CN105085677A - Anti-VEGFR2 human source nano antibody NTV1 and preparation method therefor and use thereof - Google Patents
Anti-VEGFR2 human source nano antibody NTV1 and preparation method therefor and use thereof Download PDFInfo
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Abstract
The invention discloses an anti-VEGFR2 human source nano antibody NTV1 which comprises a frame region and a complementarity-determining region, wherein the nano antibody has an amino acid sequence shown in SEQ ID NO: 1. The invention further provides a preparation method for the anti-VEGFR2 human source nano antibody NTV1. The preparation method comprises the following steps: a, screening a nano antibody combined with VEGFR2 by adopting a phage display technology; b, carrying out expression and purification of the nano antibody; and c, determining the interaction between an antigen and the antibody by adopting an AlphaScreen technology to further screen the antibody with the highest affinity . The human source nano antibody NTV1 with higher affinity and good anti-tumor activity is obtained. The VEGFR2 nano antibody egg white which is high in affinity and stable and uniform is obtained by expression and optimization, a human source anti-VEGFR3 nano antibody drug is successfully developed, and a novel field of drug research and development of a targeted VEGFR2 antibody is opened up.
Description
Technical field
The present invention relates to field of biological pharmacy.More specifically, the present invention relates to a kind of VEGFR2 nano antibody, the preparation method of this antibody and this antibody and prepare the purposes in anticancer medicine.
Background technology
Malignant tumour is that current serious affects one of human health, the principal disease threatening human life, and end 2012, the whole world has dies from cancer more than 8,200,000 people, and the World Health Organization and hygiene department of national governments are all classified as capture cancer as a top priority.But the treatment of tumour remains a difficult problem for current medical circle, researchist is finding the new direction of oncotherapy always.Found by research tumour, vasculogenesis is the prerequisite of tumorigenesis, and vascular endothelial growth factor receptor (vascularendothelialgrowthfactorreceptor, VEGFR) also becomes the study hotspot of the target of antitumor drug as the acceptor of vasculogenesis key factor VEGF.
Tumor vessel is that the growth of tumour provides sufficient nutrient supply, also promotes diffusion and the transfer of cancer cells simultaneously.VEGF is the somatomedin mainly acting on vascular endothelial cell, has and promotes endothelial cell proliferation, several functions such as increase microvascular permeability, induction of vascular generation etc.Existing research confirms the high expression level of VEGF and the disease close relation such as the formation of noumenal tumour and transfer, rheumatic arthritis, psoriasis, diabetic retinopathy, arteriosclerosis.VEGF is combined activation signal conduction by the specific receptors VEGFR on endothelial cellular membrane.The member of VEGFR family comprises: VEGFR1, VEGFR2 and VEGFR3.They are transmembrane protein, are made up of one section of cross-film sequence of the tyrosine kinase domain in the functional domain of the immunoglobulin-like of 7 outside born of the same parents, born of the same parents and centre.Wherein VEGFR2 is positioned people 4q11-12, by 1356 Amino acid profiles, can promote vascular endothelial cell proliferation, migration and increase capillary permeability.Study verified, the mitotic division of tumor vascular endothelial cell and the perviousness of tumor-microvessel are mainly occurred by the interaction of VEGF and VEGFR2.In the one-tenth person of health, expression level is very low does not even express for VEGFR2; And in many tumours, such as, be high expression level in the tumor vascular endothelial cell of mammary cancer, lung cancer, bladder cancer, colorectal carcinoma, cervical cancer, hepatocellular carcinoma, hemangioblastoma, cerebral glioma etc.In addition, VEGFR2 extracellular part deletion experiments confirms: VEGF is mainly by intracellular signaling occurring with I-III district specific binding outside VEGFR2 born of the same parents.
In decades since Kohler and Milstein invention hybridoma technology prepares monoclonal antibody, antibody has been widely used in the Diagnosis and Treat of clinical disease.The advantages such as current therapeutic monoclonal antibodies medicine has become fastest-rising field in bio-pharmaceuticals, and monoclonal antibody medicine is strong with targeting, clear curative effect, side effect are little are widely used in the field such as autoimmune disorder, cancer.Wherein, Antineoplastic angiogenesis antibody has the characteristics such as targeting, specificity, specificity, can specific inhibition is tumor vascular develops, and reduces the lethal effect to its hetero-organization.Antibody drug rhuMAb-VEGF (bevacizumab, Avastin) at present for clinical Antineoplastic angiogenesis was used for the treatment of nonsmall-cell lung cancer in 2004 by U.S. FDA approval, and showed good curative effect in the treatment of other tumours.But, because it also has restraining effect to VEGFR1 and VEGFR3, block the associated signal paths of VEGF mediation comprehensively, caused patient to occur the serious adverse reactions such as hypertension, nasal bleeding, proteinuria.The heavy chain antibody of natural existence disappearance light chain in camel body, its variable region is minimum functional antigen binding fragment.In general, the relative molecular mass of this antibody-like is 15KDa, is only 1/10 of conventional antibody, and its numberator height is 4.8nm, and diameter is 2.2nm, therefore is called nano antibody (nanobody).Nano antibody is as minimum functional antigen binding fragment, and unique biological structure feature imparts their many advantages, as strong in stability, solubility is good, immunogenicity is low, easy expression etc.Nano antibody contains 3 complementary determining regions (complementaritydeterminingregions, CDRs).Find from the three-dimensional arrangement of nano antibody, longer CDR3 and adjacent CDR1 or CDR2 connects into ring by disulfide linkage and carrys out Stable conformation.Dished or the two dimensional structure of the antigen binding domain shape of conventional antibodies Fab fragment and single-chain antibody ScFv antibody, the site being positioned at antigenic surface can only be identified, and the CDR3 of heavy chain antibody is longer, stable large bulge loop structure can be formed, the antigen deeply with crack sunk structure is inner, the avtive spot, virus and host cellular binding sites etc. of such as enzyme.Therefore nano antibody has antigen bonding force more widely.Secondly, there is cysteine residues (Cys) in the CDR1 district of nano antibody and skeleton district, can form disulphide bridges, add stability and the structural changes of variable region with the Cys residue in CDR3 district.In addition, nano antibody has that molecular weight is little, penetration power is comparatively strong and can be prepared into multipurpose antibody or carry the features such as chemical drug, makes this antibody-like to solid tumor with need to have significant advantage in the treatment through hemato encephalic barrier disease.In addition, the introducing of the technology such as random mutation and phage display also makes the screening process of the structure in nano antibody library and specific nano antibody become more convenient, efficient.VEGFR2 is one of most important drug target, has wide Prospect of R & D and important realistic meaning.But, the research and development limiting VEGFR2 antibody drug that the technical bottleneck of conventional antibodies affinity maturation, protein expression and purification and biological effect is serious.
Summary of the invention
In order to solve the problems of the technologies described above, an object of the present invention be to provide a kind of have high-affinity and good anti-tumor activity, efficient, toxic side effect is low and be easy to the anti-vegf R2 people source nano antibody produced.
The present invention's object is on the other hand to provide a kind of method preparing above-mentioned antibody.
The object of another aspect of the invention is to provide above-mentioned antibody for the preparation of the purposes in treatment antitumor drug.
In order to realize foregoing invention object, the present invention adopts following technical scheme:
The invention provides a kind of anti-vegf R2 people source nano antibody, comprise framework region and complementary determining region, it is characterized in that, described nano antibody has the aminoacid sequence shown in SEQIDNO:1.
The present invention also provides a kind of preparation method preparing anti-vegf R2 nano antibody, comprises the following steps:
A. adopt display technique of bacteriophage to filter out the nano antibody be combined with VEGFR2, and by gene compare of analysis, pick out and repeat the highest sequence;
B. the nano antibody that previous step filters out is rebuild in expression vector, and adopt procaryotic cell expression system expression, obtain high purity protein; And
C. the antibody that the avidity adopting AlphaScreen technology to filter out further from the nano antibody that previous step filters out is the highest.
Below above-mentioned steps is described in more detail.
A. display technique of bacteriophage is adopted to screen the nano antibody be combined with VEGFR2
Preferably, first adopt biopanning technique screening positive bacteriophage, and enrichment that positive colony is increased in Host Strains TG1; Then carry out positive colony determined dna sequence, gene compare of analysis, select and repeat the highest sequence.
More preferably, first adopt liquid phase panning technique that antigen is fixed on magnetic bead surfaces, add containing nano antibody storehouse, people source in conjunction with in liquid, screening positive bacteriophage, and enrichment that positive colony is increased in Host Strains TG1, preferably, described amplification is carried out 4 and is taken turns; Then in the end two-wheeled elutriation, random choose positive colony carries out gene sequencing and gene compare of analysis, selects the sequence that multiplicity is the highest.
B. the expression and purification of nano antibody
Preferably, the pcr amplification product after the sequence application NotI picked out and BamHI restriction enzyme and DNA recombinant technology being cut by enzyme inserts and carries in the pET carrier of the linearization for enzyme restriction of 6His-sumo label, construction expression plasmid; Logarithmic phase is cultured to, abduction delivering by Plastid transformation to Host Strains; Collect bacterial precipitation, the abundant cracking bacterium of ultrasonic method, gets supernatant, is thick leach protein; Thick leach protein, through nickel affinity column and molecular sieve column, obtains purified protein samples; With the expression of electrophoresis testing goal albumen, the clone higher to the frequency of occurrences carries out prokaryotic system expression and purification.
C. the screening of nano antibody avidity preferably, adopts the interaction between AlphaScreen technical measurement antigen and antibody, the antibody that the avidity filtered out further is the highest.
The present invention also provides above-described anti-vegf R2 people source nano antibody for the preparation of the purposes in anticancer medicine.
Wherein, described cancer comprises mammary cancer, lung cancer, bladder cancer, colorectal carcinoma, cervical cancer, hepatocellular carcinoma, hemangioblastoma, cerebral glioma.
The present inventor, by furtheing investigate the mechanism of VEGFR2 and even whole VEGFRs receptor family part identification and receptor activation, adopts display technique of bacteriophage filter out enrichment humanization nano antibody and carry out gene sequencing and protein expression and purification to it first; The nano antibody had with antigen compared with high-bond is gone out further by AlphaScreen technology screening; Obtain the people source nano antibody NTV1 with more high-affinity and good anti-tumor activity.By the dynamic behavior of this antibody of surface plasma resonance (SPR) characterized by techniques; Cell experiment demonstrates it and suppresses vascular endothelial cell proliferation ability and angiogenesis inhibiting activity.The present invention expresses and optimizes and obtains the high and VEGFR2 nano antibody albumen of stable uniform of avidity, people source anti-vegf R2 nano antibody medicine is researched and developed in success, open the frontier of the medicament research and development of target VEGFR2 antibody, there is far-reaching social effect and wide potential applicability in clinical practice.Further, the present invention is that relevant subsequent medicament research and development provides the foundation, and can research and develop accordingly and carry chemical drug or merge the antibody of Fc to increase curative effect.
Accompanying drawing explanation
Fig. 1: display NTV (1-4) four kinds of nano antibody amino acid alignment.The gene order of antibody is translated into aminoacid sequence, carries out Multiple Sequence Alignment.Unlabelled is complementary determining region, mark be framework region.
Fig. 2: the expression and purification of display NTV (1-4) albumen.The polyacrylamide gel electrophoresis of 10% detects purity of protein, and standard molecular weight is marked on the left of gel.From left to right be respectively NTV1, NTV2, NTV3, NTV4.
Fig. 3: AlphaScreen detects the reaction between VEGFR2D3 and NTV (1-4).(A) AlphaScreen reacts schematic diagram, the AlphaScreen detected result of (B-E) NTV1, NTV2, NTV3, NTV4 and VEGFR2D3 reaction.Fig. 4: SPR analyzes NTV1 and VEGFR2D3 reaction kinetics.
Fig. 5: NTV1 effect to human umbilical vein cell proliferation.
Fig. 6: NTV1 vitro inhibition HUVEC vascularization effect.
Embodiment
Following examples and experimental example laboratory apparatus used and experiment material information as follows:
Reagent and test kit
Plasmid little extraction reagent kit hundred Tyke
PCR primer reclaims test kit hundred Tyke
Sepharose reclaims test kit hundred Tyke
The raw work of agarose
Peptone sigma
Yeast extract sigma
Tris-basesigma
Vitamin H sigma
Imidazoles sigma
Restriction endonuclease BamH1NEB
Restriction endonuclease Not1NEB
T4DNA ligase enzyme Fermentas
Q5-HighFidelityDNApolymeraseNEB
Maltose sigma
Phage display library HuSdLTMCreativeBioLabs
Helper phage M13K07helperphageGEHealthcare
Magnetic bead
myOne
tMstreptavidinC1lifetechnologies
The raw work of IPTG
PEG8000sigma
SDSsigma
Tween-20 traditional Chinese medicines
BiotinCAPtureKitSeriesSGEHealthcare
AlphascreenHistidineDetectionKitPerkinElmer
Plant and instrument
AKTAFPLC system (GE)
NanoVuespectrophotometer(GE)
centrifuges5415R(Eppendorf)
SorvallRC12BPcentrifuge(Thermo)
avantiJ-26xp(BECKMANCOULTER)
Incubator/shakerfor2Lbacterialcultures(Thermo)
Chromatography freezer (moral sky, Beijing is helped)
PCRmachines(Eppendorf)
High pressure cell cracker
Ultraviolet spectrophotometer (BECKMANCOULTER)
Bioradimager(BIA-Rad)
Pure water instrument (Milli-Q)
Bechtop (AIRTECH)
High-pressure steam sterilizing pan (MIURA)
Liquid-transfering gun (device) (Eppendorf)
BiacoreT200(GEHealthcare)
Envision microplate reader (PerkinElmer)
CO2 incubator (Thermo)
Flat Tissue Culture Plate (NunclonDenmark)
Crystallography instrument (FORMULATRIX)
ArtRobbinsInstruments(PHOENIX)
Crystal incubator (MolecularDimensions)
Microscope (OLYMPUS)
Constant-temperature table (her Fu Sen Bioisystech Co., Ltd)
Preparation embodiment
The present embodiment provides a kind of preparation method preparing anti-vegf R2 nano antibody, comprises the following steps:
A. display technique of bacteriophage screening antibodies is adopted
Biotin-VEGFR2 fused antigen is coated on magnetic bead surfaces, hatches 2 hours for 4 DEG C.After PBS washes 3 times, close 1 hour with the PBS containing 2% skim-milk in 37 DEG C.Be 1 × 10 by capacity
12the people source nano antibody phage antibody library of clone's number adds in the immune pipe after closing, and 37 DEG C in conjunction with 1 hour.Wash 10 times with PBST, PBS washes 5 times.Add 1mL glycine elution damping fluid (pH2.2), room temperature shake 10min, sucking-off elutriant, is neutralized to pH7.4 with Tris.The sucking-off elutriant TG1 room temperature of 10 times of volumes infects 10min, measures titre and proceed to 37 DEG C to add helper phage M13KO7 jolting overnight incubation.Above process carries out four-wheel.Thus filter out nano antibody VEGFR2 being had to avidity.Select 300 clones from the culture plate after last two-wheeled screening at random, carry out gene sequencing and gene comparison.Pick out the clone standby that sequence repetition number is higher.Experimental result shows: obtain 8 nano antibodies that may be combined with VEGFR2 altogether, finds through gene comparison, and the frequency that in 8 clones, NTV1 and NTV2 occurs is the highest, and each complementary determinant conservative property not high (as shown in table 1 and Fig. 1).Prompting display technique of bacteriophage can filter out the nano antibody be combined with VEGFR2.
B. the expression and purification of nano antibody
Pcr amplification product after the sequence application NotI picked out and BamHI restriction enzyme and DNA recombinant technology being cut by enzyme inserts and carries in the pET carrier of the linearization for enzyme restriction of 6His-sumo label, construction expression plasmid.With the sequence gene filtered out (i.e. NTV1-4) for masterplate, introduce BamH1, Not1 restriction enzyme site, goal gene is introduced and carries in the Pet-duet expression vector of 6His-sumo label, construction expression plasmid.By cut NTV1-4 gene fragment and carrier that glue reclaims in molar ratio 5:1 set up 10 μ l linked systems, T4DNA ligase enzyme 25 DEG C connection 10min.Connection product is proceeded to competent cell TOP10, and the bacterium liquid got after 150 μ l conversions coats the LB solid medium flat board containing 50 μ g/mlAmp, is inverted quiescent culture for 37 DEG C and spends the night.Picked clones, proceeds to e. coli bl21 competent cell with reference to above step by expression plasmid, and secondary morning receives LB flat board.100ml sterilizing LB liquid nutrient medium is loaded 250ml shaking flask, and extremely wherein, 37 DEG C, 180rpm/min shakes overnight incubation to the BL21 bacterial strain mono-clonal on picking LB flat board; Survey the bacterium liquid OD value of incubated overnight, be seeded in 4 × 2LLB (OD value 0.15) shaking flask, 24 DEG C, 180rpm/min shakes cultivation; Survey its OD600 after about 4 hours, when between it is to 0.6 ~ 0.8, shaking table temperature is adjusted to 16 DEG C, and lowering the temperature and continuing to be cultured to OD value is between 0.8 ~ 1.2; Adding IPTG (0.5M), is 50 ~ 100 μMs to ultimate density, 16 DEG C of overnight induction (16 ~ 18h), collected by centrifugation bacterial sediment (4000rpm/min, 30 minutes); Be transferred in 50ml centrifuge tube after the abundant suspendible of every 2L bacterium liquid 30mlHisBufferA, ice bath.Use high pressure cracker smudge cells.16000rpm/min, high speed centrifugation 20 minutes, collects supernatant and is thick leach protein.Thick leach protein is crossed nickel post.Nickel post is loaded onto FPLC system, and setting elution program carries out albumen wash-out.By volume (slightly larger than 10ml) extremely suitable for the protein concentration collected, cross molecular sieve column and be further purified target protein, obtain purified protein samples.With the expression of 12%SDS-PAGE electrophoresis detection target protein.The clone NTV1-4 higher to the frequency of occurrences carries out prokaryotic system expression and purification, and as shown in Figure 2, result prompting adopts present method can obtain the albumen of stable homogeneous.
C. adopt AlphaScreen to screen further and and adopt SPR technique to measure the avidity of nano antibody and VEGFR2
Because false-positive result may appear in the positive colony be enriched to by biopanning technique, therefore application AlphaScreen technology filters out the nano antibody the highest with VEGFR2 avidity further from the antibody that previous step filters out.Get 1mg luminous particle and add centrifuge tube respectively containing antigen or antibody response liquid, normal temperature lucifuge hatches 1h.By two kinds of particulate mixing (concentration is biotin-NTV (1-4) and the mixing of His8-VEGFR2D3 equal proportion of 20nM, 50nM, 100nM, 200nM); to hatch after 15min sensed light signal in nanometer homogeneous phase time discrimination fluorescence detector, each concentration in triplicate.As shown in Figure 3, the visible AlphaScreen technology that adopts has filtered out the nano antibody NYV1 the highest with VEGFR2 avidity to AlphaScreen detected result further.
Application is based on the BIAcoreT200 systematic study nano antibody of Applications of surface plasmon resonance and the binding ability of VEGFR2.CAP sensor chip arranges 2 passages, and a coupling VEGFR2 is as sense channel, and another does not fix VEGFR2 as blank reference channel.Using HBS solution as working fluid, flow velocity is 2 μ L/min; Activation and closed chip; Again with the flow velocity of 2 μ L/min2 respectively with gradient concentration sample introduction nano antibody, each concentration rank detects 2 times; Acquisition, in conjunction with dynamic collection of illustrative plates, carries out calculation of parameter by software module after process of fitting treatment.As shown in Figure 4, injection concentration is 10 μMs of biotinylated NTV1, makes signal reach about 3500RU, is 20nM, 60nM, 180nM, 550nM, the VEGFR2D3 of 1.6 μMs, 5 μMs flow through chip surface, draw kinetic curve according to signal intensity by concentration.By BiacoreT200 assessment software calculations incorporated constant (ka) and dissociation constant (kd).Dissociation equilibrium constant (K is obtained by kd/ka
d).SPR technique confirms the binding ability of NTV1 and VEGFR2, its dissociation constant (K
d) be 4.9nM, prompting nano antibody NTV1 and VEGFR2 has very high avidity, infers that it may possess anti-tumor function.
Experimental example: nano antibody biological activity determination
This experimental example adopts MTT experiment to measure suppression Human umbilical vein endothelial cells (HUVECs) ability of cell proliferation of nano antibody, uses microtubule to form measuring nano antibody to the impact of HUVECs angiogenesis.
MTT experiment: the nano antibody adding respective concentration after hatching HUVECs24 hour with the substratum (5%FBS-ECM) that HUVECs cell is special at 37 DEG C, in Tissue Culture Plate, add MTT dyestuff after continuing to hatch 72 hours, after 4 hours, read absorbancy with under microplate reader 570nm.
Be that to be added to HUVECs concentration be 5 × 10 for the NTV1 of 1nM, 10nM, 100nM and 1000nM by concentration
3in 96 orifice plates of cells/well, in 570nm wavelength place reading after 72 hours, blank is set.Each concentration repeats 3 times, and calculate standard deviation, carry out the statistical analysis of P<0.05, result as shown in Figure 5.As seen from Figure 5, compared with control group, NTV1 has significant restraining effect when concentration is 10nM, 100nM, 1000nM to HUVECs propagation, and presents dose-dependent relation.
Micro-tube formation assay: by 4.5 × 10
3hUVECs is inoculated into and spreads in advance by 37 DEG C of overnight incubation in 96 orifice plates of Geltrix.Get culture plate next day under inverted microscope, observe each hole microtubule generation situation.
Be that to be added to HUVEC concentration be 4.5 × 10 for the NTV1 of 0nM, 10nM, 100nM, 1000nM by concentration
3night incubation in 96 orifice plates of cells/well, form photographic analysis by inverted microscope Human Umbilical Vein Endothelial Cells official jargon, artificial counting is carried out in 100 times of amplifications, does not add NTV1 as a control group, counts 100%.Each concentration repeats 3 times, calculates standard deviation, carries out the statistical analysis of P<0.05.As shown in Figure 6, compared with blank (0nMNTV1), NTV1, under 100nM, 1000nM concentration, significantly can suppress the formation of endotheliocyte official jargon to result, and along with the rising of concentration, and this restraining effect has and becomes large trend.
Above-mentioned experiment in vitro shows that NTV1 can effectively suppress HUVECs cell proliferation and Angiogenesis.
Above embodiment is only enumerate as the example of embodiment of the present invention, does not form any restriction to the present invention, it will be appreciated by those skilled in the art that the amendment in the scope not departing from essence of the present invention and design all falls into protection scope of the present invention.
Claims (8)
1. an anti-vegf R2 people source nano antibody, comprises framework region and complementary determining region, it is characterized in that, described nano antibody has the aminoacid sequence shown in SEQIDNO:1.
2. prepare a preparation method for anti-vegf R2 nano antibody, comprise the following steps:
A. adopt display technique of bacteriophage to filter out the nano antibody be combined with VEGFR2, and by gene compare of analysis, pick out and repeat the highest sequence; ;
B. the nano antibody that previous step filters out is rebuild in expression vector, and adopt procaryotic cell expression system expression, obtain high purity protein; And
C. the antibody that the avidity adopting AlphaScreen technology to filter out further from the nano antibody that previous step filters out is the highest.
3. method according to claim 2, is characterized in that, in step a, first adopts biopanning technique screening positive bacteriophage, and enrichment that positive colony is increased in Host Strains TG1; Then carry out positive colony determined dna sequence, gene compare of analysis, select and repeat the highest sequence.
4. method according to claim 3, is characterized in that, in step a, adopt liquid phase panning technique that antigen is fixed on magnetic bead surfaces, add containing nano antibody storehouse, people source in conjunction with in liquid, screening positive bacteriophage, and enrichment that positive colony is increased in Host Strains TG1, preferably carry out 4 and take turns; Then in the end two-wheeled elutriation, random choose positive colony carries out gene sequencing and gene compare of analysis, selects the sequence that multiplicity is the highest.
5. method according to claim 2, it is characterized in that, in stepb, pcr amplification product after the sequence application NotI picked out and BamHI restriction enzyme and DNA recombinant technology being cut by enzyme inserts and carries in the pET carrier of the linearization for enzyme restriction of 6His-sumo label, construction expression plasmid; Logarithmic phase is cultured to, abduction delivering by Plastid transformation to Host Strains; Collect bacterial precipitation, the abundant cracking bacterium of ultrasonic method, gets supernatant, is thick leach protein; Thick leach protein, through nickel affinity column and molecular sieve column, obtains purified protein samples; With the expression of electrophoresis testing goal albumen, the clone higher to the frequency of occurrences carries out prokaryotic system expression and purification.
6. method according to claim 2, is characterized in that, in step c, adopts the interaction between AlphaScreen technical measurement antigen and antibody, the antibody that the avidity filtered out further is the highest.
7. antibody according to claim 1 is preparing the purposes in anticancer medicine.
8. purposes according to claim 7, is characterized in that, described cancer comprises mammary cancer, lung cancer, bladder cancer, colorectal carcinoma, cervical cancer, hepatocellular carcinoma, hemangioblastoma and cerebral glioma.
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| WO2018233574A1 (en) * | 2017-06-20 | 2018-12-27 | 华兰生物工程股份有限公司 | Anti-pd-l1 humanized nanobody and use thereof |
| CN110382540A (en) * | 2017-01-05 | 2019-10-25 | 赫利克斯生物药品公司 | VEGFR-2 antibody |
| CN110407924A (en) * | 2019-07-24 | 2019-11-05 | 暨南大学 | Target binding protein and its application of CD133 |
| CN111139264A (en) * | 2020-01-20 | 2020-05-12 | 天津达济科技有限公司 | Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules |
| CN111763255A (en) * | 2020-07-01 | 2020-10-13 | 江苏莱森生物科技研究院有限公司 | Genetically modified VEGFA protein, monoclonal antibody thereof and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108289966A (en) * | 2015-09-24 | 2018-07-17 | 北卡罗来纳-查佩尔山大学 | Method and composition for reducing transfer |
| CN110382540A (en) * | 2017-01-05 | 2019-10-25 | 赫利克斯生物药品公司 | VEGFR-2 antibody |
| WO2018233574A1 (en) * | 2017-06-20 | 2018-12-27 | 华兰生物工程股份有限公司 | Anti-pd-l1 humanized nanobody and use thereof |
| CN110407924A (en) * | 2019-07-24 | 2019-11-05 | 暨南大学 | Target binding protein and its application of CD133 |
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| CN111139264A (en) * | 2020-01-20 | 2020-05-12 | 天津达济科技有限公司 | Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules |
| CN111763255A (en) * | 2020-07-01 | 2020-10-13 | 江苏莱森生物科技研究院有限公司 | Genetically modified VEGFA protein, monoclonal antibody thereof and application |
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