CN101134777A - Method for preparing humanized immune globulin capable of antagonizing vessel endothelium cell growth factor and combined uses thereof - Google Patents

Abstract

The present invention relates to biotechnology, belongs to the field of recombinant gene engineering, and is six kinds of humanized fused immunoglobulins prepared through recombinant gene engineering and cell culturing process and capable of recognizing and combining with vascular endothelial cell growth factor specifically and their application. These six kinds of fused immunoglobulins feature that each of them has immunoglobulin-like structure regions capable of being combined with VEGF in the N-end and human immunoglobulin constant region in the C-end. These fused immunoglobulins have the bioactivity of high combination with VEGF and the bioactivity of immunoglobulin constant region, and can neutralize the VEGF inside adsorbed body to antagonize and inhibit fission and proliferation of vascular endothelial cell. These fused immunoglobulins are applied as VEGF neutralizing adsorbent alone or in combination.

Description

But the preparation method of the humanized immunoglobulin (Ig) of antagonizing vessel endothelium cell growth factor and applied in any combination thereof

Technical field

The invention belongs to biotechnology-recombination engineering field.More particularly, but the present invention reported and prepare six kinds of humanized fusion immunoglobulin (Ig)s of specific recognition and combining with vascular endothelial cell somatomedin with the method for recombination engineering and cell cultures (wherein three kinds for IgM; Three kinds is IgG) and applied in any combination.These six kinds common traits that merge immunoglobulin (Ig)s be its N-end contain several can be in conjunction with the immunoglobulin-like structural area of VEGF, and the C-end contains the human normal immunoglobulin constant region.Such fusion immunoglobulin (Ig) remains with the double activity with VEGF height bonded biological activity and constant region for immunoglobulin.Such merges immunoglobulin (Ig) can bring into play the effect that antagonism suppresses the vascular endothelial cell division and proliferation by the intravital VEGF of neutralization absorption.These six kinds merge immunoglobulin (Ig) as among the VEGF and sorbent material, both can one independent the use, also can severally be used in combination, to bring into play maximum drug action.

Background technology

Blood vessel hyperplasia or new life (angiogenesis) are 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.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.EndogenousInhibitors of Angiogenesis, Cancer Research, 2005,65:3967).Wherein with vascular endothelial growth factor (vascular endothelial growth factor, VEGF) important in modulating vascular new life's effect.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 when growing to 11 to 12 days (Nature 1996 such as Carmeliet P, 380:435; Nature1996 such as Ferrara N, 380:439).

VEGF by be expressed in vascular endothelial cell on the height of acceptor combine and mediate out the mitotic division of stimulation vascular endothelial cell and outgrowth activity (Science 1989 such as Leung DW, 246:1306; Science such as Keck PJ, 1989,246:1309).The acceptor of present known vascular endothelial growth factor mainly contains three kinds: i.e. VEGFR1 (fms-liketyrosine kinase, be called for short FLT-1) (Oncogene such as Shibuya M, 1990,5:519; Science1992 such as De Vries, 255:989); (Kinase-insert Domain Receptor is called for short KDR to VEGFR2; In mouse, be called Flk-1) (Biochem Biophys Res Commun 1992 such as Terman BI, 187:1579-1586) and VEGFR3 (being called for short FLT-4).VEGF and acceptor thereof such as KDR and Flt-1 in multiple solid tumor tissue (as cancer of the stomach, liver cancer, knot, the rectum cancer, ovarian cancer, mammary cancer etc.) express positive, and its expression level height becomes high-positive correlation (FolkmanJ.Nat Med with growth of tumor with transfer and relapse, 1995,1:27; Carmel iet P.Nat Med, 2003,9:653; FerraraN.Endocrine Reviews 2004,25:581).At present, be the key areas that the research and development of the antineoplastic vascular hyperplastic inhibitory agent of target spot have become tumor pharmacother with VEGF and acceptor thereof.

It with VEGF and acceptor thereof the medicament research and development field of target spot 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 in the short vascular endothelial cell division and proliferation of vegf receptor mediation, if the drug main of such research and development various small molecules thing inhibitor such as PTK787/ZK222584 (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 such research and development comprises the antibody of various anti-VEGF or anti-vegf receptor, (Nature 1993 such as Kim KJ, 362:841 such as antisense oligonucleotide and micromolecular inhibitor; Cancer Res 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).At present more noticeable with the exploitation of the antineoplastic vascular hyperplasia medicine that is combined into purpose that suppresses VEGF and its acceptor.

Two kinds of acceptors of VEGF 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 a film inner region that contains 2 Tyrosylprotein kinases are formed.The outer immunoglobulin-like structural area of born of the same parents wherein is to keep highly bonded key position (EMBO J such as Davis-Smyth T, 1996,15:4919 of vegf receptor and its part VEGF; J Biol Chem such as Fuh G, 1998,273,11197).。The acceptor on being expressed in cytolemma, also have another kind in the body and wander about as a refugee in the vegf receptor of extracellular matrix with the soluble proteins form.Such soluble VEGF-receptor is the vegf receptor of brachymemma: promptly only contain the outer immunoglobulin-like structural area of born of the same parents, and do not contain Tyrosylprotein kinase district in transmembrane domains and the film.The same with complete VEGF membrane receptor, the soluble VEGF-receptor of brachymemma remains with and VEGF height bonded biological activity.But different with complete VEGF membrane receptor is: the soluble VEGF-receptor of brachymemma is not because it contains the tyrosine protein kinase district, so with after VEGF combines, can not mediate out that the stimulation vascular endothelial cell divides and the activity of angiogenesis.The soluble VEGF-receptor of present known brachymemma be detected in the Flt-1 acceptor (Kendall RL and Thomas KA 1993 (and Kendall RL and Thomas KA Proc Natl Acad Sci USA, 1993,90:1070).But the sFlt-1 acceptor gene expression level of wandering about as a refugee in the body is low, thus be difficult to extract with the protein that separates q.s to do further research; And solubility KDR acceptor does not see also that up till now report is arranged.

The after birth outskirt gene that the present invention then chooses vegf receptor Flt-1 and KDR is for making up target spot, and (wherein three kinds is IgM but adopt the recombination engineering and the method for cell cultures to prepare six kinds of humanized fusion immunoglobulin (Ig)s of specific recognition and combining with vascular endothelial cell somatomedin; Three kinds is IgG).These six kinds common traits that merge immunoglobulin (Ig)s be its N-end contain several can be in conjunction with the immunoglobulin-like structural area of VEGF, and the C-end contains the human normal immunoglobulin constant region.So such fusion immunoglobulin (Ig) remains with the double activity with VEGF height bonded biological activity and immunoglobulin (Ig).Such merges immunoglobulin (Ig) can bring into play the effect that antagonism suppresses the vascular endothelial cell division and proliferation by the intravital VEGF of neutralization absorption.These six kinds merge immunoglobulin (Ig) as among the VEGF and sorbent material, both can one independent the use, also can severally be used in combination, to bring into play maximum drug action.

Summary of the invention

The present invention chooses the after birth outer structure district of two the principal recipient Flt-1 of VEGF and KDR for making up target spot, adopt will the encode gene in such after birth outer structure district of the method for recombination engineering to link to each other the immunoglobulin gene expression vector that acquisition is recombinated with the constant region gene of coding human normal immunoglobulin.Give expression to the humanized fusion immunoglobulin (Ig) of such reorganization again by the method for gene transfection and cell cultures.The present invention has reported six kinds of such fusion immunoglobulin (Ig)s, and wherein three kinds is that IgM merges immunoglobulin (Ig); Three kinds are IgG fusion immunoglobulin (Ig).These six kinds such fusion immunoglobulin (Ig)s remain with and VEGF height bonded biological activity.These six kinds merge immunoglobulin (Ig) as among the VEGF and sorbent material, both can one independent the use, also can severally be used in combination, to bring into play maximum drug action.

Six kinds of code name and titles that merge immunoglobulin (Ig) of among the present invention this are summarized as table 1:

Table 1.Merge the code name and the title of immunoglobulin (Ig)

	The code name of immunoglobulin (Ig)	The title of immunoglobulin (Ig)
			1. 2. 3. 4. 5. 6.	PF1 PF2 PK1 PK2 PF1K1 PF2K2	Flt1-D123-IgM Flt1-D123-IgG KDR-D123-IgM KDR-D123-IgG Flt1-D123-IgM/KDR-D123-IgM Flt1-D123-IgG/KDR-D123-IgG

The constitutional features of six kinds of immunoglobulin (Ig)s of this among the present invention is as follows:

1。Recombination immunoglobulin PF1 (Flt1-D123-IgM) is by the 1st in the after birth outer structure district of vegf receptor Flt-1, the the 2nd and the 3rd immunoglobulin-like born of the same parents structural area (is held from N-, down with) with the fusion of recombinating mutually of the Fc section (containing CH2, CH3 district and CH4 district) of the IgM of human normal immunoglobulin.

2。Recombination immunoglobulin PF2 (Flt1-D123-IgG) is by the 1st in the after birth outer structure district of vegf receptor Flt-1, the Fc section of the IgG of the 2nd and the 3rd immunoglobulin-like born of the same parents structural area and human normal immunoglobulin (contains hinge area, CH2, and CH3 district) reorganization fusion mutually.

3。Recombination immunoglobulin PK1 (KDR-D123-IgM) is by the fusion of recombinating mutually of the Fc section (containing CH2, CH3 district and CH4 district) of the IgM of the the 1st, the 2 and the 3rd immunoglobulin-like born of the same parents structural area in the after birth outer structure district of vegf receptor KDR and human normal immunoglobulin.

4。Recombination immunoglobulin PK2 (KDR-D123-IgG) is by the fusion of recombinating mutually of the Fc section (containing hinge area, CH2 and CH3 district) of the IgG of the the 1st, the 2 and the 3rd immunoglobulin-like born of the same parents structural area in the after birth outer structure district of vegf receptor KDR and human normal immunoglobulin.

5。Recombination immunoglobulin PF1K1 (Flt1-D123-IgM/KDR-D123-IgM) is the allodiploid of recombination immunoglobulin PF1 (Flt1-D123-IgM) and recombination immunoglobulin PK1 (KDR-D123-IgM).

6。Recombination immunoglobulin PF2K2 (Flt1-D123-IgG/KDR-D123IgG) is the allodiploid of recombination immunoglobulin PF2 (Flt1-D123-IgG) and recombination immunoglobulin PK2 (KDR-D123-IgG).

Six kinds of recombination immunoglobulins of among the present invention this are all expressed production with the method for genetically engineered and cell cultures.

Expressing the host cell first-selection of producing these six kinds of immunoglobulin (Ig)s is mammalian cell (as CHO, 293 cells etc.).Protein expression production comprise three big steps: 1) will contain recombination immunoglobulin expression of gene plasmid transfection and go into mammalian cell such as Chinese hamster ovary celI; 2) screening of the evaluation of recombinant gene expression positive cell and high expressing cell strain; 3) separation and purification of fusion immune globulin.

Go into mammalian cell as the expression vector that will contain the Flt1-D123-IgM recombination and the expression vector such as the cotransfection that contain the KDR-D123-IgM recombination, but then the cell secreting, expressing after the transfection goes out three kinds of recombination immunoglobulins: i.e. Flt1-D123-IgM; KDR-D123-IgM; And the allodiploid of Flt1-D123-IgM/KDR-D123-IgM.

In like manner, the expression vector that will contain the Flt1-D123-IgG recombination is gone into mammalian cell with the expression vector cotransfection that contains the KDR-D123-IgG recombination, and then the cell after the transfection also goes out secreting, expressing three kinds of recombination immunoglobulins: i.e. Flt1-D123-IgG; KDR-D123-IgG; And the allodiploid of Flt1-D123-IgG/KDR-D123-IgG.

Another big content of the present invention merges immunoglobulin (Ig)s as among the VEGF and sorbent material with these six kinds, is used in the body and external antagonism suppresses the vascular endothelial cell division and proliferation.As previously mentioned, these the six kinds common traits that merge immunoglobulin (Ig) are outer several immunoglobulin-like structural areas of after birth that its N-end contains vegf receptor, can remain with and VEGF height bonded biological activity.But because it does not contain the tyrosine protein kinase district, so with after VEGF combines, can not mediate out that the stimulation vascular endothelial cell divides and the activity of angiogenesis.Opposite, these six kinds of recombination immunoglobulins are because its C-end all is with immunoglobulin (Ig)-Fc section, so have the part biological activity of immunoglobulin (Ig) again.The present invention's its constant section in concrete enforcement comes from people's the Immunoglobulin IgM heavy chain and the heavy chain of Immunoglobulin IgG1.IgG1 and constant section fusion rotein thereof have as the long half time in serum (14 days), are convenient to features such as detection, isolation and purification; IgM and constant section fusion rotein thereof then have can form pentaploid or hexaploid constitutional features, and the cytotoxicity of strong activating complement mediation.Recombination immunoglobulin can be by its immunoglobulin-mediated immunization (as Antibody-dependent cell-mediated with after VEGF combines

Cytotoxicity, ADCC, the cytotoxicity of antibody-dependant cell mediation, and the cytotoxicity of complement-mediated) and quicken the removing of VEGF.

Of the present invention another is characterised in that greatly these six kinds merge immunoglobulin (Ig)s as among the VEGF and sorbent material, both can one independent the use, also can severally be used in combination, to bring into play maximum drug action.As the preparation of single use, these several fusion immunoglobulin (Ig)s are not quite similar in the drug effect effect of neutralization absorption VEGF.As sorting from high to low be: then be: Flt1-D123-IgM by its action effect〉KDR-D123-IgM〉Flt1-D123-IgG〉KDR-D123-IgG.And various fusion immunoglobulin (Ig)s are used in combination, can reach the drug action that neutralization is more up hill and dale adsorbed VEGF and intervened the angiogenesis of VEGF mediation.

Particular content of the present invention is as follows:

1。The clonal expansion of the recombination of the recombination immunoglobulin in the code book invention and the structure of expression vector thereof.

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

Guarantee that recombination immunoglobulin is to need a segment signal peptide (signalpeptide) to penetrate endocytoplasmic reticulum to guide new synthetic protein in eukaryotic cell secretion expression's a big key factor, and finally be secreted into extracellular matrix.The gene of coded signal peptide can come from the vegf receptor gene, immunoglobulin gene, or genes such as other foreign genes such as IL-2.

In the after birth outer structure district of PF1 (Flt1-D123-IgM) in the code book invention and the vegf receptor Flt-1 of PF2 (Flt1-D123-IgG) the 1st, the D123 district of the 2nd and the 3rd immunoglobulin-like born of the same parents structural area can (human umbilical vein endothelial cells, HUVEC) middle clonal expansion obtains from healthy Human umbilical vein endothelial cells from adopting the RT-PCR method.The upstream and downstream primer that is used for PCR can be external synthetic with reference to the Flt-1 nucleotide sequences of having reported.

In like manner, in the after birth outer structure district of PK1 (KDR-D123-IgM) in the code book invention and the vegf receptor KDR of PK2 (KDR-D123-IgG) the 1st, the D123 district of the 2nd and the 3rd immunoglobulin-like born of the same parents structural area can (human umbilical vein endothelial cells, HUVEC) middle clonal expansion obtains from healthy Human umbilical vein endothelial cells from adopting the RT-PCR method.The upstream and downstream primer that is used for PCR can be external synthetic with reference to the KDR nucleotide sequences of having reported.

The constant section of immunoglobulin gene among 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 people's the Immunoglobulin IgM heavy chain and the heavy chain of Immunoglobulin IgG1.IgG1 and constant section fusion rotein thereof have as the long half time in serum (14 days), are convenient to features such as detection, isolation and purification; IgM and constant section fusion rotein thereof then have can form pentaploid or hexaploid constitutional features, and the cytotoxicity of strong activating complement mediation.

The structure of recombination immunoglobulin expression vector of the present invention can be divided into two portions: 1) clone the immunoglobulin-like structural area gene on the vegf receptor, and its orientation is inserted into eukaryotic expression vector, acquisition contains the carrier of vegf receptor gene, 2) clone human immunoglobulin gene's CH section, and it is linked to each other with the carrier people immunity ball that contains the vegf receptor gene, thereby acquisition contains the immunoglobulin gene expression vector of reorganization.

The clonal expansion of the immunoglobulin-like structural area gene on (1a.Flt1 being vegf receptor 1)

(human umbilical vein endothelialcells, HUVEC) middle clone obtains from Human umbilical vein endothelial cells to adopt the RT-PCR method.In clonal expansion obtain to contain the cDNA that can discern with the immunoglobulin-like structural area gene of the Flt1 that combines VEGF.The upstream and downstream primer that is used for PCR can be external synthetic with reference to the Flt1 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 expression vector again, thus the carrier of acquisition band Flt1 receptor fragments gene.

The clonal expansion of the immunoglobulin-like structural area gene on (1b.KDR being vegf receptor 2)

(human umbilical vein endothelialcells, HUVEC) middle clone obtains from Human umbilical vein endothelial cells to adopt the RT-PCR method.In clonal expansion obtain to contain the cDNA that can discern with the immunoglobulin-like structural area gene of the KDR that combines VEGF.The upstream and downstream primer that is used for PCR can be external synthetic with reference to the KDR 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 expression vector again, thus the carrier of acquisition band KDR receptor fragments gene.

1c. the clonal expansion of constant region for immunoglobulin 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 nucleotide 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 (Invi trogen), 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.

To contain again Flt1 receptor fragments gene or contain KDR receptor fragments genophore and link to each other with the expression vector reorganization that contains the constant region for immunoglobulin fragment gene, promptly obtain to be with the expression vector of recombination Flt1-D123-Ig or recombination KDR-D123-Ig.Recombination Flt1-D123-Ig and recombination KDR-D123-Ig maintain unique open reading frame (open-reading frame, ORF).The coding of this ORF originates in the signal peptide of Flt1 or KDR, ends at heavy chain immunoglobulin CH3 district.

2. the expression production of recombination immunoglobulin in mammalian cell

The expression production of recombination immunoglobulin 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 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 recombinant protein 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 the separation and Extraction recombination immunoglobulin.

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

Recombination immunoglobulin can be used 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 the separation and Extraction recombination immunoglobulin has protein-A or protein-G affinity column.As with Protein-G immune-affinity chromatography post separation and Extraction recombination immunoglobulin, 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 Protein G-affinity chromatographic column, recombination immunoglobulin 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 pH2.7, 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 of separation and Extraction can be used 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.

One big feature of recombination immunoglobulin 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 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 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, and cell should not have colour developing in the hole of contrast immunoglobulin (Ig) and add, and proves that recombination immunoglobulin maintains the biological activity of specific combination mutually with VEGF.

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

Recombination immunoglobulin 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: earlier with the recombination immunoglobulin bag by 96 hole elisa plates, 4 ℃ are spent the night, behind 1x PBS liquid rinsing and 2%BSA liquid chamber temperature sealing 1～2h, add respectively with concentration biotin-VEGF and hatch 1-2h, behind PBS liquid wash-out at 4 ℃, 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 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 of different concns combine with recombination immunoglobulin competition on being fixed on 96 orifice plates, measure the variation of coating protein and biotin-VEGF binding capacity, obtain free/bag and competed in conjunction with the biotin-VEGF curve by recombination immunoglobulin.Can infer from this curve and various recombination immunoglobulins and the external bonded activity intensity of VEGF.

5. the compound applied in any combination of recombination immunoglobulin

Several immunoglobulin (Ig) 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 to intervene the angiogenesis of VEGF mediation in the body and tumor propagation etc.Of the present invention another is characterised in that greatly these six kinds merge immunoglobulin (Ig)s as among the VEGF and sorbent material, both can one independent the use, also can severally be used in combination, to bring into play maximum drug action.As the preparation of single use, these several fusion immunoglobulin (Ig)s are not quite similar in the drug effect effect of neutralization absorption VEGF.As sorting from high to low be: then be: Flt1-D123-IgM by its action effect〉KDR-D123-IgM〉Flt1-D123-IgG〉KDR-D123-IgG.And various fusion immunoglobulin (Ig)s are used in combination, can reach the effect of neutralization absorption VEGF more up hill and dale.Therefore, merge immunoglobulin (Ig) and be used to intervene the angiogenesis of VEGF mediation and tumor proliferative and transfer etc. to bring into play maximum drug action as the blood-vessels target medicine.

5a. recombination immunoglobulin is used for inside and outside absorption and separates vegf expression positive cell and tissue

Recombination immunoglobulin 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 Flt1-D123-IgM proteoplast be organized as example, can earlier recombination immunoglobulin Flt1-D123-IgM 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.

5b. recombination immunoglobulin is used for the blood vessel chrotoplast proliferation function of external antagonism VEGF mediation

From healthy people's human umbilical vein) or extract vascular endothelial cell endotheliocyte (human umbilical vein endothelialcells, HUVEC) after, place the culture dish that contains people VEGF to cultivate in the HUVEC cell, again recombination immunoglobulin is joined respectively in 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 vascular cell multiple fission ability of the external antagonism VEGF mediation of recombination immunoglobulin.According to external antagonistic effect result, can from various recombination immunoglobulins, filter out the best conduct of antagonism VEGF effect and select new drug, become to be used for interior therapeutic vascular proliferation venereal disease (as the blood vessel hyperplasia in the antitumor zone of antagonism etc.).

Description of drawings

Fig. 1 is the structural representation of monomer Flt1-D123-IgM (3) and monomer Flt1-D123-IgG (4).

D1 wherein, D2 and D3 represent the the 1st, the 2 and the 3rd immunoglobulin-like born of the same parents structural area (from the N-end, same down) in the after birth outer structure district of vegf receptor Flt-1 respectively.The IgM-Fc section of human normal immunoglobulin contains CH2, CH3 district and CH4 district; The IgG-Fc section of human normal immunoglobulin contains hinge, CH2, and CH3 district.

Fig. 2.Structural representation for monomer KDR-D123-IgM (3) and monomer KDR-D123-IgG (4).

D1 wherein, D2 and D3 represent the the 1st, the 2 and the 3rd immunoglobulin-like born of the same parents structural area in the after birth outer structure district of vegf receptor KDR respectively.The IgM-Fc section of human normal immunoglobulin contains CH2, CH3 district and CH4 district; The IgG-Fc section of human normal immunoglobulin contains hinge, CH2, and CH3 district.

Fig. 3. be Flt1-D123-IgM and the amphiploid structural representation of KDR-D123-IgM allos.

Fig. 4. be Flt1-D123-IgG and the amphiploid structural representation of KDR-D123-IgG allos.

The specific embodiment of the invention

Embodiment one: the structure that merges the expression vector of immunoglobulin (Ig) PF1 (Flt1-D123-IgM) and fusion immunoglobulin (Ig) PF2 (Flt1-D123-IgG)

Albumen PF1 (Flt1-D123-IgM) among the present invention and the monomer structure of albumen PF2 (Flt1-D123-IgG) are as shown in Figure 1.The common trait of these two kinds of immunoglobulin monomers is that their N-section contains the outer immunoglobulin D 1 of born of the same parents of people Flt1 acceptor, D2 and D3 district; And its C-section contains Immunoglobulin IgM-Fc or IgG-Fc section.Encode these two kinds the gene that merges immunoglobulin (Ig)s the clone and to reach the structure of carrier as follows:

1. the clone of human Flt1-D123 gene fragment.

The outer immunoglobulin domain D1 of born of the same parents of human Flt1 acceptor encodes, the gene fragment of D2 and D3 adopts polymerase chain reaction (RT-PCR) method, and (human umbilical vein endothelial cells, HUVEC) middle clone obtains from healthy Human umbilical vein endothelial cells.Its concrete enforcement is as follows: extract total RNA according to a conventional method with Trizol from the HUVEC cell that obtains.Get the total RNA of 1 μ g again, join that (cumulative volume is 20 μ in the reaction solution of reversed transcriptive enzyme, comprise 5 * buffer4 μ L, Oligo (dT) primer 2 μ L and 10mmol/L dNTP 8 μ L, RNasin1 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.Again with the human Flt1-D123 gene fragment of pcr amplification.The primer of this pcr amplification is:

Upstream primer 1.fltl-NcoATG:gcg AagcttGagacc atg gtc agc tac tgg

Downstream primer 2.flt1-RD3a332:gcg GgatccTgt ttt atc ata tat atg cac

The Taq enzyme that pcr amplification is used is available from Dalian TAKARA biotech firm.The reaction cumulative volume of PCR is 50 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 ℃.Obtain the dna fragmentation of 1.0kb behind the pcr amplification

PCR product purification and enzyme are cut: get PCR product (1.0kb band) and cut with HinD III and BamH I 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, get 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 and enzyme again and are cut the transition plasmid that the evaluation back obtains to contain the Flt1D123 gene fragment.

2.IgM-Fc the structure of the expression vector of segmental clone of fragment gene and PF2 (Flt1-D123-IgM)

Length is that the IgM-Fc section cDNA (containing the CH2 district, CH2 district and CH4 district) of the human immunoglobulin of 1.1kb 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 human normal immunoglobulin IgM-Fc section is as follows:

Upstream primer: hIgM-F:-27bases-GCG GGA TCCATT GCT GAG CTG CCT CCC AAA

Downstream primer: hIgM-R1279:-27bases-CAG CAG GGT CAG TAG CAG GTG CCA GCT

The pcr amplification reaction cumulative volume is 50 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 ℃.Obtain the dna fragmentation of 1.2kb behind the pcr amplification

PCR product purification and enzyme are cut: get 10ul PCR product (1.2kb) 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 IgG1-Fc section 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 Flt1-D123 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.Enzyme is cut the plasmid (pQY-Flt1-D123-IgM) that proof contains the Flt1-D123-IgM recombination to be identified through order-checking again.Sequencing result shows that recombinant plasmid contains direction and all correct recombinant plasmid of sequence.The nucleotide sequence of recombination Flt1-D123-IgM is wherein seen sequence 1.

3.IgG1-Fc the structure of the expression vector of segmental clone of fragment gene and PF2 (Flt1-D123-IgG):

Length is that the Fc section cDNA (containing hinge area hinge, CH2 district and CH3 district) of the human immunoglobulin IgG1 of 0.7kb 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 human normal immunoglobulin IgG1-Fc section is as follows:

Upstream primer (hIgG1-F): GAG GGA TCC CCC AAA TCT TGT GAC AAA ACT reaches

Downstream primer (hIgG1-R): GTC AGA TCT TCA TTT ACC CGG AGA CAG GGA.

The pcr amplification reaction cumulative volume is 50 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 IgG1-Fc section 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 Flt1-D123 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.,, 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.Enzyme is cut the plasmid that proof contains recombination identify that through order-checking the result shows that recombinant plasmid contains direction and all correct Flt1-D123-IgG recombination of sequence again.Sequencing result shows that recombinant plasmid contains direction and all correct recombinant plasmid of sequence.The nucleotide sequence of contained recombination Flt1-D123-IgG recombination is seen appended gene order 2.

Embodiment two: the structure of the expression vector of PK1 (KDR-D123-IgM) and PK2 (KDR-D123-IgG)

The same with albumen PF2 (Flt1-D123-IgG) with aforesaid albumen PF1 (Flt1-D123-IgM), PK1 among the present invention (KDR-D123-IgM) and PK2 (KDR-D123-IgG) are also for merging immunoglobulin (Ig).The monomer structure of these two kinds of immunoglobulin (Ig)s as shown in Figure 2, its common trait is that their N-section contains the outer immunoglobulin D 1 of born of the same parents of human KDR acceptor, D2 and D3 structural area; And its C-section contains Immunoglobulin IgM-Fc or IgG-Fc section.Encode these two kinds the recombination that merges immunoglobulin (Ig)s the clone and to reach the structure of carrier as follows.

1. the clone of human KDR-D123 gene fragment.

The outer immunoglobulin domain D1 of the born of the same parents of human KDR, the gene fragment of D2 and D3 also adopts polymerase chain reaction (PCR) method clone to obtain.The primer of the PCR that pcr amplification is used is:

Upstream primer 1.KDR-ATG:gcg AagcttCc atg gag agc aag gtg

Downstream primer 2.kdrRD3a328:gcgctcgagagg ttt ttc atg gac cct

The pcr amplification reaction cumulative volume is 50 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 ℃.Obtain the dna fragmentation of 1.0kb behind the pcr amplification.

PCR product purification and enzyme are cut: get 10ul PCR product (1.0kb) after 2% agarose electrophoresis is separated, downcut amplified fragments. and 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.Extract recombinant plasmid dna and enzyme again and cut the transition plasmid that the evaluation back obtains to contain the KDR-D123 gene fragment.

2.PK1 (KDR-D123-IgM) structure of expression vector:

Adopt the method for T4DNA ligase enzyme that the expression vector that contains human IgM-Fc fragment gene section that contains the same double digestion of human KDR-D123 gene Duan Yujing after the BamH1/NotI enzyme is cut is linked to each other.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.Through centrifugal collection bacterium, adopt alkaline lysis to extract plasmid DNA in a small amount.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.Enzyme is cut the plasmid that proof contains recombination to be identified through order-checking again.Sequencing result shows that recombinant plasmid contains direction and all correct recombinant plasmid of sequence.The nucleotide sequence of contained KDR-D123-IgM recombination is seen appended gene order 3.

3.PK1 (KDR-D123-IgG) structure of expression vector:

Adopt the method for T4DNA ligase enzyme that the expression vector that contains IgG-Fc fragment gene section that contains the same double digestion of human KDR-D123 gene Duan Yujing after the BamH1/NotI enzyme is cut is linked to each other.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.Through centrifugal collection bacterium, adopt alkaline lysis to extract plasmid DNA in a small amount.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.Enzyme is cut the plasmid that proof contains recombination to be identified through order-checking again.

Embodiment three: the expression production and the separation and purification of recombination immunoglobulin.

To have a molecular weight big in view of the recombination immunoglobulin among the present invention, contains disulfide linkage S_S and glycosyl, its express with at mammalian cell for well.In the invention process, the expression production in mammalian cell is example with immunoglobulin (Ig) KDR-D123-IgM and immunoglobulin (Ig) KDR-D123-IgG in choosing.The expression production of merging immunoglobulin (Ig) PF1 (Flt1-D123-IgM) and fusion immunoglobulin (Ig) PF2 (Fl t1-D123-IgG) should be routine therewith roughly the same.

Immunoglobulin (Ig) KDR-D123-IgM and the KDR-D123-IgG expression production in mammalian cell comprises three big steps: 1) will contain the expression plasmid that merges the immunoglobulin (Ig) recombination and be transfected into Chinese hamster ovary celI; 2) evaluation of the positive Chinese hamster ovary celI of recombinant gene expression and the screening of high expressing cell strain; 3) separation and purification of fusion immune globulin.

1。Expression plasmid is transfected into the recombination immunoglobulin in Chinese hamster ovary celI and the detection cell culture supernatant:

Transfection CHO 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 recombinant plasmid and negative control empty carrier DNA (the 1-2 μ g) mixing of 6 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, 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 (IgG or IgM) with the ELISA method.As measuring recombination immunoglobulin IgM with the ELISA method, its operation steps is as follows: with goat-anti people IgM-Fc monoclonal antibody (2 μ g/ml, 50 μ L/ holes, antibody is available from Sigma,) wrap 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 KDR-D123-IgM or the Chinese hamster ovary celI supernatant liquor of empty carrier transfection, 37 ℃ of 2h, PBS-0.1%tween20 washes plate * 3, and (antibody is available from Sigma to add the anti-people IgM of the goat polyclonal antibody of horseradish peroxidase-labeled again, dilution in 1: 1000), hatch 1h for 37 ℃; Wash plate * 3 with PBS-0.1%tween20, add colour developing liquid (O-Phenylene Diamine)-3%H202 then, room temperature 10min, 1mol/L HCL termination reaction, measure with microplate reader (Microplate reader 550, Bio-Rad) light absorption value in mensuration each hole, 490nm wavelength place with the enzyme linked immunological instrument.

2。The separation of recombination immunoglobulin KDR-D123-IgM is purified

The separation of recombination immunoglobulin KDR-D123-IgM is purified and is adopted immune affinity chromatographic column method separation and Extraction from the transfectional cell nutrient solution of collecting.The chromatography column weighting material that adopts is through the crosslinked grain pearl (antibody grain pearl is available from Sigma) of goat-anti people IgM polyclonal antibody.Its operation steps of separating purification KDR-D123-IgM with this immune affinity chromatographic column is as follows: get the cell culture supernatant 50ml that contains recombination immunoglobulin of collection, through centrifugal and with 0.45 μ m membrane filtration after, filtrate is splined on the 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 (pH2.7) the liquid wash-out of 2ml0.1M again; Again through regulating the recombination immunoglobulin KDR-D123-IgM that promptly obtains purifying after pH to 7.0 reaches the PBS dialysis.

3。Recombination immunoglobulin IgM physics and chemistry activity identification

The recombination immunoglobulin KDR-D123-IgM that above-mentioned separation is purified can use the SDS-polyacrylamide gel electrophoresis, and (polyacrylamide gel electrophoresis, PAGE), binding immunoassay trace test method is identified.Its operation steps is as follows: the recombination immunoglobulin KDR-D123-IgM 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-people IgM of horseradish peroxidase (HRP) mark-goat polyclonal antibody (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.The deducibility as a result of the acute western blot test of root separates the molecular weight of the recombination immunoglobulin KDR-D123-IgM that purifies, indexs such as polyploid number and immune globulin activity.

Embodiment four: the expression production of immunoglobulin (Ig) allos amphiploid (Flt1-D123-IgM/KDR-D123-IgM).

The expression production of the immunoglobulin (Ig) allos amphiploid (Flt1-D123-IgM/KDR-D123-IgM) among the present invention obtains after can going into Chinese hamster ovary celI by expression plasmid that will contain Flt1-D123-IgM and the expression plasmid cotransfection that contains KDR-D123-IgM.But the cell secreting, expressing of two kinds of expression plasmid cotransfections goes out the recombination immunoglobulin of three kinds of various combinations: i.e. Flt1-D123-IgM/Flt1-D123-IgM duploid; The KDR-D123-IgM/KDR-D123-IgM duploid; And Flt1-D123-IgM/KDR-D123-IgM allos amphiploid.The same with duploid, the albumen sepn of allos amphiploid (Flt1-D123-IgM/KDR-D123-IgM) is purified and is also adopted immune affinity chromatographic column method separation and Extraction from the transfectional cell nutrient solution of collecting.

In like manner, Flt1-D123-IgG among the present invention and KDR-D123-IgG such as cotransfection are gone into cell (as Chinese hamster ovary celI), but then the cell after the transfection also secreting, expressing go out other three kinds of recombination immunoglobulins: i.e. Flt1-D123-IgG/Flt1-D123-IgG duploid; The KDR-D123-IgG/KDR-D123-IgG duploid; And Flt1-D123-IgM/KDR-D123-IgM allos amphiploid.

The same with duploid, allos amphiploid (Flt1-D123-IgM/KDR-D123-IgM) and allos amphiploid (Flt1-D123-IgM/KDR-D123-IgM) can be used as VEGF in be used to separate absorption VEGF molecule and VEGF secreting, expressing positive cells and tissue with sorbent material; And be used for the new life etc. that the inside and outside antagonism suppresses the tumor region blood vessel of VEGF mediation.

Attached: sequence table

Sequence table

＜110〉Shanghai Stainwei Biotechnology Inc

＜120〉but the preparation method of the humanized immunoglobulin (Ig) of antagonizing vessel endothelium cell growth factor and applied in any combination thereof

<160>3

<170>PatantIn Vesion 3.3

<210>1

<211>2117

<212>DNA

＜213〉artificial sequence

<400>1

ccatggtcag ctactgggac accggggtcc tgctgtgcgc gctgctcagc tgtctgcttc 60

tcacaggatc tagttcaggt tcaaaattaa aagatcctga actgagttta aaaggcaccc 120

agcacatcat gcaagcaggc cagacactgc atctccaatg caggggggaa gcagcccata 180

aatggtcttt gcctgaaatg gtgagtaagg aaagcgaaag gctgagcata actaaatctg 240

cctgtggaag aaatggcaaa caattctgca gtactttaac cttgaacaca gctcaagcaa 300

accacactgg cttctacagc tgcaaatatc tagctgtacc tacttcaaag aagaaggaaa 360

cagaatctgc aatctatata tttattagtg atacaggtag acctttcgta gagatgtaca 420

gtgaaatccc cgaaattata cacatgactg aaggaaggga gctcgtcatt ccctgccggg 480

ttacgtcacc taacatcact gttactttaa aaaagtttcc acttgacact ttgatccctg 540

atggaaaacg cataatctgg gacagtagaa agggcttcat catatcaaat gcaacgtaca 600

aagaaatagg gcttctgacc tgtgaagcaa cagtcaatgg gcatttgtat aagacaaact 660

atctcacaca tcgacaaacc aatacaatca tagatgtcca aataagcaca ccacgcccag 720

tcaaattact tagaggccat actcttgtcc tcaattgtac tgctaccact cccttgaaca 780

cgagagttca aatgacctgg agttaccctg atgaaaaaaa taagagagct tccgtaaggc 840

gacgaattga ccaaagcaat tcccatgcca acatattcta cagtgttctt actattgaca 900

aaatgcagaa caaagacaaa ggactttata cttgtcgtgt aaggagtgga ccatcattca 960

aatctgttaa cacctcagtg catatatatg ataaagcagg atccattgct gagctgcctc 1020

ccaaagtgag cgtcttcgtc ccaccccgcg acggcttctt cggcaacccc cgcagcaagt 1080

ccaagctcat ctgccaggcc acgggtttca gtccccggca gattcaggtg tcctggctgc 1140

gcgaggggaa gcaggtgggg tctggcgtca ccacggacca ggtgcaggct gaggccaaag 1200

agtctgggcc cacgacctac aaggtgacca gcacactgac catcaaagag agcgactggc 1260

tcagccagag catgttcacc tgccgcgtgg atcacagggg cctgaccttc cagcagaatg 1320

cgtcctccat gtgtgtcccc gatcaagaca cagccatccg ggtcttcgcc atccccccat 1380

cctttgccag catcttcctc accaagtcca ccaagttgac ctgcctggtc acagacctga 1440

ccacctatga cagcgtgacc atctcctgga cccgccagaa tggcgaagct gtgaaaaccc 1500

acaccaacat ctccgagagc caccccaatg ccactttcag cgccgtgggt gaggccagca 1560

tctgcgagga tgactggaat tccggggaga ggttcacgtg caccgtgacc cacacagacc 1620

tgccctcgcc actgaagcag accatctccc ggcccaaggg ggtggccctg cacaggcccg 1680

atgtctactt gctgccacca gcccgggagc agctgaacct gcgggagtcg gccaccatca 1740

cgtgcctggt gacgggcttc tctcccgcgg acgtcttcgt gcagtggatg cagagggggc 1800

agcccttgtc cccggagaag tatgtgacca gcgccccaat gcctgagccc caggccccag 1860

gccggtactt cgcccacagc atcctgaccg tgtccgaaga ggaatggaac acgggggaga 1920

cctacacctg cgtggtggcc catgaggccc tgcccaacag ggtcaccgag aggaccgtgg 1980

acaagtccac cgagggggag gtgagcgccg acgaggaggg ctttgagaac ctgtgggcca 2040

ccgcctccac cttcatcgtc ctcttcctcc tgagcctctt ctacagtacc accgtcacct 2100

tgttcaaggt gaaatga 2117

<210>2

<211>1703

<212>DNA

＜213〉artificial sequence

<400>2

ccatggtcag ctactgggac accggggtcc tgctgtgcgc gctgctcagc tgtctgcttc 60

tcacaggatc tagttcaggt tcaaaattaa aagatcctga actgagttta aaaggcaccc 120

agcacatcat gcaagcaggc cagacactgc atctccaatg caggggggaa gcagcccata 180

aatggtcttt gcctgaaatg gtgagtaagg aaagcgaaag gctgagcata actaaatctg 240

cctgtggaag aaatggcaaa caattctgca gtactttaac cttgaacaca gctcaagcaa 300

accacactgg cttctacagc tgcaaatatc tagctgtacc tacttcaaag aagaaggaaa 360

cagaatctgc aatctatata tttattagtg atacaggtag acctttcgta gagatgtaca 420

gtgaaatccc cgaaattata cacatgactg aaggaaggga gctcgtcatt ccctgccggg 480

ttacgtcacc taacatcact gttactttaa aaaagtttcc acttgacact ttgatccctg 540

atggaaaacg cataatctgg gacagtagaa agggcttcat catatcaaat gcaacgtaca 600

aagaaatagg gcttctgacc tgtgaagcaa cagtcaatgg gcatttgtat aagacaaact 660

atctcacaca tcgacaaacc aatacaatca tagatgtcca aataagcaca ccacgcccag 720

tcaaattact tagaggccat actcttgtcc tcaattgtac tgctaccact ccct tgaaca 780

cgagagttca aatgacctgg agttaccctg atgaaaaaaa taagagagct tccgtaaggc 840

gacgaattga ccaaagcaat tcccatgcca acatattcta cagtgttctt actattgaca 900

aaatgcagaa caaagacaaa ggactttata cttgtcgtgt aaggagtgga ccatcattca 960

aatctgttaa cacctcagtg catatatatg ataaagcagg atccaagccc aaatcttgtg 1020

acaaaac tcacacatgccca ccgtgcccag cacctgaact cctgggggga ccgtcagtct 1080

tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacat 1140

gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg 1200

gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtacc 1260

gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag gagtacaagt 1320

gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag 1380

ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga 1440

accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt 1500

gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg 1560

acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg cagcagggga 1620

acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc 1680

tctccctgtc tcccgggaaa tga 1703

<210>3

<211>2103

<212>DNA

＜213〉artificial sequence

<400>3

atgcagagca aggtgctgct ggccgtcgcc ctgtggctct gcgtggagac ccgggccgcc 60

tctgtgggtt tgcctagtgt ttctcttgat ctgcccaggc tcagcataca aaaagacata 120

cttacaatta aggctaatac aactcttcaa attacttgca ggggacagag ggacttggac 180

tggctttggc ccaataatca gagtggcagt gagcaaaggg tggaggtgac tgagtgcagc 240

gatggcctct tctgtaagac actcacaatt ccaaaagtga tcggaaatga cactggagcc 300

tacaagtgct tctaccggga aactgacttg gcctcggtca tttatgtcta tgttcaagat 360

tacagatctc catttattgc ttctgttagt gaccaacatg gagtcgtgta cattactgag 420

aacaaaaaca aaactgtggt gattccatgt ctcgggtcca tttcaaatct caacgtgtca 480

ctttgtgcaa gatacccaga aaagagattt gttcctgatg gtaacagaat ttcctgggac 540

agcaagaagg gctttactat tcccagctac atgatcagct atgctggcat ggtcttctgt 600

gaagcaaaaa ttaatgatga aagttaccag tctattatgt acatagttgt cgttgtaggg 660

tataggattt atgatgtggt tctgagtccg tctcatggaa ttgaactatc tgttggagaa 720

aagcttgtct taaattgtac agcaagaact gaactaaatg tggggattga cttcaactgg 780

gaataccctt cttcgaagca tcagcataag aaacttgtaa accgagacct aaaaacccag 840

tctgggagtg agatgaagaa atttttgagc accttaacta tagatggtgt aacccggagt 900

gaccaaggat tgtacacctg tgcagcatcc agtgggctga tgaccaagaa gaacagcaca 960

tttgtcaggg tccatgaaaa acctggatcc attgc tgagctgcctcccaa agtgagcgtc 1020

ttcgtcccac cccgcgacgg cttcttcggc aacccccgca gcaagtccaa gctcatctgc 1080

caggccacgg gtttcagtcc ccggcagatt caggtgtcct ggctgcgcga ggggaagcag 1140

gtggggtctg gcgtcaccac ggaccaggtg caggctgagg ccaaagagtc tgggcccacg 1200

acctacaagg tgaccagcac actgaccatc aaagagagcg actggctcag ccagagcatg 1260

ttcacctgcc gcgtggatca caggggcctg accttccagc agaatgcgtc ctccatgtgt 1320

gtccccgatc aagacacagc catccgggtc ttcgccatcc ccccatcctt tgccagcatc 1380

ttcctcacca agtccaccaa gttgacctgc ctggtcacag acctgaccac ctatgacagc 1440

gtgaccatct cctggacccg ccagaatggc gaagctgtga aaacccacac caacatctcc 1500

gagagccacc ccaatgccac tttcagcgcc gtgggtgagg ccagcatctg cgaggatgac 1560

tggaattccg gggagaggtt cacgtgcacc gtgacccaca cagacctgcc ctcgccactg 1620

aagcagacca tctcccggcc caagggggtg gccctgcaca ggcccgatgt ctacttgctg 1680

ccaccagccc gggagcagct gaacctgcgg gagtcggcca ccatcacgtg cctggtgacg 1740

ggcttctctc ccgcggacgtct tcgtgcag tggatgcaga gggggcagcc cttgtccccg 1800

gagaagtatg tgaccagcgc cccaatgcct gagccccagg ccccaggccg gtacttcgcc 1860

cacagcatcc tgaccgtgtc cgaagaggaa tggaacacgg gggagaccta cacctgcgtg 1920

gtggcccatg aggccctgcc caacagggtc accgagagga ccgtggacaa gtccaccgag 1980

ggggaggtga gcgccgacga ggagggcttt gagaacctgt gggccaccgc ctccaccttc 2040

atcgtcctct tcctcctgag cctcttctac agtaccaccg tcaccttgtt caaggtgaaa 2100

tga

Claims (6)

1. but six kinds of specific recognition and combining with vascular endothelial cell somatomedin (vascular endothelial growth factor, humanized fusion immunoglobulin (Ig) VEGF).The common trait of these six kinds of immunoglobulin (Ig)s be its N-end contain several can be in conjunction with the immunoglobulin-like structural area of VEGF, and its C-end contains the human normal immunoglobulin constant region.This immunoglobulin like protein remains with the double activity with VEGF height bonded biological activity and immunoglobulin (Ig).These six kinds code name and the titles that merge immunoglobulin (Ig) are respectively:

The code name of fusion rotein The title of fusion rotein 1.PF1 2.PF2 3.PK1 4.PK2 5.PF1K1 6.PF2K2 Flt1 D123-IgM Flt1-D123-IgG KDR-D123-IgM， KDR-D123-IgG Flt1-D123-IgM/KDR-D123-IgM Flt1-D123-IgG/KDR-D123-IgG

Immunoglobulin (Ig) PF1 (Flt1-D123-IgM) wherein and the N-of PF2 (Flt1-D123-IgG) hold the the 1st, the 2 and the 3rd immunoglobulin-like born of the same parents structural area (being Flt1-D123) in the after birth outer structure district that contains vegf receptor Flt-1; And the N-of immunoglobulin (Ig) PK1 (KDR-D123-IgM) and PK2 (KDR-D123-IgG) holds the the 1st, the 2 and the 3rd structural area (being KDR-D123) in the after birth outer structure district that contains vegf receptor KDR.And the allodiploid that immunoglobulin (Ig) PF1K1 (Flt1-D123-IgM/KDR-D123-IgM) combines with PK1 (KDR-D123-IgM) for PF1 (Flt1-D123-IgM); The allodiploid that immunoglobulin (Ig) PF2K2 (Flt1-D123-IgG/KDR-D123-IgG) combines with PK2 (KDR-D123-IgG) for PF2 (Flt1-D123-IgG).

2. method for preparing six kinds of immunoglobulin (Ig)s of claim 1, its step comprises: a) make up the expression vector that contains the recombination that Flt-1 or KDR after birth outer structure district D123 link to each other with human normal immunoglobulin constant region (the Fc-fragment gene of IgM or IgG) with polymerase chain reaction (PCR) and recombination engineering method; Such expression vector is characterised in that it has as gene order table 1 to the recombination nucleotide sequence shown in the table 3; B) expression vector is transfected into mammalian cell and evaluation, the secretion expression of detection recombination immunoglobulin in transfectional cell; C) recombination immunoglobulin is expressed 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 from the cell culture supernatant of collecting.

3. the recombination immunoglobulin 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 of claim 1, the method for VEGF and vegf expression positive cell or tissue is separated or removed in the inside and outside.

5. the described every kind of immunoglobulin (Ig) of claim 1 is as among the VEGF and sorbent material, single independent use with in and VEGF inside and outside the adsorbent.The effect size that the described several immunoglobulin (Ig)s of claim 1 adsorb the VEGF effect by its neutralization sorts from high to low and is: Flt1-D123-IgM＞KDR-D123-IgM＞Flt1-D123-IgG＞KDR-D123-IgG

6. the described various immunoglobulin (Ig)s of claim 1 are used in combination, adsorb the medicinal method of the angiogenesis of VEGF and antagonism inhibition VEGF mediation to reach neutralization more up hill and dale.

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