CN109134651A

CN109134651A - A kind of monoclonal antibody of anti-vegf and its preparation method and application

Info

Publication number: CN109134651A
Application number: CN201811466201.5A
Authority: CN
Inventors: 何虹霖; 姜伟东
Original assignee: Shanghai Fu Hong Han Lin Bio Pharmaceutical Co Ltd; Shanghai Fugrand Hilink Biotechnology Ltd By Share Ltd
Current assignee: Shanghai Fu Hong Han Lin Bio Pharmaceutical Co Ltd; Shanghai Fugrand Hilink Biotechnology Ltd By Share Ltd; Shanghai Henlius Biotech Inc
Priority date: 2018-12-03
Filing date: 2018-12-03
Publication date: 2019-01-04
Anticipated expiration: 2038-12-03
Also published as: CN109134651B

Abstract

The invention belongs to biological medicine antibody arts, more specifically, the invention discloses monoclonal antibodies of a kind of anti-vegf and its preparation method and application.Anti-X activity of the invention has good bioactivity, can effectively combine vegf protein, and can effectively close vegf protein in protein level and cellular level and combine with its receptor.The monoclonal antibody can have the good prospect of the drugs such as preparation treatment tumour, anti-autoimmune disease individually or with other anti-tumor drug use in conjunction in the diagnosis and screening of immunotherapy of tumors and tumour.

Description

A kind of monoclonal antibody of anti-vegf and its preparation method and application

Technical field

The present invention relates to biomedicine fields, and in particular to a kind of monoclonal antibody of anti-vegf and preparation method thereof and answers With.

Background technique

The generation of tumour and development and new vascular generation have it is close contact, usual tumour starts from single abnormal cell, Due to very close at a distance from using capillary bed, and dormant state can be kept in long period of time.Some Tumour cell may turn to angiogenic phenotype to activate endothelial cell, the blood capillary of the endothelial cell proliferation and maturation Cheng Xin Pipe.The blood vessel of these new formation not only promotes primary tumor continued growth, and make metastatic cancer cell propagate and again Build group.For solid tumor compared with normal cell, neovascularization allows tumour cell to obtain the growth vigor compared with normal cell. In breast cancer and some other tumours, observing that there are one between total microvessel density and patient's survival in tumor tissue section Fixed correlation (Weidner et al, N. Eng1. Med. 324 (1991) 1-8:Horak et al, Lancet 340 (1992) 1120-1124 and Macchiarini, etc. Lancet340 (1992) 145-146).Although being not fully understood The precise mechanism that new blood vessel occurs is controlled, but the formation of tumour correlation new blood vessel is originated from numerous angiogenic stimulators and inhibits The net balance of object, referring to Folkman, NatMed (1): 27-31 (1995).In addition to tumour, the generation and generation of new blood vessel have May be related to the mechanism of a variety of diseases, such as: intraocular new blood vessel syndrome, proliferating retinopathy or age-dependent Macular degeneration (AMD), rheumatoid arthritis and psoriasis Folkman, etal, Bio1.Chem267 (1992) 10931- 10934；53 (1991) 217-239 of Klagsbrun, et al, Annu. Rev.Physio1. etc..

Vascular endothelial growth factor (vascular endothelial growth factor, VEGF), is a kind of height Its receptor on the rush vascular endothelial growth factor of specificity, VEGF and endothelial cell membrane, vascular endothelial growth factor Receptor (Vascular Endothelial Growth Factor Receptor) combines, and causes the autophosphorylation of receptor, from And Mitogen activated protein kinase (MAPK) is activated, realize mitogen characteristic, inducing endothelial cell hyperplasia.VEGF is One of preferably characterize and most strength angiogenesis positive regulator, Ferrara, N.;Kerbel, R.S Nature438: 967-74(2005).C other than playing the role of occurrence factor in angiogenesis and angiogenesis (vasculogenesis), VEGF also presents the various biological effect in other physiological processes as pleiotropic growth factor, such as in adjusting Endothelial cell survival, vasopermeability and vasodilation, monocyte chemotaxis and calcium current such as enter at functions, the Ferraraand et Al., (1997) EndocrineRev.18:4-25.There are also researchs to have reported VEGF to several non-endothelial cells types, such as regards The mitogenesis effect of retinal pigment epithelial cell, pancreatic ductal cell and schwann cell, Sondell et al., J.Neurosci.19:5731-5740(1999)。

The main regulator that VEGF occurs as pathological conditions medium vessels has become a kind of very valuable medicine Object target spot has carried out a large amount of work for the target spot.But when being treated using VEGF as target spot, it can cause to be related to a variety of The case where cell factor, occurs.For example, inflammatory cell can participate in angiogenesis by secretion inflammatory cytokine, the inflammatory is thin Intracellular cytokine can influence activated endothelial cell, proliferation, migration and survival, and Reviews are shown in Albini, A., Tosetti, F., Benelli, R. Noonan, D.M..Combination with targeting VEGF and the compound of fibroblast growth factor signal transduction is controlled In treatment, not only increases antitumor effect and delay the generation of drug resistance in late stage tumour.Some researches show that tumour leachings Lubricant nature stromal fibroblast cells can become one of the source for promoting angiogenesis factor Dong, J.etal. EmboJ23:2800- 10 (2004) etc..

Anti-vegf neutralizing antibody can inhibit in nude mice it is various difference human tumor cell lines growth (Kim et al., (Nature) 362:841-844 (1993) etc.), for treating tumour or other diseases due to caused by new blood vessel, anti-vegf Monoclonal antibody or other inhibitor are a kind of relatively good selections.Using monoclonal antibody combination VEGF, can not only remove Fall the VEGF in blood, vascular endothelial cell proliferation signal can be prevented with blocking VEGF in conjunction with VEGFR, it is swollen to play inhibition The effect of tumor angiogenesis reaches the purpose for inhibiting tumour growth.The monoclonal antibody bevacizumab of humanization is that one kind is used for The anti-VEGF antibody of oncotherapy, and be a kind of approval angiogenesis inhibitors use for cancer treatment.However, bevacizumab Price costly, in the Collective Tender for Purchases of Medicines of the tissue of Chinese each province -2017 years 2016, minimum middle marked price is the drug It is more than 5000 yuan (100mg/ bottles), dosage 3-4 bottles each (100mg/ bottles) if certain colorectal cancer patients need to be administered once every two weeks, Nearly three, 40,000 yuan need to be just spent on Avastin by then patient individual's only one moon.

Therefore, that there is also types is few, selectivity is not strong, affinity is lower and price for existing Anti-X activity The defects of expensive.Therefore novel Anti-X activity is researched and developed, makes it have lower toxic side effect, more preferably clinical medicine Effect, and can reduce cost, it can be born by cancer patient in price, it has also become urgent problem to be solved.

Summary of the invention

Technical problem to be solved by the invention is to provide a kind of new Anti-X activities, with good Biological activity has good affinity to vegf protein, and there is good ligand-receptor combination rejection ability and cell to increase Rejection ability is grown, in low concentration, cell inhibitory effect ability is better than control antibodies bevacizumab.It is provided by the invention New Anti-X activity can be used in preparing antitumor, anti-infectious disease, anti-autoimmune disease and resisting exempting from The drugs such as epidemic disease repulsion, so as to complete the present invention.

Therefore, the first purpose of this invention is to provide a kind of amino acid sequence of Anti-X activity.

Second object of the present invention is to provide the nucleic acid molecule for encoding the Anti-X activity.

Third object of the present invention is to provide the expression vector comprising the nucleic acid molecule.

Fourth object of the present invention is to provide the host cell comprising the expression vector.

Of the invention the 5th is designed to provide a kind of preparation method of Anti-X activity.

Of the invention the 6th is designed to provide the composition comprising the Anti-X activity.

Of the invention the 7th is designed to provide the monoclonal antibody of the anti-vegf in preparation VEGF molecule blocking agent Purposes in object.

To achieve the goals above, this invention takes following technical solutions:

One of the technical solution that the present invention takes are as follows: a kind of monoclonal antibody of anti-vegf, which includes heavy chain can Become area and light chain variable region, the amino acid sequence of the heavy chain variable region is described light as shown in SEQ ID NO:2 in sequence table The amino acid sequence of chain variable region is as shown in SEQ ID NO:8 in sequence table.

The monoclonal antibody of anti-vegf of the present invention is conventional monoclonal antibody comprising two heavy chain of antibody and two Antibody light chain, including heavy chain variable region and light chain variable region.Preferably, the monoclonal antibody of anti-vegf of the present invention Heavy chain variable amino acid sequence as shown in SEQ ID NO:2 in sequence table, the amino acid sequence of light chain variable region such as sequence In list shown in SEQ ID NO:8.More preferably, the heavy chain variable region of the monoclonal antibody further includes CDR1, CDR2 and CDR3 Area, amino acid sequence is respectively as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.More preferably, the monoclonal The light chain variable region of antibody further includes CDR1 ', CDR2 ' and the area CDR3 ', amino acid sequence respectively such as SEQ ID NO:10, Shown in SEQ ID NO:11, SEQ ID NO:12.More preferably, the Anti-X activity is source of people.

The Anti-X activity can be the full length sequence of antibody, be also possible to the piece of anti-vegf human antibody Section, the segment be Fab, Fab ', F (ab ')₂, Fv or scFv etc..Preferably, the anti-vegf human antibody is source of people.More Preferably, the anti-vegf human antibody is IgG₁、IgG₂Or IgG₄Type antibody.

The present invention further provides the derivative of the anti-vegf human antibody, the derivative is VEGF human antibody Segment, antibody/antibody fragment-factor fusion protein, antibody/antibody fragment-chemical coupling thing；The anti-vegf human antibody Segment be Fab, Fab ', F (ab ')₂, Fv or scFv etc..

Preferably, antibody/antibody fragment-the factor fusion protein is specially antibody-factor fusion protein or antibody piece Section-factor fusion protein.Preferably, antibody/antibody fragment-the chemical coupling thing is specially antibody-chemical coupling thing or resists Body segment-chemical coupling thing.

Monoclonal antibody of the present invention can be prepared by this field routine techniques, including hybridoma technology, phage display technology Show technology, single lymphocyte gene clone technology etc., preferably passes through hybridoma technology from wild type or transgenic mice system Standby monoclonal antibody.

The two of the technical solution that the present invention takes are as follows: a kind of isolated nucleic acid molecule encodes the anti-vegf Dan Ke The nucleotide sequence of grand antibody heavy chain variable region encodes the core of the monoclonal light chain variable region as shown in SEQ ID NO:3 Nucleotide sequence is as shown in SEQ ID NO:9.

Preferably, SEQ ID NO:1 in the heavy chain coding nucleotide sequence of the Anti-X activity such as sequence table Shown, light chain coding nucleotide sequence is as shown in SEQ ID NO:7 in sequence table.

The preparation method of the nucleic acid molecule is this field customary preparation methods, is preferably comprised following preparation method: By gene clone technology such as PCR method etc., the nucleic acid molecule for encoding the monoclonal antibody is obtained, or by artificial Complete sequence synthetic method obtains the nucleic acid molecule of coding said monoclonal antibody.

As known to those skilled in the art, the nucleotide sequence for encoding the amino acid sequence of said monoclonal antibody can be appropriate Replacement is introduced, missing, changes, be inserted into or increase to provide the homologue or its conservative series of variation of a polynucleotide. The homologue of polynucleotide or its conservative series of variation in the present invention, can be by encoding the monoclonal antibody gene One or more bases are replaced, lack or increase within the scope of holding antibody activity to be made.

The three of the technical solution that the present invention takes are as follows: a kind of expression vector contains the list for encoding anti-vegf as described above The nucleic acid molecule of clonal antibody.

Expression vector of the present invention is the expression vector of this field routine, is referred to comprising regulating and controlling sequence appropriate, such as Promoter sequence, terminator sequence, polyadenylation sequences, enhancer sequence, marker gene and/or sequence and other are appropriate Sequence expression vector.The expression vector can be virus or plasmid, such as bacteriophage appropriate or phasmid, Geng Duoji Art details is referred to such as Sambrook, Molecular Cloning:A Laboratory Manual, the second edition, Cold Spring Harbor Laboratory Press, 1989.Known technology and scheme of many for nucleic-acid manipulation refer to Current Protocols in Molecular Biology, the second edition, Ausubel etc. write.Expression in the present invention carries Body refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenopathy Poison, retrovirus or other carriers.

Preferably, the expression vector is selected from one of pHLX101, pEE14.4, pCHO 1.0 or pcDNA3.1 or more The combination of kind, is more preferably pcDNA3.1.The present invention screens the gene for obtaining purpose antibody from the cell strain of Colony Culture Sequence can rebuild the activity of antibody to construct carrier for expression of eukaryon after expression, obtain Anti-X activity.

The four of the technical solution that the present invention takes are as follows: a kind of host cell, the host cell include table as described above Up to carrier.The expression vector contains the nucleic acid molecule for encoding the monoclonal antibody of anti-vegf as described above.

Host cell of the present invention is the host cell of this field routine, as long as being able to satisfy makes above-mentioned recombinant expression carrier It steadily voluntarily replicates, and the entrained nucleotide can be by effective expression.Host cell in the present invention can be Prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or higher eucaryotic cells, as mammal is thin Born of the same parents.The host cell is preferably comprised: COS, CHO, HeLa cell line, myeloid cell series for example SP2/0 cell line, NS0, sf9, Sf21, DH5 α, BL21 (DE3) orE.coliIn TG1, YB2/0 cell line etc. and the B- cell or hybridoma of conversion One or more combinations.More preferably it isE.coliTG1, BL21 cell (expression single-chain antibody or Fab antibody) or CHO-K1 Cell (expression overall length IgG antibody).

Foregoing expression vectors are converted into host cell, currently preferred recombinant expression transformants can be obtained.Wherein The method for transformation is this field conventional transformation methods, preferably chemical transformation, heat shock method or electric robin.

The five of the technical solution that the present invention takes are as follows: the preparation method of the monoclonal antibody of above-mentioned anti-vegf comprising with Lower step:

A) under expression condition, host cell as described above is cultivated, expresses the monoclonal antibody of anti-vegf；

B) monoclonal antibody of separation and purification step a) gained anti-vegf.

The cultural method of host cell of the present invention, the Isolation and purification method of the monoclonal of the anti-vegf are this Field conventional method, concrete operation method please refers to corresponding cell culture technology handbook and monoclonal antibody isolates and purifies skill Art handbook.

Host cell used in the present invention is the prior art, can be directly acquired by commercial sources, used in culture Culture medium be also various conventional mediums, those skilled in the art can rule of thumb select applicable culture medium, be suitable for place It is cultivated under conditions of chief cell growth.After host cell growth is to cell density appropriate, with suitable method (such as temperature Degree conversion or chemical induction) promoter that induces selection, cell is further cultured for a period of time.Recombination in the above methods is more Peptide can be expressed in cells, or on the cell membrane, or secreted outside the cell.If desired, can using its physics, it is chemical and Other characteristics are separated by various separation methods and purify the albumen of recombination.These methods are well known to those skilled in the art 's.The example of these methods includes but is not limited to: conventional renaturation process, with protein precipitant processing (salting-out method), from The heart permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, efficient liquid phase Chromatograph the combination of (HPLC) and various other liquid chromatography technologies and these methods.

The six of the technical solution that the present invention takes are as follows: a kind of composition, it includes a effective amount of anti-vegf as described above Monoclonal antibody and pharmaceutically acceptable carrier.

Monoclonal antibody of the invention can by any of mode compounding pharmaceutical composition in this field come using.This Kind composition is using the monoclonal antibody as active constituent, in addition one or more pharmaceutically acceptable carriers, diluent, filling Agent, bonding agent and other excipient, this depends on administration mode and designed dosage form.Known to the branch art personnel of this field Treat inert inorganic or organic carrier include but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, Wax, fat, polyol such as polyethylene glycol, water, sucrose, ethyl alcohol, glycerol, such, various preservatives, lubrication Agent, dispersing agent, corrigent.Moisturizer, antioxidant, sweetener, colorant, stabilizer, salt, buffer are such to be added Enter wherein, these substances be used to help as needed formula stability or help to improve activity or it biological effectiveness or Acceptable mouthfeel or smell are generated in the case where oral, inhibitor can be used in such a composition can be its primitivation The form of object itself is closed, or optionally uses the form of its pharmaceutically acceptable salt, monoclonal antibody of the invention can be single Solely administration, or with various combination medicine-feedings, and the combining form administration together with other healing potions.The composition root so prepared Inhibitor is administered according to needing may be selected any mode appropriate well known by persons skilled in the art.

Anti-X activity provided by the invention can form drug system with pharmaceutically acceptable carrier together For agent composition to more stably play curative effect, these preparations can guarantee the ammonia of Anti-X activity disclosed by the invention The conformation integrality of base acid core sequence, while the polyfunctional group for going back protected protein matter prevents its degradation (including but not limited to solidifying Poly-, deamination or oxidation).Under normal conditions, for liquid preparation, it can usually be saved under the conditions of 2 °C -8 °C and at least stablize one Year, for lyophilized preparation, stablize in 30 °C of holdings at least six months.The Anti-X activity preparation can be pharmaceutical field The preparations, preferably water needle or lyophilized preparation such as common suspension, water needle, freeze-drying.

For the water needle or lyophilized preparation of the monoclonal antibody of anti-vegf disclosed by the invention, pharmaceutically acceptable is carried Body is preferably comprised but is not limited to: one or a combination set of surfactant, solution stabilizer, isotonic regulator and buffer.Its Middle surfactant is preferably comprised but is not limited to: nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters (are spat Temperature 20 or 80)；Poloxamer(such as poloxamer 188)；Triton；Lauryl sodium sulfate (SDS)；Sodium Laurylsulfate；Ten Tetraalkyl, sub- oil base or octadecyl sarcosine；Pluronics；MONAQUAT^TMDeng additional amount should make anti-vegf monoclonal The granulating trend of antibody is minimum.Solution stabilizer one or a combination set of is preferably comprised but is not limited to be exemplified below: carbohydrate, example Such as, reducing sugar and nonreducing sugar；Amino acids, for example, monosodium glutamate or histidine；Alcohols, such as: trihydroxylic alcohol, height Grade sugar alcohol, propylene glycol, polyethylene glycol etc., the additional amount of solution stabilizer should make the preparation eventually formed in the technology of this field Personnel think to reach stable time interior holding stable state.Isotonic regulator is preferably comprised but is not limited to sodium chloride, sweet dew One or a combination set of alcohol.Buffer is preferably comprised but is not limited to: one of Tris, histidine buffering liquid, phosphate buffer or A combination thereof.

The seven of the technical solution that the present invention takes are as follows: the monoclonal antibody of anti-vegf as described above is in preparation VEGF molecule resistance Purposes in stagnant drug.

The purposes is more preferably the purposes prepared on VEGF molecule blocking medicine, it is preferred that the preparation VEGF molecule Purposes in blocking medicine is specially the purposes prepared on oncotherapy or diagnosing tumor class drug.

Drug of the present invention is preferably antitumor, treatment autoimmune disease, treatment infectious diseases and/or The drug of resisting transplant rejection reaction is more preferably anti-tumor drug, treatment autoimmune disease drug, it is therefore preferable to antitumor Drug.The monoclonal antibody of anti-vegf of the present invention can be used alone or be used in combination with other anti-tumor drugs.It is described Other anti-tumor drugs are this field conventional anticancer drugs, including antibody class drug or small molecule, anti-tumor drug.It is described anti- Body class drug is this field conventional antibody class drug, is preferably comprised anti-PD-1 monoclonal antibody, her monoclonal antibody etc..Described small point Sub- anti-tumor drug is this field conventional medicine, including taxol, 5-Fu pyrimidine etc..

Wherein the targeted tumour of the anti-tumor drug is preferably comprised but is not limited to: lung cancer, liver cancer, oophoroma, palace Neck cancer, cutaneum carcinoma, colon cancer, glioma, bladder cancer, breast cancer, kidney, cancer of the esophagus, gastric cancer, oral squamous cell carcinoma, One of urothelial cell cancer, cancer of pancreas and/or H/N tumors are a variety of.

The so-called anti-tumor drug of the present invention, refers to the drug for inhibiting and/or treating tumour, may include with tumour The delay of related symptoms development and/or the reduction of these severity of symptom, further comprise already present tumour with disease The mitigation of shape and the appearance for preventing other symptoms further include the transfer etc. for reducing or preventing tumour.

Monoclonal antibody of anti-vegf and combinations thereof gives medicament when to animal administration including people in the present invention It measures age because of patient and weight, disease traits and seriousness and administration route and different, the result of zoopery can be referred to With various situations, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 1-1800mg/ days.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is that: Anti-X activity provided by the invention has good biology living Property, expression quantity with higher, has significant affinity, suppression to VEGF in larger concentration range in mammalian cells The ability and cell inhibitory effect ability that ligand-receptor processed combines, and (about 0.1 μ g/mL -1 μ g/ in the low concentration range ML), the cell inhibitory effect ability of present invention gained Anti-X activity is better than control antibodies bevacizumab.The list Clonal antibody can be independent, or with other anti-tumor drug use in conjunction in immunotherapy of tumors and diagnosis and screening, energy It is enough efficiently applied in the preparations of drugs such as treatment tumour, infectious diseases, autoimmune disease and anti-immunity repulsion, and Novel monoclonal antibody class drug may be provided for vast tumor patient, so that drug cost be effectively reduced, significant alleviate is swollen The economic pressures of tumor sufferer.

Detailed description of the invention

Fig. 1 is that the SDS-PAGE for the VEGF recombinant protein that the present invention is expressed and purified analyzes result.

Fig. 2 is the affinity experimental result of Anti-X activity of the present invention.

Fig. 3 is the ligand-receptor combination inhibition assay result of Anti-X activity of the present invention.

Fig. 4 is that Anti-X activity of the present invention inhibits HUVEC cell proliferation experiment result.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.Room temperature as described in the examples be this field routine room temperature, generally 10 ~ 30 DEG C.

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text In addition explicitly point out, singular "one", " one " and " this " include plural form.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc. CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor of MOLECULAR Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The expression of embodiment 1 and Purification of Human VEGF extracellular region recombinant protein

It synthesizes people VEGF-Fc fusion (being synthesized by Nanjing Genscript Biotechnology Co., Ltd.), wherein VEGF gene order It is SEQ ID NO:14 for SEQ ID NO:13, Fc gene order, two sections of sequence connections are VEGF-Fc fusion, design Forward and reverse primer is respectively as follows:

5 '-atccgccggcaagccgccaccatgaactttctgctgtcttgg-3 ' (SEQ ID NO:15) and

5 '-tcactacgtatcatttacccggagacagggagaggctc-3 ' (SEQ ID NO:16) are compiled for expanding The gene of code VEGF-Fc fusion protein, introduces restriction endonuclease sites Ngo MIV and Sna in the upstream and downstream of gene respectively BI, PCR condition are 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 15s, and 35 recycle, 72 DEG C of extension 10min, and archaeal dna polymerase is TAKARA Products.

PCR product recycles (QIAquick Gel Extraction Kit is Omega bio-tek Products) after purification through Ago-Gel, is added Restriction enzyme Ngo MIV and Sna BI carries out digestion, and through DNA reclaim reagent, (Omega bio-tek company produces digestion products Product) after purification with the carrier pHLX101(of identical digestion with restriction enzyme by Shanghai Henlius Biotech Co. Ltd. Building, referring to Chinese patent 201210211812.1) it is attached reaction.Connection product is transformed into DH5 α Escherichia coli, coating On the 2YT agar medium containing 50 μ g/ml carbenicillins.By the positive colony of acquisition green containing 50 μ g/ml carboxylic benzyls It is cultivated in the 2YT fluid nutrient medium of mycin, after Invitrogen company sequence verification, with the big pumping kit (Omega of plasmid Bio-tek Products) extract positive colony plasmid.

Using the method for polyethyleneimine (Polyethylenimine, PEI) by plasmid DNA transfection into 293F cell (Invitrogen), after transfecting 6 hours, cell is centrifuged, replaces 293 fresh freestyle Medium culture solutions (GIBCO Products) continue shaking table culture.Cell can be observed, be started within the 3rd day after transfection daily after 24 and 48 hours To cell count, expression time is usually 7-10 days, harvests cell culture supernatant when viable cell density is lower than 30%.It will be thin With Protein A affinity column, (purchase purifies born of the same parents' culture solution supernatant from Invitrogen), finally obtains people's VEGF extracellular region Recombinant protein.The SDS-PAGE analysis result for purifying obtained VEGF recombinant protein is as shown in Figure 1.

Building, expression, purifying and the identification of 2 anti-vegf human antibody carrier for expression of eukaryon of embodiment

Heavy chain of antibody (SEQ ID NO:1) and light chain (SEQ ID NO:7) gene are by the limited public affairs of Nanjing Jin Sirui biotechnology Department's synthesis.The DNA sequences encoding of above-mentioned heavy chain and light chain is expanded using PCR reaction, in the base of heavy chain of antibody and light chain Because both ends introduce restriction enzyme site appropriate, wherein introducing Ngo MIV and Sna BI respectively at the end of antibody heavy chain gene 5 ' and 3 ' ends Restriction enzyme site, forward and reverse primer sequence are respectively as follows:

5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ ID NO:17) and

5 '-GCTCTACGTATCATTTACCTGGAGACAGGGAGAGGC-3 ' (SEQ ID NO:18), in antibody's light chain variable region Gene 5 ' end and 3 ' ends introduce Ngo MIV and SnaB I respectively, and forward and reverse primer sequence is respectively as follows:

5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ ID NO:17)

With 5 '-GCATCTTACGTATTATTAACACTCTCCCCTGTTGAAG -3 ' (SEQ ID NO:19).It is anti-using PCR It answers, condition is 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 10s, and 35 circulations, 72 DEG C of extension 10min, archaeal dna polymerase is TAKARA company Product.After PCR amplification, by PCR product through agarose gel electrophoresis recovery purifying.PCR product is cloned into pMD18-T carrier, It transfers to Sheng Gong company to be sequenced and analyzes variable region gene sequence.Sequencing result are as follows: coding Anti-X activity weight chain variable The nucleotide sequence in area encodes the nucleotide sequence such as SEQ ID NO:9 institute of its light chain variable region as shown in SEQ ID NO:3 Show.The amino acid sequence of anti-VEGF mAb heavy chain variable region is as shown in SEQ ID NO:2 in sequence table, the ammonia of light chain variable region Base acid sequence is as shown in SEQ ID NO:8 in sequence table.The heavy chain variable region of the monoclonal antibody further include CDR1, CDR2 and The area CDR3, amino acid sequence is respectively as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.Its light chain variable Area further includes CDR1 ', CDR2 ' and the area CDR3 ', amino acid sequence respectively such as SEQ ID NO:10, SEQ ID NO:11, Shown in SEQ ID NO:12.

Light-chain variable region gene is added restriction enzyme Ngo MIV and SnaB I and carries out digestion, heavy chain variable region gene Restriction enzyme Ngo MIV and Sna BI is added and carries out digestion, is purified after digestion through DNA purification kit, and with phase With the carrier pHLX101 of digestion with restriction enzyme.Connection product is transformed into DH5 α Escherichia coli, is coated on containing 50 μ g/ml On the 2YT agar medium of carbenicillin.The positive colony of acquisition is trained in the 2YT liquid containing 50 μ g/ml carbenicillins It supports and is cultivated in base, after Invitrogen company sequence verification, extract positive colony plasmid with the big pumping kit of plasmid.

Using the Neon system of Invitrogen company, by the plasmid DNA transfection of linearisation to Chinese hamster ovary celI, (purchase is certainly ATCC).Chinese hamster ovary celI after transfection is diluted colonized culture, is containing 50 μm of ol methionine imino groups for sulfone It is sieved in 94113 culture mediums (Irvine Scientific Products) of (methionine sulfoximine, MSX) Choosing, to obtain monoclonal cell system, is named as 2VA7.

By the monoclonal cell system 2VA7 of acquisition in containing 94113 culture mediums of 50 μm of ol methionine imino groups for sulfone Shaking flask culture is carried out, expression time is usually 7-14 days, harvests cell culture supernatant when viable cell density is lower than 50%.Benefit With goat anti-human igg Fd(Meridian Products) and the goat-anti people constant region of light chain of horseradish peroxidase-labeled carry out The content of antibody in double sandwich-ELISA method detection expression supernatant, using untransfected supernatant as negative control, the IgG sterling of people is made For standard items.Experimental result shows that the destination protein for expressing acquisition can be identified there is human IgG antibody's feature by two antibody, And the human antibody expression quantity constructed is in 200 ~ 500 μ g/ml, expression with higher.Using the affine layer of Protein A Analysis column isolates and purifies purpose monoclonal antibody 2VA7 from cells and supernatant.

The identification of 3 anti-vegf human antibody affinity of embodiment

Affinity of antibody identification.(the work into ELISA Plate by 2 μ g/ml coating of VEGF recombinant protein will be prepared in embodiment 1 30 μ L of volume), 4 DEG C stand overnight.It is washed 3 times with the conventional phosphoric acid salt buffer (PBST) of the Tween 20 containing 0.05%.With 5% skim milk room temperature is closed 1 hour.It is washed 3 times with PBST, the monoclonal antibody 2VA7 that embodiment 2 prepares is configured to 26.5 μ g/ml concentration, and 3 times of gradient dilutions are carried out, totally 8 gradients, every hole are added 30 μ l, are stored at room temperature 1 hour.It is washed with PBST 3 times, goat-anti people's constant region of light chain secondary antibody (Millipore public affairs of the 30 diluted horseradish peroxidase-labeleds of μ l 1:4000 are added Take charge of product), it is stored at room temperature 1 hour.It is washed 4 times with PBST, TMB colour developing is added, and with the H of 2M₂SO₄Terminate reaction.Existed with microplate reader Read under 450nm, bought using bevacizumab(from Roche Holding Ag) as control.The affinity of Anti-X activity For experimental result as shown in Fig. 2, can be seen that from result, the present invention screens gained monoclonal antibody 2VA7 in larger concentration range (about 0.01 μ g/mL-10 μ g/mL) has significant affinity, affinity and positive control antibodies to VEGF recombinant protein Bevacizumab is similar.

Embodiment 4 expresses 2 extracellular region recombinant protein of human VEGFR-3

It synthesizes human VEGFR-3 2-AP fusion (being synthesized by Nanjing Genscript Biotechnology Co., Ltd.), wherein VEGFR2 gene Sequence is that SEQ ID NO:20, AP gene order is SEQ ID NO:21, and two sections of sequence connections are that VEGFR2-AP merges base Cause, design forward and reverse primer are respectively as follows:

5 '-TAACGGGGCCGGCAAGCCGCCACCATGCAGAGCAAGGTGCTGCTGGCC-3 ' (SEQ ID NO:22) and

5 '-TCACTACGTATCAGTGGTGGTGGTGGTGGTGGCCTG-3 ' (SEQ ID NO:23) are encoded for expanding The gene of VEGFR2-Fc fusion protein introduces restriction endonuclease sites Ngo MIV and Sna in the upstream and downstream of gene respectively BI, PCR condition are 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 30s, and 35 recycle, 72 DEG C of extension 10min, and archaeal dna polymerase is TAKARA Products.

Using the method for polyethyleneimine (Polyethylenimine, PEI) by plasmid DNA transfection into 293F cell, After transfecting 6 hours, cell is centrifuged, replaces 293 fresh freestyle Medium culture solutions (GIBCO Products), Continue shaking table culture.Cell can be observed, be started within the 3rd day after transfection daily to cell count, expression after 24 and 48 hours Time is usually 7-10 days, harvests cell culture supernatant when viable cell density is lower than 50%.

5 ligands of embodiment-antibody, which combines, inhibits Function Identification

VEGF recombinant protein will be prepared in embodiment 1 by 2 μ g/ml coating into ELISA Plate, 4 DEG C stand overnight.Use PBST It washes 3 times.It is closed 1 hour with 5% skim milk room temperature.It is washed 3 times with PBST.The monoclonal antibody that embodiment 2 is prepared 2VA7 is configured to 2 μ g/ml concentration, and carries out 3 times of gradient dilutions, totally 8 gradients.The VEGFR2 that will be prepared in embodiment 4 Cell expresses supernatant, is diluted with PBST by 1:400, then mixes, be added in ELISA Plate by 1:1 with the antibody diluted, room Temperature stands 1 hour.It is washed 6 times with PBST.Overnight with pNPP colour developing, bought using bevacizumab(from Roche Holding Ag) as right According to.The ligand-receptor combination inhibition assay result of Anti-X activity is as shown in figure 3, from the results, it was seen that the present invention Screening gained monoclonal antibody 2VA7 in larger concentration range (about 0.1 μ g/mL-10 μ g/mL), can effectively inhibit VEGF with The combination of its receptor VEGFR2, ligand-receptor combination rejection ability is similar to control antibodies bevacizumab, can effectively press down The combination of VEGF processed and its receptor VEGFR2.

Inhibition Function Identification of 6 antibody of embodiment for vascular endothelial cell proliferation

(purchase is from BCRC) with added with EGM for HUVEC cell^TM- 2 SingleQuots(buy the EBM from Lonza)^®- 2 cultures Base (cultivated from Lonza), is inoculated into overnight incubation in 96 well culture plates by 6000, every hole cell/100 μ l by purchase.Carefully Supernatant is sucked, is added in 96 well culture plates by every 2.4 μ g/ml VEGF/50 μ l of hole.The antibody 2VA7 that embodiment 2 is prepared 15 μ g/ml concentration are configured to, and carry out 2 times of gradient dilutions, totally 8 gradients, then are added in 96 well culture plates by every 50 μ l of hole.It will The tissue culture plate that sample has been added is placed in CO₂In incubator, 37^oC, 5% CO₂, it is incubated for 70 ~ 74 h.20 μ l are added in every hole Cell culture is placed in CO by MTS₂In incubator, 37^oC, 5% CO₂, it is incubated for 3.5 h.It is bought using bevacizumab(from sieve Family name company) as control, it is read at 490nm with microplate reader.Anti-X activity inhibits HUVEC cell proliferation experiment knot Fruit is as shown in figure 4, from the results, it was seen that the present invention screens gained monoclonal antibody 2VA7 to HUVEC cell with significant Inhibit the ability of cell Proliferation, at high concentrations (about 10 μ g/mL), cell inhibitory effect ability and control antibodies Bevacizumab is similar, in the low concentration range (about 0.1 μ g/mL -1 μ g/mL), the cell Proliferation suppression of monoclonal antibody 2VA7 Ability processed is significantly better than control antibodies bevacizumab.

It should be understood that those skilled in the art can make the present invention various after having read above content of the invention Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.

Sequence table

<110>Shanghai Fu Honghan continuous heavy rain Biotechnology Ltd.

Shanghai Fu Honghan continuous heavy rain Biology Pharmacy Co., Ltd

<120>a kind of monoclonal antibody of anti-vegf and its preparation method and application

<141> 2018-12-03

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1404

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt ccactcccag 60

gtgcagctgg tggagtctgg ggctgaggtg aagaagcctg ggtcctcggt gaaggtctcc 120

tgcaaggctt ctggaggcac cttcagcagc tatgctatca gctgggtgcg acaggcccct 180

ggacaagggc ttgagtggat gggggggatc atccctatct ttggtacagc aaactacgca 240

cagaagttcc agggcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 300

gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag ggcactgggg 360

aacgttgctt ttgatatctg gggccaaggg acaatggtca ccgtctcaag cgctagcacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgaccgtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc cctgctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagcagagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accatcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccaggtaa atga 1404

<210> 2

<211> 115

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Leu Gly Asn Val Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Met Val Thr

115

<210> 3

<211> 345

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

caggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatggggggg atcatcccta tctttggtac agcaaactac 180

gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagggcactg 300

gggaacgttg cttttgatat ctggggccaa gggacaatgg tcacc 345

<210> 4

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Ser Tyr Ala Ile Ser

1 5

<210> 5

<211> 17

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 6

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Ala Leu Gly Asn Val Ala Phe Asp Ile

1 5

<210> 7

<211> 708

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt ccactccctt 60

gaaattgtga tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 180

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 240

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgta cacttttggc 360

caggggacca agctggagat cagacgaact gtggctgcac catctgtctt catcttcccg 420

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttaa 708

<210> 8

<211> 108

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Arg

100 105

<210> 9

<211> 324

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gaaattgtga tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgta cacttttggc 300

caggggacca agctggagat caga 324

<210> 10

<211> 12

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 11

<211> 7

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

Gly Ala Ser Ser Arg Ala Thr

1 5

<210> 12

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Gln Gln Tyr Gly Ser Ser Pro Tyr Thr

1 5

<210> 13

<211> 573

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat 60

gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca tcacgaagtg 120

gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac 180

atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg 240

atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 300

aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg 360

agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa 420

aatccctgtg ggccttgctc agagcggaga aagcatttgt ttgtacaaga tccgcagacg 480

tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac 540

gaacgtactt gcagatgtga caagccgagg cgg 573

<210> 14

<211> 753

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ggagggaatt cggatccccg tcgtgcatct atcgaaggtc gtggagatcc cgaggagccc 60

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 120

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 180

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 240

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 300

agcacgtacc gggtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 360

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 420

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 480

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 540

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 600

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 660

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 720

cagaagagcc tctccctgtc tccgggtaaa tga 753

<210> 15

<211> 42

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

atccgccggc aagccgccac catgaacttt ctgctgtctt gg 42

<210> 16

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

tcactacgta tcatttaccc ggagacaggg agaggctc 38

<210> 17

<211> 48

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

acctgccggc aagccgccac catgggttgg agcctcatct tgctcttc 48

<210> 18

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

gctctacgta tcatttacct ggagacaggg agaggc 36

<210> 19

<211> 37

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gcatcttacg tattattaac actctcccct gttgaag 37

<210> 20

<211> 2292

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

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 accttttgtt gcttttggaa gtggcatgga atctctggtg 1020

gaagccacgg tgggggagcg tgtcagaatc cctgcgaagt accttggtta cccaccccca 1080

gaaataaaat ggtataaaaa tggaataccc cttgagtcca atcacacaat taaagcgggg 1140

catgtactga cgattatgga agtgagtgaa agagacacag gaaattacac tgtcatcctt 1200

accaatccca tttcaaagga gaagcagagc catgtggtct ctctggttgt gtatgtccca 1260

ccccagattg gtgagaaatc tctaatctct cctgtggatt cctaccagta cggcaccact 1320

caaacgctga catgtacggt ctatgccatt cctcccccgc atcacatcca ctggtattgg 1380

cagttggagg aagagtgcgc caacgagccc agccaagctg tctcagtgac aaacccatac 1440

ccttgtgaag aatggagaag tgtggaggac ttccagggag gaaataaaat tgaagttaat 1500

aaaaatcaat ttgctctaat tgaaggaaaa aacaaaactg taagtaccct tgttatccaa 1560

gcggcaaatg tgtcagcttt gtacaaatgt gaagcggtca acaaagtcgg gagaggagag 1620

agggtgatct ccttccacgt gaccaggggt cctgaaatta ctttgcaacc tgacatgcag 1680

cccactgagc aggagagcgt gtctttgtgg tgcactgcag acagatctac gtttgagaac 1740

ctcacatggt acaagcttgg cccacagcct ctgccaatcc atgtgggaga gttgcccaca 1800

cctgtttgca agaacttgga tactctttgg aaattgaatg ccaccatgtt ctctaatagc 1860

acaaatgaca ttttgatcat ggagcttaag aatgcatcct tgcaggacca aggagactat 1920

gtctgccttg ctcaagacag gaagaccaag aaaagacatt gcgtggtcag gcagctcaca 1980

gtcctagagc gtgtggcacc cacgatcaca ggaaacctgg agaatcagac gacaagtatt 2040

ggggaaagca tcgaagtctc atgcacggca tctgggaatc cccctccaca gatcatgtgg 2100

tttaaagata atgagaccct tgtagaagac tcaggcattg tattgaagga tgggaaccgg 2160

aacctcacta tccgcagagt gaggaaggag gacgaaggcc tctacacctg ccaggcatgc 2220

agtgttcttg gctgtgcaaa agtggaggca tttttcataa tagaaggtgc ccaggaaaag 2280

acgaacttgg aa 2292

<210> 21

<211> 1521

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

cgaattcctt acgtaagatc ttccggaatc atcccagttg aggaggagaa cccggacttc 60

tggaaccgcg aggcagccga ggccctgggt gccgccaaga agctgcagcc tgcacagaca 120

gccgccaaga acctcatcat cttcctgggc gatgggatgg gggtgtctac ggtgacagct 180

gccaggatcc taaaagggca gaagaaggac aaactggggc ctgagatacc cctggccatg 240

gaccgcttcc catatgtggc tctgtccaag acatacaatg tagacaaaca tgtgccagac 300

agtggagcca cagccacggc ctacctgtgc ggggtcaagg gcaacttcca gaccattggc 360

ttgagtgcag ccgcccgctt taaccagtgc aacacgacac gcggcaacga ggtcatctcc 420

gtgatgaatc gggccaagaa agcagggaag tcagtgggag tggtaaccac cacacgagtg 480

cagcacgcct cgccagccgg cacctacgcc cacacggtga accgcaactg gtactcggac 540

gccgacgtgc ctgcctcggc ccgccaggag gggtgccagg acatcgctac gcagctcatc 600

tccaacatgg acattgacgt gatcctaggt ggaggccgaa agtacatgtt tcccatggga 660

accccagacc ctgagtaccc agatgactac agccaaggtg ggaccaggct ggacgggaag 720

aatctggtgc aggaatggct ggcgaagcgc cagggtgccc ggtatgtgtg gaaccgcact 780

gagctcatgc aggcttccct ggacccgtct gtgacccatc tcatgggtct ctttgagcct 840

ggagacatga aatacgagat ccaccgagac tccacactgg acccctccct gatggagatg 900

acagaggctg ccctgcgcct gctgagcagg aacccccgcg gcttcttcct cttcgtggag 960

ggtggtcgca tcgaccatgg tcatcatgaa agcagggctt accgggcact gactgagacg 1020

atcatgttcg acgacgccat tgagagggcg ggccagctca ccagcgagga ggacacgctg 1080

agcctcgtca ctgccgacca ctcccacgtc ttctccttcg gaggctaccc cctgcgaggg 1140

agctccatct tcgggctggc ccctggcaag gcccgggaca ggaaggccta cacggtcctc 1200

ctatacggaa acggtccagg ctatgtgctc aaggacggcg cccggccgga tgttaccgag 1260

agcgagagcg ggagccccga gtatcggcag cagtcagcag tgcccctgga cgaagagacc 1320

cacgcaggcg aggacgtggc ggtgttcgcg cgcggcccgc aggcgcacct ggttcacggc 1380

gtgcaggagc agaccttcat agcgcacgtc atggccttcg ccgcctgcct ggagccctac 1440

accgcctgcg acctggcgcc ccccgccggc accaccgacg ccgcgcaccc gggttcaggc 1500

caccaccacc accaccactg a 1521

<210> 22

<211> 48

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

taacggggcc ggcaagccgc caccatgcag agcaaggtgc tgctggcc 48

<210> 23

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

tcactacgta tcagtggtgg tggtggtggt ggcctg 36

Claims

1. a kind of monoclonal antibody of anti-vegf, which is characterized in that the monoclonal antibody includes heavy chain variable region and light chain variable Area, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2 in sequence table, the amino of the light chain variable region Acid sequence is as shown in SEQ ID NO:8 in sequence table.

2. the monoclonal antibody of anti-vegf as described in claim 1, which is characterized in that the heavy chain variable region of the monoclonal antibody is also Including the area CDR1, CDR2 and CDR3, amino acid sequence is respectively such as SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 It is shown.

3. the monoclonal antibody of anti-vegf as described in claim 1, which is characterized in that the light chain variable region of the monoclonal antibody is also Including CDR1 ', CDR2 ' and the area CDR3 ', amino acid sequence is respectively such as SEQ ID NO:10, SEQ ID NO:11, SEQ ID Shown in NO:12.

4. a kind of isolated nucleic acid molecule, which is characterized in that the nucleotide of monoclonal antibody heavy described in coding claim 1 Sequence encodes the nucleotide sequence of its light chain as shown in SEQ ID NO:7 as shown in SEQ ID NO:1.

5. nucleic acid molecule as claimed in claim 4, which is characterized in that monoclonal antibody heavy described in coding claim 1 The nucleotide sequence of variable region encodes the nucleotide sequence such as SEQ ID NO:9 of its light chain variable region as shown in SEQ ID NO:3 It is shown.

6. a kind of expression vector, which is characterized in that the expression vector contains the Dan Ke for encoding anti-vegf as described in claim 1 The nucleic acid molecule of grand antibody.

7. a kind of host cell, which is characterized in that the host cell contains expression vector as claimed in claim 4.

8. a kind of preparation method of the monoclonal antibody of anti-vegf as described in claim 1, which is characterized in that the preparation method The following steps are included:

A) under expression condition, host cell as claimed in claim 5 is cultivated, expresses the monoclonal antibody of anti-vegf；

B) monoclonal antibody of separation and purification step a) gained anti-vegf.

9. a kind of composition, which is characterized in that the composition includes the Dan Ke of a effective amount of anti-vegf as described in claim 1 Grand antibody and pharmaceutically acceptable carrier.

10. the monoclonal antibody of anti-vegf is preparing the purposes in VEGF molecule blocking medicine.