela, HEK-293 etc.The key factor that guarantees the high efficiency stable expression of recombination immunoglobulin in mammalian cell is 1) efficiently expression vector is transfected in the cell; 2) screen and amplify the high expressing cell strain.

Expression vector is transfected into mammalian cell (as Chinese hamster ovary celI) two kinds of main modes are arranged: be i.e. transient transfection and stable transfection.The cell of stable transfection is preferred in the invention process because of being further used for screening and the strain of amplification high expressing cell.The method bag calcium phosphate method that stable transfection is commonly used; The cationic-liposome method; Virus-mediated method and electroporation etc.

The expression of recombination immunoglobulin S2345 gene in transfectional cell can detect with the immunohistochemical methods method; Also can be by the recombination immunoglobulin in western blot test and the ELISA method detection by quantitative cell culture fluid.After confirming that after testing transfectional cell is expressed this recombination immunoglobulin, cells transfected then changes in the nutrient solution that contains screening of medicaments such as G418 and screens.The expression cell line that filters out is amplification cultivation again, and collects its culture supernatant.The nutrient solution of collecting is used for separation and Extraction recombination immunoglobulin S2345.

3. the separation and Extraction of recombination immunoglobulin S2345 and physics and chemistry thereof and biological activity are identified

Recombination immunoglobulin S2345 can use immune affinity chromatographic column method separation and Extraction from the transfectional cell nutrient solution of collecting.The immune affinity chromatographic column commonly used that is used for separation and Extraction recombination immunoglobulin S2345 has protein-A or protein-G affinity column.As with Protein-G immune-affinity chromatography post separation and Extraction recombination immunoglobulin S2345, its operation steps is as follows: the cell culture supernatant that gets collection, through centrifugal and with 0.45 μ m membrane filtration after, filtrate is gone up sample put the ProteinG-affinity chromatographic column, recombination immunoglobulin S2345 is inhaled in the post by the special affinity layer that is adsorbed in according to the non-covalent coupling connection of its heavy chain Fc section and protein-G.Layer suction post is removed foreign protein with the PBS wash-out earlier, again the albumen that is adsorbed with low pH liquid (as pH 2.7, the 0.1M glycine) wash-out.Again through regulating the recombination immunoglobulin S2345 that promptly obtains purifying after pH to 7.0 reaches the PBS dialysis.

The recombination immunoglobulin S2345 of separation and Extraction can use the SDS-polyacrylamide gel electrophoresis, and (polyacrylamide gelelectrophoresis PAGE), is identified in conjunction with methods such as coomassie brilliant blue staining or western blot test and ELISA.

The big feature of recombination immunoglobulin S2345 of the present invention is to maintain the biological activity of specific combination mutually with VEGF.A kind ofly identify that the method for recombination immunoglobulin S2345 and VEGF specific combination is as follows: be transfected into mammalian cell such as Chinese hamster ovary celI to contain the VEGF expression carrier earlier, behind the 24-48h, the Chinese hamster ovary celI of transfection is through the rinsing of 1x PBS liquid and with after fixing, add respectively and contain recombination immunoglobulin S2345 or contrast immunoglobulin (Ig), hatch 1-2h for 25 ℃, with PBS liquid wash-out, anti-human IgG-Fc the antibody that adds enzyme labelling again, hatch 1-2h for 25 ℃, again with PBS liquid wash-out. add colour developing liquid then, room temperature is observed the colour developing result to colour developing in microscopically.As there is the colour developing positive cells in the hole that adds recombination immunoglobulin S2345, and cell should not have colour developing in the hole of contrast immunoglobulin (Ig) and add, and proves that recombination immunoglobulin S2345 maintains the biological activity of specific combination mutually with VEGF.

4. the application of recombination immunoglobulin 82345

Another major part of content of the present invention is the application of recombination immunoglobulin S2345.

Recombination immunoglobulin S2345 of the present invention is because of having the activity with the VEGF specific combination, so can be widely used in the expression level that detects VEGF as the inside and outside; Be used for the inside and outside and separate absorption VEGF molecule and VEGF secreting, expressing positive cells and tissue; Be used for intervening in the body angiogenesis of VEGF mediation and tumor propagation etc.Its concrete application note is as follows:

4a. recombination immunoglobulin 82345 is used for the expression level that VEGF is detected in the inside and outside as reagent

Recombination immunoglobulin S2345 of the present invention can be used as reagent and is used for the expression level that VEGF is detected in the inside and outside.Detected VEGF molecule comprises and is present in the shla molecule of wandering about as a refugee in body fluid (as serum, blood plasma, saliva etc.), the cell culture supernatant and is expressed in albumen on the cell/tissue film.

As detection reagent, recombination immunoglobulin S2345 can exist by two kinds of principal modes underlined processing and unmarked processing.The marker that is usually used in the mark recombination immunoglobulin comprises enzyme (as horseradish peroxidase, alkaline phosphatase, glucose oxidase, microperoxisome); Fluorescein (fluorescein isothiocyanate, FITC); Vitamin H-antibiotin and Radioactive colloidal gold etc.Through the recombination immunoglobulin S2345 of mark, can be used as reagent and be directly used in the expression level that tests such as immunoenzyme groupization, immunofluorescence or immunoblotting come inside and outside detection VEGF molecule.

The recombination immunoglobulin S2345 albumen of unmarked processing also can with other the reagent of mark merge as the test kit composition, be used for the expression of vitro detection VEGF molecule.The reagent of other marks can be enzyme mark or fluorescently-labeled anti-immunoglobulin Fc section antibody, enzyme mark or fluorescently-labeled protein-A/protein-G albumen, enzyme mark or fluorescently-labeled anti-people VEGF antibody etc.Merge the composition test kit as mouse-anti people VEGF monoclonal antibody with unlabelled recombination immunoglobulin S2345 fusion rotein and enzyme labelling, detect body fluid (as serum with the sandwich ELISA method, cell suspension, cell culture supernatant) expression level of the VEGF in is an example, it is as follows that it detects step: wrap by elisa plate with recombination immunoglobulin S2345 earlier, 4 ℃ are spent the night, behind 1x PBS liquid rinsing and 2%BSA liquid chamber temperature sealing 1～2h, add sample to be checked such as serum respectively, hatch 1-2h for 37 ℃, behind PBS liquid wash-out, the mouse-anti people VEGF antibody that adds enzyme labelling again, hatch 1-2h for 37 ℃, behind the PBS liquid wash-out, add colour developing liquid, room temperature is measured the light absorption value in each hole of certain wave strong point again to colour developing with the enzyme linked immunological instrument.Compare according to the OD value and with the normal object of reference of VEGF, can estimate the VEGF level in the sample to be checked.

4b. recombination immunoglobulin S2345 is used for inside and outside absorption and separates vegf expression positive cell and tissue

Recombination immunoglobulin S2345 can be used for inside and outside absorption and separates vegf expression positive cells and tissue.As with the outer fractionation by adsorption vegf expression positive cells of S2345 proteoplast be organized as example, can earlier recombination immunoglobulin S2345 be adsorbed on that (solid phase carrier comprises as Tissue Culture Plate on the suitable solid phase carrier, Tissue Culture Dish, nitrocellulose filter etc.), add again and contain vegf expression positive cells or tissue, hatch 1-2h for 4 ℃, with 1x PBS liquid wash-out, promptly reach absorption and the purpose of separating vegf expression positive cell or structural constituent again.

4c. recombination immunoglobulin S2345 is used for the blood vessel chrotoplast proliferation function of antagonism VEGF mediation

From healthy human peripheral blood monocyte (PBMCs), extract vascular endothelial cell or Human umbilical vein endothelial cells (humanumbilical vein endothelial cells, HUVEC), place the Tissue Culture Dish that contains people VEGF to cultivate, again recombination immunoglobulin S2345 is joined respectively in vascular endothelial cell or the HUVEC cultivation by various dose (high, medium and low dosage), to add normal immunoglobulin (Ig), hatched 2-3 days for 37 ℃ as control group.Use flow cytometer and observe the situation of blood vessel chrotoplast propagation and apoptosis.The result is compared with control group, can judge the blood vessel chrotoplast multiple fission ability of recombination immunoglobulin S2345 antagonism VEGF mediation.Such antagonistic effect result can be immunoglobulin (Ig) S2345 and treats the vascular proliferation venereal disease in vivo and become (as the blood vessel hyperplasia in the antitumor zone) the experiment in vitro foundation is provided.

4d. external test recombination immunoglobulin S2345 is active with combining of VEGF

Recombination immunoglobulin S2345 and VEGF combine activity can by with ¹²⁵The VEGF of the VEGF of I mark or biotin mark measures in conjunction with experiment.As the VEGF with the biotin mark is example: wrap by 96 hole elisa plates with recombination immunoglobulin S2345 earlier, 4 ℃ are spent the night, behind 1x PBS liquid rinsing and 2%BSA liquid chamber temperature sealing 1～2h, add respectively with concentration biotin-VEGF4 ℃ and hatch 1-2h, behind PBS liquid wash-out, the Avidin that adds horseradish peroxidase (HRP) mark again, hatch 1-2h for 4 ℃, behind the PBS liquid wash-out, add colour developing liquid, room temperature is measured the light absorption value in each hole of certain wave strong point again to colour developing with the enzyme linked immunological instrument.Can measure by the VEGF amount of recombination immunoglobulin absorption on 96 orifice plates according to the OD value.

Recombination immunoglobulin S2345 combines the competition of the VEGF of biotin mark with VEGF: the VEGF of biotin mark that can fixed concentration and the free recombination immunoglobulin S2345 of different concns combine with recombination immunoglobulin S2345 competition on being fixed on 96 orifice plates, measure the variation of coating protein and biotin-VEGF binding capacity, obtain free S2345/ bag and competed in conjunction with the biotin-VEGF curve by S2345.Can infer from this curve and recombination immunoglobulin S2345 and the external bonded activity intensity of VEGF.

Description of drawings

Fig. 1 is for containing recombination immunoglobulin S2345 expression carrier

Fig. 2 is that the enzyme that contains recombination immunoglobulin S2345 expression carrier is cut the result

Fig. 3 is the content with the recombination immunoglobulin S2345 in the Chinese hamster ovary celI supernatant of ELISA detection transfection

Fig. 4 is with immunoblotting analysis of experiments recombination immunoglobulin S2345

Fig. 5 immunohistochemical methods method detection recombination immunoglobulin S2345 combines with vegf protein

The specific embodiment of the invention

Embodiment one: the structure of recombination immunoglobulin S2345 expression vector

1. the clone of human immunoglobulin constant region gene fragment.

Human immunoglobulin constant region gene section can adopt polymerase chain reaction (RT-PCR) method to clone acquisition from healthy human peripheral blood monocyte (PBMCs).Its concrete enforcement is as follows: get the healthy human peripheral blood sub-argument and go out monocyte (PBMCs), extract total RNA according to a conventional method with Trizol from the monocyte that sub-argument goes out.Get the total RNA of 1 μ g again, join that (cumulative volume is 20 μ in the reaction solution of reversed transcriptive enzyme, bag mouthful 5 * buffer, 4 μ L, Oligo (dT) primer 2 μ L and 10mmol/L dNTP8 μ L, RNasin 1 μ L, ThermoScript II 5U～10U), through 70 ℃ of sex change 10 minutes, 42 ℃ 1 hour, 42 ℃ 30 minutes, 70 ℃ 15 minutes, reverse transcription reaction stops, the synthetic cDNA that obtains.

Pcr amplification reaction: the Taq enzyme that pcr amplification is used is available from Dalian TAKARA biotech firm.The reaction cumulative volume of PCR be 50 μ l (above-mentioned reverse transcription reaction end product cDNA 2 μ L, 10 * Taq enzyme buffer, 5 μ L, on, each 2 μ L of downstream primer, 10mmol/L dNTP 1 μ L, RNasin 1 μ L, Taq enzyme 5U, deionized water 37 μ l).The reaction conditions of PCR is: behind 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 40s then, 55 ℃ of annealing 40s, 72 ℃ are extended 45s, 35 circulations, the last circulation is extended 5min for 72 ℃.

PCR product purification and enzyme are cut: get the PCR product and cut with BamH I and Bgl II enzyme, after reaction finishes, after 1% agarose electrophoresis, downcut enzyme and cut amplified fragments, again through reclaiming purifying, behind 20 μ l deionized water dissolvings, it is standby to put-20 ℃ of preservations.

The DNA reorganization connects and transforms experiences the host bacterium: the same enzyme of DNA after employing is cut above-mentioned enzyme with the method for T4DNA ligase enzyme and warp is cut external the linking to each other of expression vector pcDNA3.1 (Invitrogen) DNA of processing.Ligation system cumulative volume is 20 μ l, connects 16h under 16 ℃ of conditions, afterwards, gets 2 μ l ligation things transformed competence colibacillus host bacterium bacterium DH5a (50 μ l/ pipe) 30 minutes under 4 ℃ of conditions.After transforming end, will experience microbionation in LB/agar culture plate (containing 100 μ g/mlAmp), 37 ℃ of overnight incubation are extracted recombinant plasmid dna again and enzyme is cut evaluation.

2. the structure of recombination immunoglobulin S2345 expression vector:

Length is that the outer partial immunity sphaeroprotein spline structure district gene of human VEGFR born of the same parents of 1.7kb also adopts the RT-PCR technology to clone from people's healthy human peripheral blood monocyte (PBMCs) cDNA gene pool.The upstream and downstream primer sequence that is used for pcr amplification is as follows:

1. upstream primer: F-R1 (27mer) gcg gga tcc tct gtg ggt ttg cct agt

2. downstream primer: R-R6n (27mer) gcg ctc gag ctc tag gac tgt gag ctg

Pcr amplification reaction: the reaction cumulative volume be 50 μ L (above-mentioned reverse transcription reaction end product cDNA 2 μ L, 10 * Taq enzyme buffer, 5 μ L, on, each 2 μ L of downstream primer, 10mmol/L dNTP 1 μ L, RNasin 1 μ L L, Taq enzyme 5U, deionized water 37 μ L).The reaction conditions of PCR is: behind 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 40s then, 55 ℃ of annealing 40s, 72 ℃ are extended 30s, 35 circulations, the last circulation is extended 5min for 72 ℃.

PCR product purification and enzyme are cut: get 10ul PCR product after 2% agarose electrophoresis is separated, downcut amplified fragments, again after reclaiming purifying and BamH1/XhoI double digestion, behind 20 μ l deionized water dissolvings, it is standby to put-20 ℃ of preservations.

The DNA reorganization connects and transforms experiences the host bacterium: the outer immunoglobulin-like structural area gene of the VEGFR born of the same parents after employing is cut above-mentioned enzyme with the method for T4DNA ligase enzyme links to each other with the expression vector of handling through same double digestion (BamH1/XhoI) that contains human immunoglobulin constant region gene section.After connecting 16h under 16 ℃ of conditions, get 2 μ l ligation thing transformed competence colibacillus host bacterium bacterium DH5a.After transforming end, will experience microbionation in LB/agar culture plate (containing 100 μ g/ml Amp), 37 ℃ of overnight incubation.

The extraction and the enzyme of recombinant plasmid dna are cut evaluation: (contain 100 μ g/ml Amp) from containing the dull and stereotyped picking colony of going up of Amp screening, being inoculated in the 3ml LB substratum, 37 ℃ of joltings are spent the night.The 2nd day,, adopt alkaline lysis to extract plasmid DNA in a small amount through centrifugal collection bacterium.The plasmid DNA of extracting is cut through Xho I enzyme, or behind HinD III and the two endonuclease digestions of Not I, carries out electrophoresis again and identify.The two endonuclease digestion results of HinD III wherein and Not I are as shown in Figure 2: sample 1 and sample 2 are for containing the plasmid of S2345Ig recombination.This enzyme is cut the result and is consistent with the pQY-S2345Ig plasmid map restriction enzyme site of Fig. 1.Enzyme is cut the plasmid that proof contains the S2345Ig recombination identify that through order-checking the result shows that recombinant plasmid contains direction and all correct pQY-S2345Ig recombination of sequence again.The nucleotide sequence of contained S2345Ig recombination is seen appended gene order 2; Its encoded protein matter aminoacid sequence is seen appended gene order 3.

Embodiment two: with the recombination immunoglobulin S2345 in the RLISA detection pQY-S2345Ig cells transfected supernatant

To have a molecular weight big in view of recombination immunoglobulin S2345, contains disulfide linkage S_S and glycosyl, its express with at mammalian cell for well.Preliminary experiment confirms that Chinese hamster ovary celI does not contain endogenous VEGF genetic expression, as can be used for in-vitro transfection and express recombinant immunoglobulin (Ig) S2345.In-vitro transfection CH0 cell can adopt the method for fugen6 mediation: the Chinese hamster ovary celI in the vegetative period of taking the logarithm, after the conventional digestion of pancreatin/5mM EDTA, with 1 * 105 cells/well inoculation 6-hole plastic culture plate, 37 ℃, the 5%CO2 overnight incubation. the 2nd day, at 100 μ L serum-frees, the DMEM of antibiotic-free adds the pQY-S2345Ig recombinant plasmid and negative control empty carrier DNA (the 1-2 μ g) mixing of 6 μ Lfugen6 and different amounts, be prepared into the fugen6-DNA mixed solution, after room temperature is placed 15min, slowly drop in the Chinese hamster ovary celI, in 37 ℃, after cultivating 4-6h under the 5%CO2 condition, add 3ml DMEM-5%fcs/ hole, behind the transfection 24h, change serum-free DMEM substratum, behind 48h～72h, collect the culturing cell supernatant liquor.

The cell culture supernatant of collecting is measured recombination immunoglobulin S2345 with the ELISA method.Its operation steps is as follows: with goat anti-human igg-Fc monoclonal antibody (2 μ g/ml, 50 μ L/ holes) bag is by the 96-well elisa plate, 4 ℃ are spent the night, behind 1x PBS liquid rinsing and 2%BSA (in PBS-0.1%tween 20 liquid) room temperature sealing 1～2h, add respectively and contain the plasmid of immunoglobulin (Ig) S2345 gene or the Chinese hamster ovary celI supernatant liquor of empty carrier transfection, 37 ℃ of 2h, PBS-0.1%tween 20 washes plate * 3, add the anti-human IgG of the goat-Fc polyclonal antibody (dilution in 1: 1000) of horseradish peroxidase-labeled again, hatch 1h for 37 ℃; Wash plate * 3 with PBS-0.1%tween 20, add colour developing liquid (O-Phenylene Diamine)-3%H2O2 then, room temperature 10min, the 1mol/LHCL termination reaction, measure with microplate reader (Microplate reader 550, Bio-Rad) light absorption value (OD490) in mensuration each hole, 490nm wavelength place with the enzyme linked immunological instrument.Show (Fig. 3: with the content of the recombination immunoglobulin S2345 in the CH0 cell conditioned medium of ELISA detection transfection) as Fig. 3, the Chinese hamster ovary celI culture supernatant of pQY-S2345Ig plasmid transfection (sample 1 and sample 2) can detect VS2345Ig albumen; And the culture supernatant result of empty carrier transfection sample (sample 3) does not see positive reaction.Compare with normal immunoglobulin (Ig) object of reference (sample 4: immunoglobulin (Ig) solubility is 20 μ g/ml), the solubility of immunoglobulin (Ig) is between 2-5 μ g/ml in the cell conditioned medium liquid of pQY-S2345Ig plasmid transfection.

Embodiment three: the purifies and separates of recombination immunoglobulin S2345 and evaluation

1。The separation of recombination immunoglobulin S2345 is purified

Recombination immunoglobulin S2345 is with protein-G immune affinity chromatographic column method separation and Extraction from the transfectional cell nutrient solution of collecting.Its operation steps is as follows: what get collection contains recombination immunoglobulin S2345 cell culture supernatant 100ml, through centrifugal and with 0.45 μ m membrane filtration after, filtrate is splined on the Protein G-affinity chromatographic column.Behind the end of the sample, earlier inhale post to the PBS of chromatography column volume liquid wash-out layer with 10 times, totally 2 times, the albumen that is adsorbed with glycine (pH 2.7) the liquid wash-out of 2ml 0.1M again; Again through regulating the recombination immunoglobulin S2345 that promptly obtains purifying after pH to 7.0 reaches the PBS dialysis.

2。The physics and chemistry of recombination immunoglobulin S2345 and biological activity are identified

The recombination immunoglobulin S2345 albumen that separation is purified can be used the SDS-polyacrylamide gel electrophoresis, and (polyacrylamidegel electrophoresis, PAGE), binding immunoassay trace test method is identified.Its operation steps is as follows: the recombination immunoglobulin S2345 albumen of purifying is splined on the 10%SDS-PAGE gel, carries out electrophoretic separation.After the SDS-PAGE electrophoresis finishes, take off gel, with the protein transfer printing to pvdf membrane (30V, 4 ℃, 16h), seal with 1%BSA again, behind room temperature 1～2h, add the anti-human IgG polyclonal antibody of horseradish peroxidase (HRP) mark-goat (antibody dilutes available from Sigma at 1: 1000), hatch 1h for 37 ℃.Film after abundant rinsing, add colour developing liquid (O-Phenylene Diamine, 3%H202), after occurring to the band that develops the color, distilled water rinsing termination reaction.Western blot test result such as Fig. 4 show: in non-sex change sample, containing the proteic sample band of recombination immunoglobulin S2345 is positive, swimming band is position about 140kd in molecular mass, and this molecular weight size with the amphiploid recombination immunoglobulin S2345 that expects conforms to.The western blot test result shows that separating the recombination immunoglobulin S2345 that purifies maintains immune globulin activity, can be discerned by anti-human IgG antibody.

Embodiment four: recombination immunoglobulin S2345 is used for detecting the expression level of the VEGF of cell/tissue as reagent

Recombination immunoglobulin S2345 of the present invention is used for the expression that VEGF is detected in the inside and outside because of having the activity with the VEGF specific combination so can be used as antibody sample reagent.In an embodiment of the present invention, be the 1st antibody with recombination immunoglobulin S2345, be the 2nd antibody with the anti-human IgG polyclonal antibody of horseradish peroxidase target goat, both merge use to detect the expression of the VEGF in the cell/tissue.Its step is as follows: the Chinese hamster ovary celI in the vegetative period of taking the logarithm, and after the conventional digestion of pancreatin/5mM EDTA, with 2 * 10 ⁴Cells/well inoculation 24-hole plastic culture plate, 37 ℃, the 5%CO2 overnight incubation.The 2nd day, contain VEGF165 plasmid and control plasmid DNA (1 μ g) mixing what the DMEM of 100 μ L serum-frees added 2 μ L Fugen6 and different amounts, be prepared into the Fugen6-DNA mixed solution, after room temperature is placed 15min, slowly drop in the Chinese hamster ovary celI, after cultivating 4-6h under 37 ℃, 5%CO2 condition, add 3ml DMEM-5% foetal calf serum/hole speed that continues and cultivate.Behind transfection 48h, with 1x PBS liquid rinsing and 4 ℃ of following fixation of C HO cell 20min of 90% methyl alcohol, add recombination immunoglobulin S2345 (dilution in 1: 100 again, the 300ul/ hole), 37 ℃ hatch 1h after, with PBS liquid wash-out 3 times, the anti-human IgG polyclonal antibody of the goat (Sigma that adds horseradish peroxidase-labeled again, 1: 200 the dilution, the 300ul/ hole), 37 ℃ hatch 1h again after, with PBS liquid wash-out 3 times, (O-Phenylene Diamine-3%H2O2), room temperature 5-10min occur to color reaction, observe the colour developing result in microscopically to add colour developing liquid then.Show as Fig. 5: contain the anti-human IgG antibody of goat of recombination immunoglobulin S2345 albumen and enzyme labelling in adding after, the cell cultures hole of VEGF165 plasmid transfection has 10% cell color reaction to be positive (Fig. 5 a), and control plasmid cells transfected culture hole does not have colour developing positive cell (Fig. 5 b).Recombination immunoglobulin S2345 among the present invention of this presentation of results maintains the biological activity with the VEGF specific combination, can be used as reagent and is used for the qualitative or intracellular VEGF of detection by quantitative or wanders about as a refugee in the expression of the VEGF of extracellular matrix tissue.

In like manner, recombination immunoglobulin S2345 is also as being used to separate absorption VEGF molecule and VEGF secreting, expressing positive cells and tissue with sorbent material among the VEGF; And be used for the new life etc. that antagonism suppresses the tumor region blood vessel of VEGF mediation.

Reference

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2.Carmeliet?P.(Review).Angiogenesis?in?health?and?disease.Nat?Med，2003，9：653-660。

3.Ferrara?N.(Review).Vascular?Endothelial?Growth?Factor：Basic?Science?andClinical?Progress.Endocrine?Reviews，2004，25：581-611。

4. Zhai Xin, Wang Yuya, (summary) equalled in the palace: with vascular endothelial growth factor and acceptor thereof is the Study in progress of tumor angiogenesis inhibitors of target spot.China's pharmaceutical chemistry magazine, 2006,16:60-64.

5.Liotta?LA，Steeg?PS，Stetle-Stevenson?WJ，et?al.Cancer?metastasis?andangiogenesis：an?imbalance?of?positive?and?negative?regulation.Cell，1989，64：327-336。

6.Nyberg?P，Xie?L，and?Kalluri?R(Review).Endogenous?Inhibitors?of?AngiogenesisCancer?Research，2005，65：3967-3979。

7.Carmeliet，P，Ferreira?V，Breier?G，Pollefeyt?S，Kieckens?L，Gertsenstein?M，Fahrig?M，Vandenhoeck?A，Harpal?K，Eberhardt?C，Declercq?C，Pawl?ing?J，Moons?L，CollenD，Risau?W，and?Nagy?A.Abnormal?blood?vessel?development?and?lethality?in?embryoslacking?a?single?VEGF?allele.Nature?1996，380：435-439。

8.Ferrara，N，Carver?Moore?K，Chen?H，Dowd?M，Lu?L，O’Shea?KS，Powell?Braxton?L，Hillan?KJ，and?Moore?MW.Heterozygous?embryonic?lethality?induced?by?targetedinactivation?of?the?VEGF?gene.Nature?1996，380：439-442。

9.Tischer，E，Mitchell?R，Hartman?T，Silva?M，Gospodarowicz?D，Fiddes?JC，andAbraham?JA.The?human?gene?for?vascular?endothelial?growth?factor.Mul?tiple?proteinforms?are?encoded?through?alternative?exon?splicing.J?Biol?Chem?1991，266：11947-11954。

10.Leung，DW，Cachianes?G，Kuang?WJ，Goeddel?DV，and?Ferrara?N.Vascular?endothelialgrowth?factor?is?a?secreted?angiogenic?mitogen.Science，1989，246：1306-1309。

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12.Shibuya?M，Yamaguchi?S，Yamane?A，Ikeda?T，Tojo?A，Matsushime?H，Sato?M.Nucleotide?sequence?and?expression?of?a?novel?human?receptor-type?tyrosine?kinasegene?(flt)closely?related?to?the?fms?family.Oncogene，1990，5：519。

13.De?Vries?C，Escobedo?JA，Ueno?H，Houck?K，Ferrara?N，and?Williams?LT.The?fms-liketyrosine?kinase，a?receptor?for?vascular?endothel?ial?growth?factor.Science?1992，255：989-991。

14.Terman，BI，Dougher Vermazen M，Carrion ME，Dimitrov?D，Armellino?DC，Gospodarowicz?D，and?Bohlen?P.Identification?of?the?KDR?tyrosine kinase?as?areceptor?for?vascular?endothelial?cell?growth?factor.Biochem?Bi?ophys?Res?Commun，1992，187：1579-1586。

15.Shalaby?F，Rossant?J，Yamaguchi?T?P.Failure?of?blood?i?sland?and?vasculogenesisin?Flk-1deficient?mice.Nature，1995，376：62～66。

16.Feng?GH，Rossant?J，Gertsenstein?M.Role?of?the?Flt-1receptor?tyrosine?kinasein?regulating?the?assembly?ofvascular?endothelium.Nature，1995，376：66～68。

17.Yves?A.Muller，Bing?Li，Hans?W.Christinger，James?A.Wells，Brian?C.Cunningham，and?Abraham?M.de?Vos：Vascular?endothelial?growth?factor：Crystal?structure?andfunctional?mapping?of?the?kinase?domain?receptor?binding?site.Proc.Natl.Acad.Sci.USA，1997，94：7192-7197。

18.Wood，JM，Bold?G，Buchdunger?E，et?al.PTK787/ZK?222584，a?novel?and?potentinhibitor?of?vascular?endothelial?growth?factor?receptor?tyrosine?kinases，impairsvascular?endothelial?growth?factor-induced?responses?and?tumor?growth?after?oraladministration.Cancer?Res，200060：2178-2189。

19.Kim，KJ，Li?B，Winer?J，Armanini?M，Gillett?N，Phill?ips?HS，and?Ferrara?N.Inhibition?of?vascular?endothelial?growth?factor-induced?angiogenesis?suppressestumor?growth?in?vivo.Nature，1993，362：841-844。

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22.Hurwitz?H；Fehrenbacher?L；Novotny?W；Cartwright?T；Hainsworth?J；Heim?W；Berlin?J；Baron?A；Griffing?S；Holmgren?E；Ferrara?N；FyfeG；Rogers?B；RossR；Kabbinavar?F.Bevacizumab?plus?irinotecan，fluorouracil，and?leucovorin?formetastatic?colorectal?cancer.N?Engl?J?Med，2004；350：2335-42。

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Attached: sequence table

＜110〉Shanghai Stainwei Biotechnology Inc

＜120〉but the expression production and the application thereof of the recombination immunoglobulin of specifically identifying blood vessel endothelial cell growth factor

<160>2

<170>PatentIn?version?3.3

<210>1

<211>743

<212>PRT

＜213〉artificial sequence

<220>

<400>1

Met?gly?arg?ala?met?val?ala?arg?leu?gly?leu?gly?leu?leu?leu?leu

1 5 10 15

ala?leu?leu?leu?pro?thr?gln?ile?tyr?ser?ser?glu?thr?thr?thr?gly

20 25 30

thr?ser?ser?asn?ser?ser?gln?ser?thr?ser?asn?ser?gly?leu?ala?pro

35 40 45

asn?pro?thr?asn?ala?thr?thr?lys?gly?ser?pro?phe?ile?ala?ser?val

50 55 60

ser?asp?gln?his?gly?val?val?tyr?ile?thr?glu?asn?lys?asn?lys?thr

65 70 75 80

val?val?lle?pro?cys?leu?gly?ser?ile?ser?asn?leu?asn?val?ser?ser

85 90 95

leu?cys?ala?arg?tyr?pro?glu?lys?arg?phe?val?pro?asp?gly?asn?arg

100 105 110

ile?ser?trp?asp?ser?lys?lys?gly?phe?thr?ile?pro?ser?tyr?met?ile

115 120 125

ser?tyr?ala?gly?met?val?phe?cys?glu?ala?lys?ile?asn?asp?glu?ser

130 135 140

tyr?gln?ser?ile?met?tyr?ile?val?val?val?val?gly?tyr?arg?ile?tyr

145 150 155 160

asp?val?val?leu?ser?pro?ser?his?gly?ile?glu?leu?ser?val?gly?glu

165 170 175

lys?leu?val?leu?asn?cys?thr?ala?arg?thr?glu?leu?asn?val?gly?ile

180 185 190

asp?phe?asn?trp?glu?tyr?pro?ser?ser?lys?his?gln?his?lys?lys?leu

195 200 205

val?asn?arg?asp?leu?lys?thr?gln?ser?gly?ser?glu?met lys?lys?phe

210 215 220

leu?ser?thrleu?thr?ile?asp?gly?val?thr?arg?ser?asp?gln?gly?leu

225 230 235 240

tyr?thr?cys?ala?ala?ser?ser?gly?leu?met?thr?lys?lys?asn?ser?thr

245 250 255

phe?val?arg?val?his?glu?lys?pro?phe?val?ala?phe?gly?ser?gly?met

260 265 270

glu?ser?leu?val?glu?ala?thr?val?gly?glu?arg?val?arg?ile?pro?ala

275 280 285

lys?tyr?leu?gly?tyr?pro?pro?pro?glu?ile?lys?trp?tyr?lys?asn?gly

290 295 300

ile?pro?leu?glu?ser?asn?his?thr?ile?lys?ala?gly?his?val?leu?thr

305 310 315 320

ile?met?glu?val?ser?glu?arg?asp?thr?gly?asn?tyr?thr?val?ile?leu

325 330 335

thr?asn?pro?ile?ser?lys?glu?lys?gln?ser?his?val?val?ser?leu?val

340 345 350

val?tyr?val?pro?pro?gln?ile?gly?glu?lys?ser?leu?ile?ser?pro?val

355 360 365

asp?ser?tyr?gln?tyr?gly?thr?thr?gln?thr?leu?thr?cys?thr?val?tyr

370 375 380

ala?ile?pro?pro?pro?his?his?ile?his?trp?tyr?trp?gln?leu?glu?glu

385 390 395 400

glu?cys?ala?asn?glu?pro?ser?gln?ala?val?ser?val?thr?asn?pro?tyr

405 410 415

pro?cys?glu?glu?trp?arg?ser?val?glu?asp?phe?gln?gly?gly?asn?lys

420 425 430

ile?glu?val?asn?lys?asn?gln?phe?ala?leu?ile?glu?gly?lys?asn?lys

435 440 445

thr?val?ser?thr?leu?val?ile?gln?ala?ala?asn?val?ser?ala?leu?tyr

450 455 460

lys?cys?glu?ala?val?asn?lys?val?gly?arg?gly?glu?arg?val?ile?ser

465 470 475 480

phe?his?val?thr?arg?gly?pro?glu?ile?thr?leu?gln?pro?asp?met?gln

485 490 495

pro?thr?glu?gln?glu?ser?val?ser?leu?trp?cys?thr?ala?asp?arg?ser

500 505 510

pro?lys?ser?cys?asp?lys?thr?his?thr?cys?pro?pro?cys?pro?ala?pro

515 520 525

glu?leu?leu?gly?gly?pro?ser?val?phe?leu?phe?pro?pro?lys?pro?lys

530 535 540

asp?thr?leu?met?ile?ser?arg?thr?9ro?glu?val?thr?cys?val?val val

545 550 555 560

asp?val?ser?his?glu?asp?pro?glu?val?lys?phe?asn?trp?tyr?val?asp

565 570 575

gly?val?glu?val?his?asn?ala?lys?thr?lys?pro?arg?glu?glu?gln?tyr

580 585 590

asn?ser?thr?tyr?arg?val?val?ser?val leu?thr?val?leu?his?gln?asp

595 600 605

trp?leu?asn?gly?lys?glu?tyr?lys?cys?lys?val?ser?asn?lys?ala?leu

610 615 620

pro?ala?pro?ile?glu?lys?thr?ile?ser?lys?ala?lys?gly?gln?pro?arg

625 630 635 640

glu?pro?gln?val?tyr?thr?leu?pro?pro?ser?arg?asp?glu?leu?thr?lys

645 650 655

asn?gln?val?ser?leu?thr?cys?leu?val?lys?gly?phe?tyr?pro?ser?asp

660 665 670

ile?ala?val?glu?trp?glu?ser?asn?gly?gln?pro?glu?asn?asn?tyr?lys

675 680 685

thr?thr?pro?pro?val?leu?asp?ser?asp?gly?ser?phe?phe?leu?tyr?ser

690 695 700

lys leu?thr?val?asp?lys?ser?arg?trp?gln?gln?gly?asn?val?phe?ser

705 710 715 720

cys?ser?val?met?his?glu?ala?leu?his?asn?his?tyr?thr?gln?lys?ser

725 730 735

leu?ser?leu?ser?pro?gly?lys.

740

<210>2

<211>2157

<212>DNA

＜213〉artificial sequence

<400>2

atgggcagag?caatggtggc?caggctgggg?ctggggctgc?tgctgctggc?actgctccta 60

cccacgcaga?tttattccag?tgaaacaaca?actggaactt?caagtaactc?ctcccagagt 120

acttccaact?ctgggttggc?cccaaatcca?actaatgcca?ccaccaaggg?atctccattt 180

attgcttctg?ttagtgacca?acatggagtc?gtgtacatta?ctgagaacaa?aaacaaaact 240

gtggtgattc?catgtctcgg?gtccatttca?aatctcaacg?tgtcatctct?ttgtgcaaga 300

tacccagaaa?agagatttgt?tcctgatggt?aacagaattt?cctgggacag?caagaagggc 360

tttactattc?ccagctacat?gatcagctat?gctggcatgg?tcttctgtga?agcaaaaatt 420

aatgatgaaa?gttaccagtc?tattatgtac?atagttgtcg?ttgtagggta?taggatttat 480

gatgtggttc?tgagtccgtc?tcatggaatt?gaactatctg?ttggagaaaa?gcttgtctta 540

aattgtacag?caagaactga?actaaatgtg?gggattgact?tcaactggga?atacccttct 600

tcgaagcatc?agcataagaa?acttgtaaac?cgagacctaa?aaacccagtc?tgggagtgag 660

atgaagaaat?ttttgagcac?cttaactata?gatggtgtaa?cccggagtga?ccaaggattg 720

tacacctgtg?cagcatccag?tgggctgatg?accaagaaga?acagcacatt?tgtcagggtc 780

catgaaaaac?cttttgttgc?ttttggaagt?ggcatggaat?ctctggtgga?agccacggtg 840

ggggagcgtg?tcagaatccc?tgcgaagtac?cttggttacc?cacccccaga?aataaaatgg 900

tataaaaatg?gaatacccct?tgagtccaat?cacacaatta?aagcggggca?tgtactgacg 960

attatggaag?tgagtgaaag?agacacagga?aattacactg?tcatccttac?caatcccatt 1020

tcaaaggaga?agcagagcca?tgtggtctct?ctggttgtgt?atgtcccacc?ccagattggt 1080

gagaaatctc?taatctctcc?tgtggattcc?taccagtacg?gcaccactca?aacgctgaca 1140

tgtacggtct?atgccattcc?tcccccgcat?cacatccact?ggtattggca?gttggaggaa 1200

gagtgcgcca?acgagcccag?ccaagctgtc?tcagtgacaa?acccataccc?ttgtgaagaa 1260

tggagaagtg?tggaggactt?ccagggagga?aataaaattg?aagttaataa?aaatcaattt 1320

gctctaattg?aaggaaaaaa?caaaactgta?agtacccttg?ttatccaagc?ggcaaatgtg 1380

tcagctttgt?acaaatgtga?agcggtcaac?aaagtcggga?gaggagagag?ggtgatctcc 1440

ttccacaacc?tcgagagatc?ccccaaatct?tgtgacaaaa?ctcacacatg?cccaccgtgc 1500

ccagcacctg?aactcctggg?gggaccgtca?gtcttcctct?tccccccaaa?acccaaggac 1560

accctcatga?tctcccggac?ccctgaggtc?acatgcgtgg?tggtggacgt?gagccacgaa 1620

gaccctgagg?tcaagttcaa?ctggtacgtg?gacggcgtgg?aggtgcataa?tgccaagaca 1680

aagccgcggg?aggagcagta?caacagcacg?taccgtgtgg?tcagcgtcct?caccgtcctg 1740

caccaggact?ggctgaatgg?caaggagtac?aagtgcaagg?tctccaacaa?agccctccca 1800

gcccccatcg?agaaaaccat?ctccaaagcc?aaagggcagc?cccgagaacc?acaggtgtac 1860

accctgcccc?catcccggga?tgagctgacc?aagaaccagg?tcagcctgac?ctgcctggtc 1920

aaaggcttct?atcccagcga?catcgccgtg?gagtgggaga?gcaatgggca?gccggagaac 1980

aactacaaga?ccacgcctcc?cgtgctggac?tccgacggct?ccttcttcct?ctatagcaag 2040

ctcaccgtgg?acaagagcag?gtggcagcag?gggaacgtct?tctcatgctc?cgtgatgcat 2100

gaggctctgc?acaaccacta?cacgcagaag?agcctctccc?tgtctccggg?taaatga 2157

Claims (6)

1. but the humanized recombination immunoglobulin S2345 of specific recognition and combining with vascular endothelial cell somatomedin.It is characterized in that a) this recombination immunoglobulin has the aminoacid sequence shown in gene order table 1; B) this recombination immunoglobulin maintains the special activity that combines with VEGF, and can suppress the short vascular endothelial proliferation effect of VEGF mediation; C) this recombination immunoglobulin is produced antibody protein for the method for reorganization genetically engineered and cell cultures.

2. method for preparing the recombination immunoglobulin S2345 of claim 1; Its step comprises: a) make up with polymerase chain reaction (PCR) and recombination engineering method and contain recombination immunoglobulin S2345 expression carrier; This expression vector is characterised in that it has the recombination nucleotide sequence shown in gene order table 2; B) expression vector is transfected into mammal cell and evaluation, the secretion expression of detection recombination immunoglobulin S2345 in transfectional cell; C) recombination immunoglobulin S2345 expresses the collection of screening, amplification cultivation and the supernatant liquor of positive cell; D) with immune affinity chromatographic column method separation and Extraction recombination immunoglobulin S2345 from the cell culture supernatant of collecting.

3. the recombination immunoglobulin S2345 with claim 1 carries out in the body or the method for vitro detection vegf expression level for the test kit composition; It is characterized in that detected VEGF be soluble substance form with the degradation of wandering about as a refugee be present in body fluid (as serum, blood plasma, saliva etc.) and the cell culture supernatant or with the membranin formal representation in cell/tissue.

4. be sorbent material with the described recombination immunoglobulin S2345 of claim 1, the method for in-vitro separation or removing VEGF and vegf expression positive cell or tissue.

5. use the described recombination immunoglobulin S2345 of claim 1 as the short vascular endothelial proliferation that suppresses the VEGF mediation among the VEGF with sorbent material at external antagonism.

6. use the described recombination immunoglobulin S2345 of claim 1 as the blood vessel hyperplasia that suppresses tumor region among the VEGF in the body with sorbent material with antagonism.

Images